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Dominant role of splenic marginal zone lipid rafts in the classical complement pathway against S. pneumoniae

Dominant role of splenic marginal zone lipid rafts in the classical complement pathway against S.... Lipid rafts (LRs) play crucial roles in complex physiological processes, modulating innate and acquired immune responses to pathogens. The transmembrane C-type lectins human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and its mouse homolog SIGN-R1 are distributed in LRs and expressed on splenic marginal zone (MZ) macrophages. The DC-SIGN-C1q or SIGN-R1-C1q complex could mediate the immunoglobulin (Ig)-independent classical complement pathway against Streptococcus pneumoniae. Precise roles of LRs during this complement pathway are unknown. Here we show that LRs are indispensable for accelerating the DC-SIGN- or SIGN-R1-mediated classical complement pathway against S. pneumoniae, thus facilitating rapid clearance of the pathogen. The trimolecular complex of SIGN-R1-C1q-C4 was exclusively enriched in LRs of splenic MZ macrophages and their localization was essential for activating C3 catabolism and enhancing pneumococcal clearance, which were abolished in SIGN-R1-knockout mice. However, DC-SIGN replacement on splenic MZ macrophage’s LRs of SIGN-R1-depleted mice reversed these defects. Disruption of LRs dramatically reduced pneumococcal uptake and decomposition. Additionally, DC- SIGN, C1q, C4, and C3 were obviously distributed in + + splenic LRs of cadavers. Therefore, LRs on splenic SIGN-R1 or DC-SIGN macrophages could provide spatially confined and optimal bidirectional platforms, not only for usual intracellular events, for example recognition and phagocytosis of pathogens, but also an unusual extracellular event such as the complement system. These findings improve our understanding of the orchestrated roles of the spleen, unraveling a new innate immune system initiated from splenic MZ LRs, and yielding answers to several long-standing problems, including the need to understand the profound role of LRs in innate immunity, the need to identify how such a small portion of splenic SIGN-R1 macrophages (<0.05% of splenic macrophages) effectively resist S. pneumoniae, and the need to understand how LRs can promote the protective function of DC-SIGN against S. pneumoniae in the human spleen. Introduction Lipid rafts (LRs) are small (10–200 nM), highly Correspondence: Young-Sun Kang (kangys1967@naver.com) dynamic, detergent-resistant membrane fractions enri- Department of Biomedical Science and Technology, Konkuk University, ched in cholesterol and glycosphingolipid content on the 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea 1,2 plasma membranes of eukaryotic cells . Although LRs Department of Obstetrics and Gynecology, Division of Maternal and Fetal Medicine, Research Institute of Medical Science, Konkuk University School of comprise only a small percentage of the cell surface area , Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea their sizes can increase by coalescence with other raft Full list of author information is available at the end of the article. 4,5 units , providing spatiotemporal platforms for many These authors contributed equally: Seung Woo Yang, Jin-Yeon Park Edited by A. Rufini © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Yang et al. Cell Death Discovery (2019) 5:133 Page 2 of 17 molecular entities . LRs play crucial roles in complex Accordingly, in this study, the role of LRs was examined physiological processes, such as phagocytosis, in C-type lectin-mediated phagocytosis in order to eluci- receptor–receptor associations, receptor–pathogen asso- date the role of DC-SIGN in the human spleen. ciations, and signal transduction in many pathological 1,2,7–9 situations , modulating innate and acquired immune Results 10 + responses . LRs on splenic MZ DC-SIGN macrophages may be The complement system is important for several innate important for DC-SIGN-mediated uptake and and adaptive resistance mechanisms and consists of a decomposition of S. pneumoniae highly regulated cascade of more than 30 serum com- DC-SIGN transfectants were immunostained for DC- plement proteins that can be triggered by the recognition SIGN and the raft protein GM-1 ganglioside with fluor- of a microbe . This system can be activated through escein isothiocyanate (FITC)/cholera toxin B (CTB) sub- classical, soluble mannose-binding lectin (MBL), and unit. Large aggregates of DC-SIGN were strongly 12,13 alternative pathways . A pivotal step in the comple- distributed in CTB-enriched vertex regions of the cells ment pathways is assembly of a C3 convertase, which (Fig. 1a). Moreover, DC-SIGN monomers and dimers were digests C3 to form microbial binding C3 fragments obviously enriched in LR fractions (fractions 4–6) com- Because C3 fragments , such as C3b, C3bi, and C3d, pared with non-LR fractions (Fig. 1a). Isolation of serve as ligands for complement receptors, their recipro- detergent-resistant raft fractions was confirmed with cal binding promotes the uptake and killing of microbes immunoblotting for flotillin-1 and caveolin-1 as repre- 15 40 by phagocytes . Thus, the complement system provides a sentative markers of planar LRs and caveolae, respectively . major extracellular defense mechanism against infectious The same experiment was performed with SIGN-R1 16 BMT organisms depletion and DC-SIGN transgenic mice (DC-SIGN / TKO Various immune receptors in LRs, such as Fc recep- SIGN-R1 ;Fig. 1b and Supplementary Fig. S1a, b) or 17,18 19–21 22 tors , cytokine receptors , B cell receptors , and T human cadavers (Supplementary Fig. S1c). DC-SIGN was 23 + cell receptors , increase their binding capacity through obviously expressed in LRs of splenic MZ DC-SIGN cells clustering and facilitate signaling to favor the clearance (Fig. 1c, d and Supplementary Fig. S1d). of intracellular pathogens . Some innate pattern recog- Disruption of LRs with methyl-β-cyclodextrin (MβCD), nition receptors, such as Toll-like receptors and trans- a cholesterol-extracting agent, did not reduce DC-SIGN membrane C-type lectins, translocate to LRs upon expression on the cellular surface, but altered its surface 25–30 stimulation with specific agonists distribution pattern (Fig. 1e). Moreover, when DC-SIGN , thus demonstrat- ing the importance of this membrane partitioning for the transfectants were incubated with carboxyfluorescein innate immune recognition of various pathogens .In succinimidyl ester (CFSE)-labeled S. pneumoniae, pneu- addition, various complement receptors (CR2, CR3, and mococcal uptake and decomposition were obvious in the 31,32 globular C1q receptor (gC1qR)) and complement cytoplasm, showing disintegrated particles of the pneu- 33,34 regulatory proteins (CD46, CD55, and CD59) are mococcal capsular polysaccharide of S. pneumoniae ser- distributed in the LRs of immune cells. otype 14 (CPS14) around the phagocytosed bacterium Transmembrane C-type lectin human dendritic cell- (Fig. 1f and Supplementary Fig. S1e). Similar results were specific intercellular adhesion molecule-3-grabbing non- observed after pretreatment with actinomycin-D or integrin (DC-SIGN, CD209) and its murine homolog cycloheximide, which did not affect the plasma membrane SIGN-R1 exhibit several common specificities, such as structure (Fig. 1f). However, pneumococcal uptake and acting as the principal receptors for the pneumococcal decomposition were dramatically reduced with disruption 35,36 capsular polysaccharide of S. pneumoniae (CPS) and of LRs using MβCD (Fig. 1f and Supplementary Fig. S1f) 37,38 human immunodeficiency virus-1 , binding to the or with inhibition of LR-dependent endocytosis using 24,39 complement C1q , and showing distribution in LRs dynamin inhibitory peptide (DIP) or transfection with 29,30 in vitro . Additionally, SIGN-R1 can initiate an dominant-negative dynamin (K44A; Fig. 1g and h, immunoglobulin (Ig)-independent classical complement respectively), only permitting microbial binding to the pathway by interacting with C1q against S. pneumoniae, cellular surface of DC-SIGN transfectants. The bacterial particularly on the cellular surface of splenic marginal decomposition ratios were quantitatively calculated zone (MZ) macrophages, facilitating their rapid clear- (Fig. 1f–h). ance . Similarly, the DC-SIGN-C1q complex may pro- vide an initiation site for the classical complement LRs on splenic MZ SIGN-R1 macrophages may be pathway under pathogenic conditions . important for SIGN-R1-mediated uptake and Although disruption of LRs significantly reduces DC- decomposition of S. pneumoniae SIGN- and SIGN-R1-mediated pneumococcal phagocy- Large aggregates of SIGN-R1 were observed in CTB- tosis, the specific mechanisms are still unknown. enriched vertex regions of SIGN-R1 transfectants and strong Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 3 of 17 Fig. 1 Distribution of DC-SIGN in splenic lipid rafts and its role in the uptake and decomposition of S. pneumoniae.a (Left) DCEK_DC-SIGN transfectants were immunostained for DC-SIGN (red), cholera toxin B (green), and DAPI (blue). Arrowheads (yellow) indicate the colocalization of DC- SIGN and cholera toxin B. (Right) After sucrose gradient ultracentrifugation of whole-cell lysates from DCEK_DC-SIGN transfectants, fractions were BMT immunoblotted for DC-SIGN, flotilin-1, and caveolin-1. b DC-SIGN /WT mice were intravenously injected with hamster IgG or 22D1 anti-SIGN-R1 monoclonal antibodies (100 μg, 24 h), and splenic cryosections were immunostained for SIGN-R1 (green), DC-SIGN (red), and SER4/CD169 (blue). c As BMT TKO in (a) (right), but spleens from DC-SIGN /SIGN-R1 mice were used, and immunoblotting for SIGN-R1 was performed. d As in a (right), but cadaver spleens were used, and representative results (#11–148) are presented. e (Left) DCEK_DC-SIGN transfectants were treated with MβCD (10 mM, 3 h), immunostained for DC-SIGN without permeabilization, and assessed by FACS. (Right) As in (left), but cells were immunostained for cholera toxin B (green) and DC-SIGN (red), followed by microscopic analysis. f–h (Left) DCEK_DC-SIGN transfectants were pretreated with f MβCD, actinomycin-D (AD; 5 μg/mL, 24 h), or cycloheximide (CH; 20 μg/mL, 24 h) or with g the myristoylated dynamin inhibitory peptide (50 μM, 1 h) and washed out, or h transfected with empty vector or dominant-negative dynamin (K44A). Samples were then incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10 , 15 h, 37 °C), followed by immunostaining for f, g DC-SIGN (green) and CPS14 (red) or h dynamin (green) and CPS14 (red). (Right) The average percentage of pneumococcal decomposition of total pneumococcal binding on DC-SIGN transfectants was calculated in five areas from each sample in five independent experiments. Data are shown as mean ± SD. n.s., Not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars a, f, g, h, 20 µm; b, 200 µm; e,10µm distribution of SIGN-R1 monomers and dimers were obvious was evident only in LRs of both tissues of WT mice with a in LR fractions (Fig. 2a). In particular, multimers of SIGN-R1 higher concentration of SIGN-R1 multimers in LRs than in were preformed in LRs (inset in Fig. 2a). In whole fractions of non-LRs (Fig. 2b), but notfromSIGN-R1-knockout (KO) spleens or lymph nodes from wild-type (WT) mice, SIGN-R1 mice (Fig. 2c and Supplementary Fig. S2a). Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 4 of 17 Fig. 2 Distribution of SIGN-R1 in splenic lipid rafts and its role in the uptake and decomposition of S. pneumoniae.a (Left) DCEK_SIGN-R1 transfectants were immunostained for SIGN-R1 (red), cholera toxin b (green), and DAPI (blue). Arrowheads (yellow) indicate the colocalization of DC- SIGN and cholera toxin B. (Right) After sucrose gradient ultracentrifugation of whole cell lysates of DCEK_SIGN-R1 transfectants, fractions were immunoblotted for DC-SIGN, flotilin-1, or caveolin-1. Multimers of SIGN-R1 are presented in the box. b, c As in a (right), but spleens from wild-type and SIGN-R1-KO mice were used and immunoblotted for SIGN-R1, flotilin-1, and caveolin-1. d (Left) DCEK_SIGN-R1 transfectants were treated with MβCD (10 mM, 3 h), immunostained for SIGN-R1 without permeabilization, and assessed by FACS. (Right) As in (left), but cells were immunostained for cholera toxin B (green) and SIGN-R1 (red) and followed by microscopic analysis. e Cells in d were further incubated with FITC-dextran (5 μg, 30 min) or CPS14 (10 μg, 2 h), and their uptake was assessed by FACS (green or pink lines for uptake without or with MβCD, respectively). f–h (Left) DCEK_SIGN-R1 transfectants were pretreated with f MβCD, actinomycin-D (AD; 5 μg/mL, 24 h), cycloheximide (CD; 20 μg/mL, 24 h) or with g myristoylated dynamin inhibitory peptide (50 μM, 1 h) and washed out, or h transfected with dominant-negative dynamin (K44A) and incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10 , 15 h, 37 °C), followed by immunostaining for f, g SIGN-R1 (green) and CPS14 (red) or (H) dynamin (green) and CPS14 (red). (Right) The average percentage of pneumococcal decomposition of total pneumococcal binding on SIGN-R1 transfectants was calculated in five areas from each sample in five independent experiments. Data are shown as mean ± SD. n.s., Not significant; *p < 0.05; **p < 0.01; ***p < 0.001., Scale bars a, f, g, h, 20 µm; d,10 µm Disruption of LRs with MβCD did not reduce SIGN-R1 binding affinity for SIGN-R1 , the uptake and decom- expression on the transfectant cellular surface, but altered position of the organism were evident under control its surface distribution pattern (Fig. 2d). However, MβCD conditions (Fig. 2f and Supplementary Fig. S2b) and in the treatment of SIGN-R1 transfectants reduced the uptake of presence of actinomycin-D or cycloheximide (Fig. 2f). 41,42 dextran or CPS14, representative ligands of SIGN-R1 However, MβCD treatment of SIGN-R1 transfectants (Fig. 2e). Similarly, when SIGN-R1 transfectants were inhibited the uptake and decomposition of S. pneumoniae, incubated with S. pneumonia type 14, which has strong only permitting microbial binding to the cellular surface Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 5 of 17 (Fig. 2f). Moreover, inhibition of LR-dependent endocy- LRs or in non-LRs (Fig. 4c). Additionally, no differences in tosis using DIP or K44A dramatically reduced the uptake IgM, MBL-C, or factor B levels were found in any fraction and decomposition of the organism (Fig. 2g, h). The (Fig. 4d and Supplementary Fig. S4d). bacterial decomposition ratios were quantitatively calcu- In SIGN-R1-KO mice, the levels of C1q and C4 in lated (Fig. 2f–h). whole-cell lysates of all tissues were the same as those in control mice (Supplementary Fig. S4e, b). However, both complements were not increased at all following LRs of splenic MZ SIGN-R1 macrophages may provide an optimal location for innate recruitment of SIGN-R1 against S. pneumoniae exposure (Fig. 4e). Moreover, increases in S. pneumoniae in vivo C1q and C4 were completely abolished in splenic LRs TKO SIGN-R1 transfectants were incubated with S. pneu- from SIGN-R1 mice exposed to S. pneumoniae moniae at 37 °C or 4 °C or in the presence of MβCD, and (Supplementary Fig. S4f). To determine whether the abundant SIGN-R1 aggregation was observed on the cell specific recognition of CPS14 by SIGN-R1 was required surface only at 37 °C (Fig. 3a, b). When these cells were for upregulation of C1q and C4 in splenic LRs, splenic then fractionated and their LR fractions were immuno- fractions were immunoblotted for C1q and C4. Both blotted for SIGN-R1, SIGN-R1 monomers and multimers complements were not upregulated at all in any splenic were obviously increased in LRs (Fig. 3c). Because SIGN- fraction following exposure to mt-Pn14 or S. aureus R1 macrophages rapidly recognized S. pneumoniae in (Fig. 4f, g). splenic MZs within 1 h (Fig. 3d), SIGN-R1 distribution in splenic LRs was examined 1 h after intravenous injection LRs from splenic MZ SIGN-R1 macrophages may provide of S. pneumoniae. SIGN-R1 complex was obviously an optimal location for dominant C3 activation in response increased only in splenic LRs following S. pneumoniae to S. pneumoniae in a SIGN-R1-dependent manner in vivo stimulation (Fig. 3e), as confirmed in separate experi- We examined C3 levels in whole-cell lysates from the ments (Supplementary Fig. S3a, cases 1–4). spleen, liver, and lung of WT mice, and the lowest Similar experiments using intravenous injection of an expression was observed in the spleen (Supplementary unencapsulated mutant of S. pneumoniae serotype 14 Fig. S5a). However, immunoblotting of their fractions for (mt-Pn14)or Staphylococcus aureus, another gram- C3 showed that constitutive distribution of C3 was the positive coccal bacterium that does not bind SIGN- highest in splenic LRs among LRs of all tissues (Fig. 5a). 41,43 R1 showed no increase in SIGN-R1 complex in Furthermore, following intravenous injection of PBS or S. pneumoniae, the initial activation of C3 was most splenic LRs (Fig. 3f, g), confirming the CPS14 dependent recruitment of SIGN-R1 complex against S. pneumoniae. dominant in splenic LRs exposed to S. pneumoniae with a Next, we examined whether MARCO, a scavenger dramatic decrease in αC3, but a relatively minor decrease receptor expressed on partial SIGN-R1 MZ macro- was observed in splenic non-LRs (Fig. 5b). These results in phages , or SER4/CD169, a cell adhesion molecule spleen fractions were confirmed in separate experiments expressed on MZ metallophils, were recruited in splenic (Supplementary S5b, c). LRs following S. pneumoniae exposure. Neither target was In sera or splenic fractions from SIGN-R1-KO or SIGN- TKO recruited in splenic LRs following exposure to S. pneu- R1 mice, SIGN-R1 deficiency did not affect C3 levels moniae (Fig. 3h). in sera (Supplementary S5g) or distribution in splenic LRs without pneumococcal challenge (Fig. 5c, top, Supple- C1q and C4 are distributed in LRs of splenic MZ SIGN-R1 mentary Fig. S5f, g, top), but abolished C3 activation with macrophages and increased following S. pneumoniae pneumococcal challenge (Fig. 5c, bottom and Supple- exposure in a SIGN-R1-dependent manner in vivo mentary Fig. S5g, bottom). Moreover, CPS14 was essential The expression levels of C1q and C4 in whole-cell for SIGN-R1-mediated C3 activation in splenic LRs lysates from the spleens, livers, and lungs of control mice because C3 activation was significantly decreased or were similar in all tissues (Supplementary Fig. S4b). abolished in all splenic fractions from WT mice following However, higher levels of C1q and C4 were found in mt-Pn14 or S. aureus challenge, yielding abundant intact splenic LRs than in hepatic or pulmonary LRs or in non- C3α and weak or no increases in 43 kDa iC3b (Fig. 5d, e). LRs from all tissues (Fig. 4a). Other traditional mediators In C3-depleted mice, C3 deficiency in splenic LRs had no in different complement pathways (IgM, MBL-C, and effect on the constitutive distribution of SIGN-R1, C1q, or factor B) were barely present in LRs from all tissues C4 in splenic LRs or their increases against S. pneumoniae (Fig. 4b), but were variable in non-LRs (Supplementary (Fig. 5f). Fig. S4c). Following intravenous injection of phosphate- When WT cells or SIGN-R1 transfectants were incu- buffered saline (PBS) or S. pneumoniae, simultaneous bated with mouse serum, fixation of C4 and C3 was increases in C1q and C4 were obvious only in splenic LRs, observed only on SIGN-R1 transfectants and was sig- whereas no changes were found in hepatic or pulmonary nificantly enhanced in SIGN-R1 transfectants in the Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 6 of 17 Fig. 3 Accumulation and multimerization of SIGN-R1 in splenic lipid rafts following exposure to CPS14 from S. pneumoniae in vivo. a DCEK_SIGN-R1 transfectants were incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10 , 10 min) at 37 °C or 4 °C and immunostained for SIGN-R1 without permeabilization. b As in a, but cells were pretreated with MβCD (10 mM, 3 h) and incubated only at 37 °C. c As in a, but cell lysates at 37 °C were fractionated with sucrose gradient ultracentrifugation, and fractions of LRs were immunoblotted for SIGN-R1, flotilin-1, or caveolin-1. Multimers of SIGN-R1 are shown in the boxes. d In total, 1 × 10 CFSE-labeled MitC-Pn14 (green) were injected intravenously into wild-type mice for 0, 15, or 60 min, and splenic sections were immunostained for SIGN-R1 (blue). The binding or uptake of organisms into splenic MZs is shown in the boxes. (E) As in (C), but spleens were used before or after intravenous injection of live S. pneumoniae (Pn14; 1 × 10 , 1 hr) into wild-type mice. (F and G) As in e, but mice were injected intravenously with PBS or 1 × 10 cells of an unencapsulated mutant of serotype 14 S. pneumoniae (mt-Pn14) or Staphylococcus aureus for 1 h, respectively. h As in e, but fractions were immunoblotted for MARCO, SER4/CD169, flotilin-1, and caveolin-1. Scale bars a, b, 20 µm; d, 250 µm Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 7 of 17 Fig. 4 Predominant distribution of complement C1q and C4 in splenic lipid rafts and their upregulation after S. pneumoniae challenge in a SIGN-R1-dependent manner in vivo. a Spleens, livers, and lungs of control mice were fractionated with sucrose gradient ultracentrifugation, and fractions were immunoblotted for C1q, C4, flotilin-1, and caveolin-1. b As in a, but fractions were immunoblotted for IgM, MBL-C, or factor B. c As in a, but wild-type mice were injected intravenously with live S. pneumoniae (Pn14; 1 × 10 , 1 h). d As in c, but fractions were immunoblotted for IgM, MBL- C, or factor B. e As in a, c, but SIGN-R1-KO mice were used, and splenic fractions were analyzed. f, g As in c, but mice were injected intravenously with an unencapsulated mutant of serotype 14 S. pneumoniae (mt-Pn14) or Staphylococcus aureus (1 × 10 , 1 h). All data are representative of five independent experiments presence of S. pneumoniae (Fig. 5g). Furthermore, fluor- Enrichment of SIGN-R1 and C1q enhances the escent microscopy revealed the colocalization of C4 and opsonization, uptake, and decomposition of S. C3 only in SIGN-R1 transfectants (Fig. 5h and Supple- pneumoniae mentary Fig. S5h), indicating that C3 activation was spe- Addition of SIGN-R1 significantly enhanced the fixa- cifically generated at the same location on SIGN-R1 cells tion of C1q on the pneumococcal surface (Fig. 6a). in which the SIGN-R1-mediated classical complement Sequentially, the increased concentration of C1q also pathway was initiated. accelerated the opsonization of iC3b in response to Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 8 of 17 Fig. 5 Dominant C3 activation in splenic lipid rafts in a SIGN-R1-dependent manner following S. pneumoniae challenge in vivo. a Spleens, livers, and lungs of control mice were fractionated with sucrose gradient ultracentrifugation, and fractions were immunoblotted for C3. b As in a,but wild-type mice were injected intravenously with PBS or live S. pneumoniae (Pn14; 1 × 10 ,1 h). c As in a, b, but SIGN-R1-KO mice were used, and splenic fractions were analyzed. d, e As in c, but mice were injected intravenously with an unencapsulated mutant of serotype 14 S. pneumoniae (mt-Pn14) or Staphylococcus aureus (1 × 10 ,1h). f As in c, but CVF-treated mice were used, and splenic fractions were immunoblotted for C3, SIGN-R1, C1q, and C4. g DCEK_WT and DCEK_SIGN-R1 were incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10 , 1 h) without or with 5% NMS in TC buffer for 15 min at 37 °C. Cells were immunostained for C4 or C3 with their respective isotype control IgGs and assessed by FACS. h As in g,but thebinding of C4 (red)orC3 (green) was assessed by fluorescence microscopy after fixing DCEK-SIGN-R1 transfectants with 1% paraformaldehyde. Arrowheads indicate colocalization of C4 and C3. Representative cells are shown in the box. All data are representative of five independent experiments. Scale bars h,10µm Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 9 of 17 Fig. 6 (See legend on next page.) S. pneumoniae and led to the formation of a membrane amount of C3 was fixed on S. pneumoniae under all attack complex on S. pneumoniae (Fig. 6b). Because the conditions. However, C3 activation was dominant only same amount of βC3 was used for fixation, the same with the addition of SIGN-R1, demonstrating the Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 10 of 17 (see figure on previous page) Fig. 6 SIGN-R1-mediated C3 activation and opsonization of S. pneumoniae enhances the uptake and decomposition of the bacteria by SIGN-R1 cells in vitro and in vivo. a Live S. pneumoniae type 14 (Pn14) was incubated with 5% NHS at 37 °C for 30 min in the presence of 10 μg bovine serum albumin, transferrin (Tfe), or purified SIGN-R1, immunostained for C1q, and analyzed by FACS. b As in a, but 10 μg Tfe or C1q was used, and organisms were immunostained for C1q, iC3b, or membrane attack complex (MAC). c As in a, but organisms were immunoblotted for C3. d In total, 1 × 10 mitomycin C-treated and PKH26-labeled S. pneumoniae (PKH26-MitC-Pn14) were incubated with CHO transfectants in the presence of 10 μg C1q or 5% NHS. The binding of organisms to cells was assessed by FACS. e DCEK_SIGN transfectants were incubated with MitC-Pn14 (1 × 10 , 15 h, 37 °C) without or with mouse sera (NMS or C3- or C1q-depleted mouse sera), and cells were immunostained for SIGN-R1 (green) or CPS14 (red), followed by microscopic analysis. Representative cells are shown in the boxes. f CFSE-MitC-Pn14 (1 × 10 , green) were injected intravenously into control, SIGN-R1-KO, or C3-KO mice for 1 h and spleen sections were immunostained for SIGN-R1 (blue) and CPS14 (red). Representative areas in splenic MZs are highlighted in the boxes (middle row). Organisms captured in the red pulp (arrowheads) are highlighted (bottom row). Scale bars e, 20 µm; f,50µm degradation of αC3 and increased fixation of small iC3b Fig. S7a). We then examined whether DC-SIGN bound (43 kDa; Fig. 6c). directly to C4, similar to SIGN-R1 (Supplementary With the addition of C1q or normal human serum Fig. S4a). DC-SIGN transfectants showed clear fixation of (NHS) as a source of C1q, pneumococcal binding was C4 (Fig. 7f). Additionally, DC-SIGN-transfected increased only on SIGN-R1 transfectants (Fig. 6d). HEK293T cells also induced the fixation of C1q or C4 Moreover, pneumococcal uptake and sequential decom- from NHS, confirming again the binding of DC-SIGN to position were observed on SIGN-R1 transfectants only C1q and C4 (Fig. 7g), and their fixation was significantly with normal mouse serum (NMS), but not without serum increased following S. pneumoniae challenge (Fig. 7g). BMT TKO or with C3- or C1q-depleted sera (Fig. 6e and Supple- In splenic fractions from DC-SIGN /SIGN-R1 mentary Fig. S6a). After intravenous injection of S. mice, upregulation of C1q and C4 was obvious following pneumoniae into WT, SIGN-R1-KO, or C3-KO mice, S. pneumoniae challenge (Fig. 7h). Moreover, after intra- BMT pneumococcal uptake and cytoplasmic decomposition venous injection of S. pneumoniae into DC-SIGN / + TKO were obvious only on splenic MZ SIGN-R1 macro- SIGN-R1 mice, systemic C3 activation reappeared in TKO phages from control mice (Fig. 6f and Supplementary Fig. SIGN-R1 mice as in control mice, demonstrating S6b), but completely abolished in splenic MZs from gradual decrease in αC3 and larger iC3b and a rapid SIGN-R1-KO and C3-KO mice and red pulp from all increase and sequential decrease in small iC3b (Fig. 7i). mice (Fig. 6f). The signal specificity of CPS14 on splenic C3 fixation was also completely recovered around splenic + + BMT MZ SIGN-R1 macrophages of control mice in Fig. 6f was MZ DC-SIGN macrophages in DC-SIGN /SIGN- TKO TKO confirmed by immunostaining with its respective isotype R1 mice from SIGN-R1-KO or SIGN-R1 mice as control immunoglobulins (Supplementary Fig. S6c). in control mice (Fig. 7j and Supplementary Fig. S7b), occurring rapidly within 15 min after challenge with S. DC-SIGN may mediate the classical complement pathway pneumoniae and disappearing at 4 h (Supplementary Fig. in LRs from splenic MZ DC-SIGN macrophages following S7c). Immunostaining with isotype control IgG for anti- S. pneumoniae challenge via interactions with C1q C3 antibodies did not yield a C3 signal (Supplementary When DC-SIGN transfectants were treated with Fig. S7d). Moreover, immunoblotting of cadaver spleens S. pneumoniae, DC-SIGN aggregates were dramatically for C1q, C4, and C3 revealed expression in human splenic increased on the cell surface (Fig. 7a). Additionally, when LRs, with predominant expression of C1q and 104-kDa LR fractions of these cells were immunoblotted for DC- αC3′ rather than 113-kDa αC3 (Fig. 7k and Supplemen- SIGN, monomers and dimers of DC-SIGN were obviously tary Fig. S7e). increased in LRs following S. pneumoniae challenge (Fig. 7b). Moreover, splenic MZ DC-SIGN macrophages Discussion BMT TKO from DC-SIGN /SIGN-R1 mice recovered the Although DC-SIGN and SIGN-R1 professionally 35,41 rapid recognition of S. pneumoniae on splenic MZs, even recognize and remove S. pneumonia , the disruption of at 15 min and 1 h after intravenous injection of the LRs on both lectin transfectants resulted in serious organisms (Fig. 7c). In spleens at 1 h, DC-SIGN mono- impairment of the uptake and sequential decomposition mers and multimers were obviously upregulated only in of the organism. Because the disruption of LRs did not splenic LRs following S. pneumoniae challenge (Fig. 7d). alter the expression levels of both lectins on the respective DC-SIGN bound well to purified human C1q or human transfectants, which would lead to normal binding of the C1q from NHS (Fig. 7e). Additionally, DC-SIGN binding organism to the cell surface, the structure of LRs may be to mouse C1q from NMS was confirmed by immuno- more important for the function of both lectins and for blotting and immunostaining (Fig. 7e and Supplementary removal of S. pneumoniae than previously thought. This Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 11 of 17 Fig. 7 (See legend on next page.) Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 12 of 17 (see figure on previous page) Fig. 7 DC-SIGN in splenic lipid rafts may mediate the classical complement pathway in response to S. pneumoniae challenge by interacting with C1q and C4. a DCEK_DC-SIGN transfectants were incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10 , 15min, 37°C) and immunostained for DC-SIGN without permeabilization. A representative cell is shown in the box. b After DCEK_DC-SIGN transfectants were incubated with MitC-Pn14 (1 × 10 , 1 h, 37 °C), lysates were fractionated with sucrose gradient ultracentrifugation, and fractions of LRs were BMT TKO immunoblotted for DC-SIGN, flotilin-1, or caveolin-1. c DC-SIGN /SIGN-R1 mice were injected intravenously with CFSE-labeled MitC-Pn14 (green; 8 BMT TKO 1× 10 , 15 min or 1 h), and splenic sections were immunostained for DC-SIGN (red). d As in b, but spleens of DC-SIGN /SIGN-R1 mice were used after intravenous injection of live S. pneumoniae (1 × 10 , 1 h). e (Left) Wild-type or DCEK transfectants were incubated with 5 μg endotoxin-free ovalbumin (efOVA), human C1q, or 3% sera (NMS, NHS, or C1q-depleted human serum) in TC buffer for 1 h at 37 °C, and lysates were immunoblotted for C1q. (Right) Wild-type or DCEK_DC-SIGN transfectants were incubated with 3% NMS for 1 h at 37 °C and immunostained for C1q, followed by microscopic analysis. Representative areas are highlighted from low-power images in Figure S7a. f Wild-type or DCEK transfectants were incubated with 5% NMS for 10 min at 37 °C, immunostained for C4 without permeabilization, and analyzed by FACS. g Empty vector or DC-SIGN-transfected HEK293T cells were incubated without or with MitC-Pn14 (1 × 10 ) in the absence or presence of 5% NHS for 15 min at 37 °C, and cell lysates were BMT TKO immunoblotted for DC-SIGN, C1q, C4, and β-actin. h As in d, but DC-SIGN /SIGN-R1 mice were used, and splenic fractions were immunoblotted TKO BMT TKO 8 for C1q or C4. i Control, SIGN-R1 , and DC-SIGN /SIGN-R1 mice were injected intravenously with Pn14 (1 × 10 ), and their sera were collected TKO BMT TKO at 0, 15, 30, or 60 min and immunoblotted for C3. j As in c, but control, SIGN-R1 KO, SIGN-R1 , and DC-SIGN /SIGN-R1 mice were intravenously injected for 15 min. Splenic sections were immunostained for C3 (red) and SIGN-R1 (green) or C3 (red) and DC-SIGN (green). Representative areas are highlighted from low-power images in Figure S7b. k As in h, but spleens of cadavers were used and immunoblotted for C1q, C4, and C3. Representative data are presented from the results of four cadavers. Scale bars a, e, 20 µm; c, 250 µm; j,50µm speculation was strongly supported by the following could accelerate the generation of a classical pathway C3 results. First, multimers of DC-SIGN or SIGN-R1 were convertase (C4bC2a), and the resulting abundant C4b and preformed in LRs of the respective transfectants, and even C4bC2a could sequentially promote fixation of C3 on the 53–56 SIGN-R1 multimers were preformed in splenic LRs. cell surface . Supporting this possibility, C3, particu- Additionally, DC-SIGN and SIGN-R1 multimers were larly C3α, was most enriched in splenic LRs, despite increased only in LRs following S. pneumoniae challenge having lowest expression in the spleen among examined in vitro or in vivo, but not in non-LRs. Therefore, LRs mouse tissues. Therefore, splenic MZ SIGN-R1 LRs may could provide a spatially confined and optimal platform be optimized to activate C3, although the C3 distribution for DC-SIGN and SIGN-R1 to facilitate clustering, was independent of the existence of SIGN-R1, probably increase their affinity and specificity to pathogens, and due to covalent fixation of C3 fragments on other splenic enhance their clearance, thus affecting their innate pro- cells . Because the complement system should be tightly 7,45 tective functions . controlled under physiological conditions to prevent sig- Raft locations for target antigens favor classical com- nificant damage to self-tissues, enrichment of SIGN-R1, plement activation by concentrating the antigen–antibody C1q, C4, and C3 in splenic MZ SIGN-R1 LRs could also complex into a comparatively small area, thus providing precisely regulate complement activation in splenic MZs; an ideal density for juxtaposed Fc regions to engage indeed, nascent C3b and C4 regulate C1 activation in 46,47 58 C1q . Similarly, both C1q and C4 were exclusively concert with C3 under normal conditions . distributed in splenic LRs among fractions from examined Pneumococcal challenge increased both C1q and C4 and mouse tissues, showing a significant dependence on the activated C3 only in splenic MZ SIGN-R1 LRs without existence of SIGN-R1. SIGN-R1 was exclusively expressed the involvement of other complement pathways. There- 42 + on splenic MZ macrophages and bound directly to C1q fore, splenic MZ SIGN-R1 LRs may provide optimal 39,48 or C4 . Additionally, there is a potential interaction locations for SIGN-R1-mediated C3 activation to accel- 49,50 between C1q and C4 . Therefore, their exclusive dis- erate pneumococcal clearance by sequentially enhancing tribution in splenic LRs is expected, and these proteins pneumococcal iC3b opsonization and rapid phagocytosis + + may be further subdivided into splenic MZ SIGN-R1 primarily in splenic MZ SIGN-R1 macrophages. In par- LRs from heterogeneous LRs of various splenic cells . ticular, S. pneumoniae capsules inhibit its recognition by These findings suggested that splenic MZ SIGN-R1 LRs natural IgM, the binding of the serum proteins to sub- may provide optimal microenvironmental platforms for capsular targets, complement activity, and neutrophil 59,60 reciprocal interactions among SIGN-R1, C1q, and C4, phagocytosis . However, enrichment of SIGN-R1, C1q, thus preforming a trimolecular complex to rapidly acti- C4, and C3 in splenic MZ SIGN-R1 LRs could drama- vate the SIGN-R1-mediated classical complement path- tically enhance the simultaneous recognition of pneumo- way in the spleen. coccal CPSs with SIGN-R1 and C1q, thus overcoming Because SIGN-R1 or C1q directly recognize S. pneu- impairment of the initial pneumococcal recognition . 41,52 moniae , the preformed trimolecular complex of The human spleen and the classical pathway are integral + 39,62–65 SIGN-R1, C1q, and C4 in splenic MZ SIGN-R1 LRs for protection against infection by S. pneumoniae . Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 13 of 17 In particular, the DC-SIGN-C1q complex could also mutant Spn14.H, and Staphylococcus aureus strain mediate a classical complement pathway against S. (RN4220) were used, all of which were kindly provided by pneumoniae in the spleen, representing a potential pro- Professor Vincent A. Fischetti of Rockefeller University tection mechanism in the human spleen . Indeed, the (New York, USA). BMT replaced DC-SIGN on splenic MZs of DC-SIGN / TKO SIGN-R1 mice completely rescued the pneumococcal Cell culture capture, the sequential activation and deposition of C3 DCEK, a mouse L-cell fibroblast line, and human against S. pneumoniae in splenic MZs, which was likely to embryonic kidney (HEK293T) cells were cultured in be related to the DC-SIGN-induced complex of mouse RPMI-1640 medium, and CHO cells were cultured in C1q and C4. Previous studies have shown that recruit- Dulbecco’s modified Eagle’s medium, supplemented with ment of DC-SIGN into LRs with binding to viral glyco- 10% fetal bovine serum, 100 U/mL penicillin G, and 29,31,66 proteins or the gC1qR strengthens the possible 100 mg/mL streptomycin. Stable DCEK transfectants formation of a tetramolecular complex of glycoproteins/ expressing complementary (cDNA) for DC-SIGN or DC-SIGN/C1q/gC1qR and thus increases binding capa- SIGN-R1 (DCEK_DC-SIGNS and DCEK_SIGN-R1 in the 24,67 city through the complex . Therefore, similar to SIGN- figures, respectively) and stable CHO transfectants R1, splenic MZ DC-SIGN LRs may also provide a expressing cDNA for Neo, or SIGN-R1 (CHO_Neo, or favorable microenvironment for the DC-SIGN-mediated CHO_SIGN-R1 in the figures, respectively) were used. classical complement pathway by forming another tetra- molecular complex of DC-SIGN, C1q, gC1qR, and C4, In vivo animal studies finally enhancing sequential C3 activation. Female C57BL/6 mice (6–10 weeks old, weighing Because DC-SIGN is exclusively expressed on human 16–20 g) were purchased (The Jackson Laboratory, Bar splenic perifollicular zone macrophages , the presence of Harbor, ME, USA) and housed under specific pathogen-free DC-SIGN in cadaver splenic LRs indicates that DC-SIGN conditions. SIGN-R1 (CD209b)-KO mice were kindly pro- is expressed in LRs from human splenic perifollicular vided by the Consortium for Functional Glycomics (http:// zone macrophages. Also, similar with the previous www.functionalglycomics.org). DC-SIGN transgenic donor 32,68,69 reports , complements C1q, C4, and C3b were mice were provided by the Rockefeller Gene Targeting enriched in human splenic LRs. Therefore, these cadaver Resource Center and identified by PCR using DC-SIGN splenic perifollicular zone DC-SIGN LRs may also be gene primers forward (5′-CgggATCCgAgTggggTgACA TgAgTgACT-3′) and reverse (5′-ACgCgTCgACAAA important for the DC-SIGN-mediated classical comple- ment system against S. pneumoniae, explaining why the AgggggTgAAgTTCTgCTACg-3′). For in vivo experiments spleen and the classical pathway are integral for protec- using animal models, all studies were approved by the 63–65 tion against infections to S. pneumoniae in humans . Institutional Animal Care and Use Committee of Konkuk The importance of LRs has been restricted to their roles University (permit number: KU11107) and performed in in receptor-mediated intracellular events from the plasma strict accordance with the approved guidelines for animal membrane, so far. However, splenic MZ LRs provide care and animal experimentation. Animal welfare was bidirectional platforms not only for usual events associated overseen by local committees. Mice were housed in a with the intracellular milieu, for example, recognition and temperature-controlled room with an automated phagocytosis of pathogens in vivo, but also unusual events darkness–light cycle system and had ad libitum access to associated with the extracellular milieu, such as the com- food and water. Mice were challenged with 1 × 10 colony- plement system, orchestrating early and complicated host forming unit of S. pneumoniae suspended in 100 mL of PBS responses to microbial infection. Thus, the spleen could be in PBS intravenously. Prior to tissue dissection, the mice equipped with the most sensitive system to target various were sacrificed via euthanasia using an overdose of CO 35,36 pathogens, such as S. pneumoniae and human with a flow rate 20% of the cage volume per minute, fol- 37,38 immunodeficiency virus-1 . These findings can explain lowing AVMA Guidelines for the Euthanasia of Animals. how such a small portion of splenic SIGN-R1 macro- phages (<0.05%) helps the spleen to efficiently protect Bacterial growth conditions and fluorescent labeling hosts against S. pneumoniae, providing insights into the Streptococcus pneumoniae capsular serotype 14 involvement of LRs in the innate immune system and the (DCC1490), the unencapsulated S. pneumoniae Tn916 roles of DC-SIGN in the human spleen. mutant Spn14.H (mt-Pn14), and Staphylococcus aureus were grown in brain heart infusion broth (DIFCO) to Materials and methods mid-logarithmic phase (18 h, 37 °C). Bacteria were inac- Bacterial strains tivated with 50 μg/mL mitomycin C (Sigma) for 1 h and Streptococcus pneumoniae capsular serotype 14 suspensions of 10 bacteria were labeled with PKH26 (DCC1490), the unencapsulated S. pneumoniae Tn916 following the manufacturer’s instructions or with 5 mM Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 14 of 17 CFSE (Sigma-Aldrich) for 30 min at 37 °C. The fluor- of pneumococcal-bound cells was calculated in five areas escent bacteria were injected intravenously into mice or for each sample from five independent experiments. incubated with cells. Cell fractionation and purification of LRs with sucrose Cellular and tissue immunofluorescence microscopy gradients Cells on coverslips or 10 μm OCT (Tissue-Tek) frozen Tissues from mice or cells were solubilized in 2 mL of spleen sections were fixed with 100% acetone (10 min, 1% Triton X-100 (Junsei Chemical Co., Ltd.) in MES- room temperature) and stained, where indicated, with buffered saline (MBS, 25 mM MES, pH 6.5, 150 mM DAPI (4′,6-diamidino-2-phenylindole) (blue) or FITC-, NaCl). After homogenizing with 10 up-and-down strokes PE-, AMCA-, or Alexa Fluor-conjugated donkey anti- of a tight-fitting Dounce homogenizer, the tissue or cel- chicken IgY, goat anti-hamster IgG, donkey anti-rabbit lular extracts were adjusted to 4 mL with sucrose con- IgG, goat anti-rat IgG, and streptavidin as secondary centrations of 45% or 40%, respectively, and overlaid with reagents (purchased from Abcam, Jackson ImmunoR- 4 mL of 30% sucrose and 4 mL of 5% sucrose in MBS. The esearch Laboratories, or Molecular Probes). Cells and sucrose gradient was formed by centrifugation at spleen sections were examined for fluorescence with a 200,000 × g for 18–20 h at 4 °C using a Beckman SW41ti deconvolution fluorescence BX61-32FDIC microscope rotor. After centrifugation, the sucrose gradients were (Olympus Corp., Tokyo, Japan). Images were acquired fractionated into 12 fractions without pelleting, and an EZ with a Coolsnap system (Roper Scientific, Inc., opaque buoyant band corresponding to the LRs was col- AZ, USA). lected at the interface between the 30% and 5% sucrose gradients. The same quantity of proteins from each frac- Flow cytometry tion was used for immunoblotting analysis. Cells used in FACS analysis were detached with 1 mM EDTA in PBS for 10 min and pre-incubated 10 min with Plasmids and transfection 2.4G2 monoclonal antibody at 4 °C to block Fc receptors. Clone pMX DC-SIGN was a gift from Dr. Chae Gyu Cells or S. pneumoniae were incubated with 3–5% mouse Park (Rockefeller University). This clone c DC-SIGN or human serum in 200 μL TC buffer (10 mM Tris-HCl, expression was monitored by immunoblotting analysis 140 mM NaCl, 2 mM CaCl , 2 mM MgCl , and 1% bovine using anti-DC-SIGN antibodies. DCEK transfectants were 2 2 serum albumin [BSA]), and complement binding to cells transiently transfected with plasmids encoding dynamin II or organisms was detected by immunostaining with K44A (dominant-negative dynamin with a point mutation respective antibodies for 30 min at 4 °C. Cytometric ana- in the nucleotide-binding site, a gift from Professor lysis was performed using a FACScan (Becton Dickinson, Seung-Jae Lee, Seoul National University College of San Jose, CA, USA) and CellQuestPro (BD Biosciences). Medicine, Seoul, Korea) or with an empty pcDNA3 vector Subsequent data analysis was performed with CellQuest- for 48 h, using Lipofectamine Plus reagent (Invitrogen Life Pro (BD Biosciences). Technologies, Carlsbad, CA, USA) according to the manufacturer’s specifications. Streptococcus pneumoniae uptake and decomposition analysis in vitro and in vivo SIGN-R1 TKO mouse generation Cells (1 × 10 ) were incubated with mitomycin SIGN-R1 TKO or isotype control mice were generated C-treated and CFSE-labeled S. pneumoniae (1 × 10 ) for by intravenous injection of 100 μg 22D1 antibody or iso- the indicated times at 37 °C, and bacterial binding to cells type hamster IgG for 48 h. The 22D1 antibody selectively and cytoplasmic decomposition of capsular poly- and transiently depleted the surface SIGN-R1 molecule, saccharides of S. pneumoniae were examined with a but not SIGN-R1 macrophages in the splenic MZ, per- fluorescence microscope. Mice were intravenously injec- mitting analysis of its function in vivo, as in a previous ted with mitomycin C-treated and CFSE-labeled report . 8 39,41 S. pneumoniae (1 × 10 ) for the indicated times , and bacterial binding and decomposition were examined on DC-SIGN transgenic mouse generation − + BMT SIGN-R1 or SIGN-R1 cells of spleen tissues. DC-SIGN transgenic (DC-SIGN /WT) mice were generated from the reconstitution of lethally irradiated Quantification of bacterial decomposition ratios C57BL/6 mice with bone marrow cells of DC-SIGN To quantify the pneumococcal decomposition ratio, the transgenic donor mice. SIGN-R1-depleted DC-SIGN BMT TKO number of pneumococcal-bound or -decomposing cells transgenic (DC-SIGN /SIGN-R1 ) mice were gen- was counted, and the average percentage of erated from the intravenous injection of 22D1 for 48 h BMT pneumococcal-decomposing cells from the total number into DC-SIGN /WT mice. Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 15 of 17 Complement C3-depleted mice polyvinylidene difluoride membranes, followed by incu- Control mice were obtained by intraperitoneally bation with antibodies. Antibody-reactive bands on the injecting 60 U/kg of cobra venom factor one day prior to blots were visualized with peroxidase-labeled secondary the experimental challenge. C3 depletion was confirmed antibodies, followed by treatment with West-ZOL plus by Western blot analysis for C3 by using sera. (Intron) or Immobilon (Millipore). Mouse infection studies Human cadaver spleen studies Mitomycin C-treated fluorescent bacteria (1 × 10 ) were Human cadaveric spleens were donated from the intravenously administered to mice for the indicated Department of Anatomy, School of Medicine, Konkuk times. Mice were sacrificed, and spleen sections were University (Seoul, Korea). examined by deconvolution fluorescence microscopy. Software used in this study Assay for in vivo and in vitro C3 processing Fluorescent images were analyzed with the MetaMorph To quantify C3 processing in tissues in vivo, 1 × 10 software (Universal Imaging). Immunoblotting signals S. pneumoniae were injected intravenously, and tissue were detected using LAS-4200 (Fuji Film). lysates were collected at the indicated times, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis Statistical analyses (SDS-PAGE), and immunoblotted with polyclonal anti- The number of cells binding to bacteria and the number mouse C3 antibodies. For in vitro C3 processing, 1 × 10 of cells decomposing bacteria were counted. The average S. pneumoniae were incubated with 5% NHS in TC buffer percentage of pneumococcal decomposition of the total (140 mM NaCl, 2 mM CaCl , 2 mM MgCl , 10 mM Tris, pneumococcal binding to cells was calculated in five areas 2 2 pH 7.5, supplemented with 1% BSA) for 30 min at 37 °C. of each sample from three independent experiments and The bacteria were washed, mixed, and boiled with 20 µL shown in the indicated graphs. Data are presented as of 2× SDS sample buffer. Bacterial lysates were separated mean ± SD. Statistical significance between groups were by SDS-PAGE and immunoblotted with polyclonal anti- determined by two-way analysis of variance, followed by mouse C3 antibodies. These antibodies detected the Tukey’s post hoc tests and unpaired Student’s t tests with components of native C3, αC3, and βC3, as well as the a two-tailed test. p Value < 0.05 was taken to indicate fragments of αC3 (molecular weights in humans: 113 kDa statistical significance (not significant, ns; *p < 0.05; for αC3, 104 kDa of αC3′, 63 kDa and 41 kDa for iC3b **p < 0.01; ***p < 0.001) fragments, and 75 kDa for βC3; molecular weights in rats: 120 kDa for αC3, 70 kDa and 43 kDa for iC3b fragments, Acknowledgements and 65 kDa for βC3) that are generated during C3 pro- This work was supported by the Basic Science Research Program through the cessing by C3 convertases by immunoblotting analysis. In National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (S201803S00049); Health Technology R & D the steady state, αC3 and βC3 are predominant, with a few Project through the Korea Health Industry Development Institute (KHIDI), Ministry larger iC3b. With activation, αC3 and larger iC3b are of Health & Welfare, Republic of Korea (HI17C1713); and Basic Science Research rapidly processed into smaller fragments, including Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2017R1C1B2010487). 43 kDa iC3b, but not βC3, which serves as a loading control for the immunoblotting analysis, resulting in loss of most of the detectable αC3 as well as larger iC3b, but Author details accumulation of the smaller iC3b in C3 activation in Department of Biomedical Science and Technology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea. immunoblotting analysis . Department of Obstetrics and Gynecology, Division of Maternal and Fetal Medicine, Research Institute of Medical Science, Konkuk University School of Western blot analysis and immunodetection Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea. Department of Anatomy, Research Institute of Medical Science, Konkuk University School of Tissues, cells, and bacteria were lysed in RIPA buffer Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea. Department (150 mM NaCl, 50 mM Tris-HCl, pH 8.0/1% Nonidet P- of Oncology, Georgetown University Medical Center, Washington, DC, USA. 40/0.5% sodium deoxycholate/0.1% SDS) supplemented Department of Pathology, New York University School of Medicine, New York, NY 10016, USA. Laboratory Animal Center, KBIO Health, Osongsarngmyon-ro with 0.2% protease inhibitor cocktail (Sigma), and lysed 123, Ghungju-si, Chungbuk, Korea. Department of Veterinary Pharmacology samples were mixed with an equal volume of 2× SDS and Toxicology, Veterinary Science Research Institute, College of Veterinary sample buffer containing 2-mercaptoethanol and boiled at Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea. Department of Infectious Diseases, College of Veterinary Medicine, 95 °C for 5 min (100 °C for 10 min). Diluted sera (1:50) or Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea fractions of LRs were mixed with 2× or 5× SDS sample buffer with 2-mercaptoethanol and boiled at 95 °C for 10 min (100 °C for 10 min). The samples were separated Conflict of interest by SDS-PAGE on 4–15% gradient gels and transferred to The authors declare that they have no conflict of interest. Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 16 of 17 Publisher’s note 25. Triantafilou, M., Morath, S., Mackie, A., Hartung, T. & Triantafilou, K. Lateral Springer Nature remains neutral with regard to jurisdictional claims in diffusion of Toll-like receptors reveals that they are transiently confined within published maps and institutional affiliations. lipid rafts on the plasma membrane. J. Cell Sci. 117,4007–4014 (2004). 26. 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Dominant role of splenic marginal zone lipid rafts in the classical complement pathway against S. pneumoniae

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Copyright © 2019 by The Author(s)
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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Abstract

Lipid rafts (LRs) play crucial roles in complex physiological processes, modulating innate and acquired immune responses to pathogens. The transmembrane C-type lectins human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and its mouse homolog SIGN-R1 are distributed in LRs and expressed on splenic marginal zone (MZ) macrophages. The DC-SIGN-C1q or SIGN-R1-C1q complex could mediate the immunoglobulin (Ig)-independent classical complement pathway against Streptococcus pneumoniae. Precise roles of LRs during this complement pathway are unknown. Here we show that LRs are indispensable for accelerating the DC-SIGN- or SIGN-R1-mediated classical complement pathway against S. pneumoniae, thus facilitating rapid clearance of the pathogen. The trimolecular complex of SIGN-R1-C1q-C4 was exclusively enriched in LRs of splenic MZ macrophages and their localization was essential for activating C3 catabolism and enhancing pneumococcal clearance, which were abolished in SIGN-R1-knockout mice. However, DC-SIGN replacement on splenic MZ macrophage’s LRs of SIGN-R1-depleted mice reversed these defects. Disruption of LRs dramatically reduced pneumococcal uptake and decomposition. Additionally, DC- SIGN, C1q, C4, and C3 were obviously distributed in + + splenic LRs of cadavers. Therefore, LRs on splenic SIGN-R1 or DC-SIGN macrophages could provide spatially confined and optimal bidirectional platforms, not only for usual intracellular events, for example recognition and phagocytosis of pathogens, but also an unusual extracellular event such as the complement system. These findings improve our understanding of the orchestrated roles of the spleen, unraveling a new innate immune system initiated from splenic MZ LRs, and yielding answers to several long-standing problems, including the need to understand the profound role of LRs in innate immunity, the need to identify how such a small portion of splenic SIGN-R1 macrophages (<0.05% of splenic macrophages) effectively resist S. pneumoniae, and the need to understand how LRs can promote the protective function of DC-SIGN against S. pneumoniae in the human spleen. Introduction Lipid rafts (LRs) are small (10–200 nM), highly Correspondence: Young-Sun Kang (kangys1967@naver.com) dynamic, detergent-resistant membrane fractions enri- Department of Biomedical Science and Technology, Konkuk University, ched in cholesterol and glycosphingolipid content on the 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea 1,2 plasma membranes of eukaryotic cells . Although LRs Department of Obstetrics and Gynecology, Division of Maternal and Fetal Medicine, Research Institute of Medical Science, Konkuk University School of comprise only a small percentage of the cell surface area , Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea their sizes can increase by coalescence with other raft Full list of author information is available at the end of the article. 4,5 units , providing spatiotemporal platforms for many These authors contributed equally: Seung Woo Yang, Jin-Yeon Park Edited by A. Rufini © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Yang et al. Cell Death Discovery (2019) 5:133 Page 2 of 17 molecular entities . LRs play crucial roles in complex Accordingly, in this study, the role of LRs was examined physiological processes, such as phagocytosis, in C-type lectin-mediated phagocytosis in order to eluci- receptor–receptor associations, receptor–pathogen asso- date the role of DC-SIGN in the human spleen. ciations, and signal transduction in many pathological 1,2,7–9 situations , modulating innate and acquired immune Results 10 + responses . LRs on splenic MZ DC-SIGN macrophages may be The complement system is important for several innate important for DC-SIGN-mediated uptake and and adaptive resistance mechanisms and consists of a decomposition of S. pneumoniae highly regulated cascade of more than 30 serum com- DC-SIGN transfectants were immunostained for DC- plement proteins that can be triggered by the recognition SIGN and the raft protein GM-1 ganglioside with fluor- of a microbe . This system can be activated through escein isothiocyanate (FITC)/cholera toxin B (CTB) sub- classical, soluble mannose-binding lectin (MBL), and unit. Large aggregates of DC-SIGN were strongly 12,13 alternative pathways . A pivotal step in the comple- distributed in CTB-enriched vertex regions of the cells ment pathways is assembly of a C3 convertase, which (Fig. 1a). Moreover, DC-SIGN monomers and dimers were digests C3 to form microbial binding C3 fragments obviously enriched in LR fractions (fractions 4–6) com- Because C3 fragments , such as C3b, C3bi, and C3d, pared with non-LR fractions (Fig. 1a). Isolation of serve as ligands for complement receptors, their recipro- detergent-resistant raft fractions was confirmed with cal binding promotes the uptake and killing of microbes immunoblotting for flotillin-1 and caveolin-1 as repre- 15 40 by phagocytes . Thus, the complement system provides a sentative markers of planar LRs and caveolae, respectively . major extracellular defense mechanism against infectious The same experiment was performed with SIGN-R1 16 BMT organisms depletion and DC-SIGN transgenic mice (DC-SIGN / TKO Various immune receptors in LRs, such as Fc recep- SIGN-R1 ;Fig. 1b and Supplementary Fig. S1a, b) or 17,18 19–21 22 tors , cytokine receptors , B cell receptors , and T human cadavers (Supplementary Fig. S1c). DC-SIGN was 23 + cell receptors , increase their binding capacity through obviously expressed in LRs of splenic MZ DC-SIGN cells clustering and facilitate signaling to favor the clearance (Fig. 1c, d and Supplementary Fig. S1d). of intracellular pathogens . Some innate pattern recog- Disruption of LRs with methyl-β-cyclodextrin (MβCD), nition receptors, such as Toll-like receptors and trans- a cholesterol-extracting agent, did not reduce DC-SIGN membrane C-type lectins, translocate to LRs upon expression on the cellular surface, but altered its surface 25–30 stimulation with specific agonists distribution pattern (Fig. 1e). Moreover, when DC-SIGN , thus demonstrat- ing the importance of this membrane partitioning for the transfectants were incubated with carboxyfluorescein innate immune recognition of various pathogens .In succinimidyl ester (CFSE)-labeled S. pneumoniae, pneu- addition, various complement receptors (CR2, CR3, and mococcal uptake and decomposition were obvious in the 31,32 globular C1q receptor (gC1qR)) and complement cytoplasm, showing disintegrated particles of the pneu- 33,34 regulatory proteins (CD46, CD55, and CD59) are mococcal capsular polysaccharide of S. pneumoniae ser- distributed in the LRs of immune cells. otype 14 (CPS14) around the phagocytosed bacterium Transmembrane C-type lectin human dendritic cell- (Fig. 1f and Supplementary Fig. S1e). Similar results were specific intercellular adhesion molecule-3-grabbing non- observed after pretreatment with actinomycin-D or integrin (DC-SIGN, CD209) and its murine homolog cycloheximide, which did not affect the plasma membrane SIGN-R1 exhibit several common specificities, such as structure (Fig. 1f). However, pneumococcal uptake and acting as the principal receptors for the pneumococcal decomposition were dramatically reduced with disruption 35,36 capsular polysaccharide of S. pneumoniae (CPS) and of LRs using MβCD (Fig. 1f and Supplementary Fig. S1f) 37,38 human immunodeficiency virus-1 , binding to the or with inhibition of LR-dependent endocytosis using 24,39 complement C1q , and showing distribution in LRs dynamin inhibitory peptide (DIP) or transfection with 29,30 in vitro . Additionally, SIGN-R1 can initiate an dominant-negative dynamin (K44A; Fig. 1g and h, immunoglobulin (Ig)-independent classical complement respectively), only permitting microbial binding to the pathway by interacting with C1q against S. pneumoniae, cellular surface of DC-SIGN transfectants. The bacterial particularly on the cellular surface of splenic marginal decomposition ratios were quantitatively calculated zone (MZ) macrophages, facilitating their rapid clear- (Fig. 1f–h). ance . Similarly, the DC-SIGN-C1q complex may pro- vide an initiation site for the classical complement LRs on splenic MZ SIGN-R1 macrophages may be pathway under pathogenic conditions . important for SIGN-R1-mediated uptake and Although disruption of LRs significantly reduces DC- decomposition of S. pneumoniae SIGN- and SIGN-R1-mediated pneumococcal phagocy- Large aggregates of SIGN-R1 were observed in CTB- tosis, the specific mechanisms are still unknown. enriched vertex regions of SIGN-R1 transfectants and strong Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 3 of 17 Fig. 1 Distribution of DC-SIGN in splenic lipid rafts and its role in the uptake and decomposition of S. pneumoniae.a (Left) DCEK_DC-SIGN transfectants were immunostained for DC-SIGN (red), cholera toxin B (green), and DAPI (blue). Arrowheads (yellow) indicate the colocalization of DC- SIGN and cholera toxin B. (Right) After sucrose gradient ultracentrifugation of whole-cell lysates from DCEK_DC-SIGN transfectants, fractions were BMT immunoblotted for DC-SIGN, flotilin-1, and caveolin-1. b DC-SIGN /WT mice were intravenously injected with hamster IgG or 22D1 anti-SIGN-R1 monoclonal antibodies (100 μg, 24 h), and splenic cryosections were immunostained for SIGN-R1 (green), DC-SIGN (red), and SER4/CD169 (blue). c As BMT TKO in (a) (right), but spleens from DC-SIGN /SIGN-R1 mice were used, and immunoblotting for SIGN-R1 was performed. d As in a (right), but cadaver spleens were used, and representative results (#11–148) are presented. e (Left) DCEK_DC-SIGN transfectants were treated with MβCD (10 mM, 3 h), immunostained for DC-SIGN without permeabilization, and assessed by FACS. (Right) As in (left), but cells were immunostained for cholera toxin B (green) and DC-SIGN (red), followed by microscopic analysis. f–h (Left) DCEK_DC-SIGN transfectants were pretreated with f MβCD, actinomycin-D (AD; 5 μg/mL, 24 h), or cycloheximide (CH; 20 μg/mL, 24 h) or with g the myristoylated dynamin inhibitory peptide (50 μM, 1 h) and washed out, or h transfected with empty vector or dominant-negative dynamin (K44A). Samples were then incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10 , 15 h, 37 °C), followed by immunostaining for f, g DC-SIGN (green) and CPS14 (red) or h dynamin (green) and CPS14 (red). (Right) The average percentage of pneumococcal decomposition of total pneumococcal binding on DC-SIGN transfectants was calculated in five areas from each sample in five independent experiments. Data are shown as mean ± SD. n.s., Not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars a, f, g, h, 20 µm; b, 200 µm; e,10µm distribution of SIGN-R1 monomers and dimers were obvious was evident only in LRs of both tissues of WT mice with a in LR fractions (Fig. 2a). In particular, multimers of SIGN-R1 higher concentration of SIGN-R1 multimers in LRs than in were preformed in LRs (inset in Fig. 2a). In whole fractions of non-LRs (Fig. 2b), but notfromSIGN-R1-knockout (KO) spleens or lymph nodes from wild-type (WT) mice, SIGN-R1 mice (Fig. 2c and Supplementary Fig. S2a). Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 4 of 17 Fig. 2 Distribution of SIGN-R1 in splenic lipid rafts and its role in the uptake and decomposition of S. pneumoniae.a (Left) DCEK_SIGN-R1 transfectants were immunostained for SIGN-R1 (red), cholera toxin b (green), and DAPI (blue). Arrowheads (yellow) indicate the colocalization of DC- SIGN and cholera toxin B. (Right) After sucrose gradient ultracentrifugation of whole cell lysates of DCEK_SIGN-R1 transfectants, fractions were immunoblotted for DC-SIGN, flotilin-1, or caveolin-1. Multimers of SIGN-R1 are presented in the box. b, c As in a (right), but spleens from wild-type and SIGN-R1-KO mice were used and immunoblotted for SIGN-R1, flotilin-1, and caveolin-1. d (Left) DCEK_SIGN-R1 transfectants were treated with MβCD (10 mM, 3 h), immunostained for SIGN-R1 without permeabilization, and assessed by FACS. (Right) As in (left), but cells were immunostained for cholera toxin B (green) and SIGN-R1 (red) and followed by microscopic analysis. e Cells in d were further incubated with FITC-dextran (5 μg, 30 min) or CPS14 (10 μg, 2 h), and their uptake was assessed by FACS (green or pink lines for uptake without or with MβCD, respectively). f–h (Left) DCEK_SIGN-R1 transfectants were pretreated with f MβCD, actinomycin-D (AD; 5 μg/mL, 24 h), cycloheximide (CD; 20 μg/mL, 24 h) or with g myristoylated dynamin inhibitory peptide (50 μM, 1 h) and washed out, or h transfected with dominant-negative dynamin (K44A) and incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10 , 15 h, 37 °C), followed by immunostaining for f, g SIGN-R1 (green) and CPS14 (red) or (H) dynamin (green) and CPS14 (red). (Right) The average percentage of pneumococcal decomposition of total pneumococcal binding on SIGN-R1 transfectants was calculated in five areas from each sample in five independent experiments. Data are shown as mean ± SD. n.s., Not significant; *p < 0.05; **p < 0.01; ***p < 0.001., Scale bars a, f, g, h, 20 µm; d,10 µm Disruption of LRs with MβCD did not reduce SIGN-R1 binding affinity for SIGN-R1 , the uptake and decom- expression on the transfectant cellular surface, but altered position of the organism were evident under control its surface distribution pattern (Fig. 2d). However, MβCD conditions (Fig. 2f and Supplementary Fig. S2b) and in the treatment of SIGN-R1 transfectants reduced the uptake of presence of actinomycin-D or cycloheximide (Fig. 2f). 41,42 dextran or CPS14, representative ligands of SIGN-R1 However, MβCD treatment of SIGN-R1 transfectants (Fig. 2e). Similarly, when SIGN-R1 transfectants were inhibited the uptake and decomposition of S. pneumoniae, incubated with S. pneumonia type 14, which has strong only permitting microbial binding to the cellular surface Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 5 of 17 (Fig. 2f). Moreover, inhibition of LR-dependent endocy- LRs or in non-LRs (Fig. 4c). Additionally, no differences in tosis using DIP or K44A dramatically reduced the uptake IgM, MBL-C, or factor B levels were found in any fraction and decomposition of the organism (Fig. 2g, h). The (Fig. 4d and Supplementary Fig. S4d). bacterial decomposition ratios were quantitatively calcu- In SIGN-R1-KO mice, the levels of C1q and C4 in lated (Fig. 2f–h). whole-cell lysates of all tissues were the same as those in control mice (Supplementary Fig. S4e, b). However, both complements were not increased at all following LRs of splenic MZ SIGN-R1 macrophages may provide an optimal location for innate recruitment of SIGN-R1 against S. pneumoniae exposure (Fig. 4e). Moreover, increases in S. pneumoniae in vivo C1q and C4 were completely abolished in splenic LRs TKO SIGN-R1 transfectants were incubated with S. pneu- from SIGN-R1 mice exposed to S. pneumoniae moniae at 37 °C or 4 °C or in the presence of MβCD, and (Supplementary Fig. S4f). To determine whether the abundant SIGN-R1 aggregation was observed on the cell specific recognition of CPS14 by SIGN-R1 was required surface only at 37 °C (Fig. 3a, b). When these cells were for upregulation of C1q and C4 in splenic LRs, splenic then fractionated and their LR fractions were immuno- fractions were immunoblotted for C1q and C4. Both blotted for SIGN-R1, SIGN-R1 monomers and multimers complements were not upregulated at all in any splenic were obviously increased in LRs (Fig. 3c). Because SIGN- fraction following exposure to mt-Pn14 or S. aureus R1 macrophages rapidly recognized S. pneumoniae in (Fig. 4f, g). splenic MZs within 1 h (Fig. 3d), SIGN-R1 distribution in splenic LRs was examined 1 h after intravenous injection LRs from splenic MZ SIGN-R1 macrophages may provide of S. pneumoniae. SIGN-R1 complex was obviously an optimal location for dominant C3 activation in response increased only in splenic LRs following S. pneumoniae to S. pneumoniae in a SIGN-R1-dependent manner in vivo stimulation (Fig. 3e), as confirmed in separate experi- We examined C3 levels in whole-cell lysates from the ments (Supplementary Fig. S3a, cases 1–4). spleen, liver, and lung of WT mice, and the lowest Similar experiments using intravenous injection of an expression was observed in the spleen (Supplementary unencapsulated mutant of S. pneumoniae serotype 14 Fig. S5a). However, immunoblotting of their fractions for (mt-Pn14)or Staphylococcus aureus, another gram- C3 showed that constitutive distribution of C3 was the positive coccal bacterium that does not bind SIGN- highest in splenic LRs among LRs of all tissues (Fig. 5a). 41,43 R1 showed no increase in SIGN-R1 complex in Furthermore, following intravenous injection of PBS or S. pneumoniae, the initial activation of C3 was most splenic LRs (Fig. 3f, g), confirming the CPS14 dependent recruitment of SIGN-R1 complex against S. pneumoniae. dominant in splenic LRs exposed to S. pneumoniae with a Next, we examined whether MARCO, a scavenger dramatic decrease in αC3, but a relatively minor decrease receptor expressed on partial SIGN-R1 MZ macro- was observed in splenic non-LRs (Fig. 5b). These results in phages , or SER4/CD169, a cell adhesion molecule spleen fractions were confirmed in separate experiments expressed on MZ metallophils, were recruited in splenic (Supplementary S5b, c). LRs following S. pneumoniae exposure. Neither target was In sera or splenic fractions from SIGN-R1-KO or SIGN- TKO recruited in splenic LRs following exposure to S. pneu- R1 mice, SIGN-R1 deficiency did not affect C3 levels moniae (Fig. 3h). in sera (Supplementary S5g) or distribution in splenic LRs without pneumococcal challenge (Fig. 5c, top, Supple- C1q and C4 are distributed in LRs of splenic MZ SIGN-R1 mentary Fig. S5f, g, top), but abolished C3 activation with macrophages and increased following S. pneumoniae pneumococcal challenge (Fig. 5c, bottom and Supple- exposure in a SIGN-R1-dependent manner in vivo mentary Fig. S5g, bottom). Moreover, CPS14 was essential The expression levels of C1q and C4 in whole-cell for SIGN-R1-mediated C3 activation in splenic LRs lysates from the spleens, livers, and lungs of control mice because C3 activation was significantly decreased or were similar in all tissues (Supplementary Fig. S4b). abolished in all splenic fractions from WT mice following However, higher levels of C1q and C4 were found in mt-Pn14 or S. aureus challenge, yielding abundant intact splenic LRs than in hepatic or pulmonary LRs or in non- C3α and weak or no increases in 43 kDa iC3b (Fig. 5d, e). LRs from all tissues (Fig. 4a). Other traditional mediators In C3-depleted mice, C3 deficiency in splenic LRs had no in different complement pathways (IgM, MBL-C, and effect on the constitutive distribution of SIGN-R1, C1q, or factor B) were barely present in LRs from all tissues C4 in splenic LRs or their increases against S. pneumoniae (Fig. 4b), but were variable in non-LRs (Supplementary (Fig. 5f). Fig. S4c). Following intravenous injection of phosphate- When WT cells or SIGN-R1 transfectants were incu- buffered saline (PBS) or S. pneumoniae, simultaneous bated with mouse serum, fixation of C4 and C3 was increases in C1q and C4 were obvious only in splenic LRs, observed only on SIGN-R1 transfectants and was sig- whereas no changes were found in hepatic or pulmonary nificantly enhanced in SIGN-R1 transfectants in the Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 6 of 17 Fig. 3 Accumulation and multimerization of SIGN-R1 in splenic lipid rafts following exposure to CPS14 from S. pneumoniae in vivo. a DCEK_SIGN-R1 transfectants were incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10 , 10 min) at 37 °C or 4 °C and immunostained for SIGN-R1 without permeabilization. b As in a, but cells were pretreated with MβCD (10 mM, 3 h) and incubated only at 37 °C. c As in a, but cell lysates at 37 °C were fractionated with sucrose gradient ultracentrifugation, and fractions of LRs were immunoblotted for SIGN-R1, flotilin-1, or caveolin-1. Multimers of SIGN-R1 are shown in the boxes. d In total, 1 × 10 CFSE-labeled MitC-Pn14 (green) were injected intravenously into wild-type mice for 0, 15, or 60 min, and splenic sections were immunostained for SIGN-R1 (blue). The binding or uptake of organisms into splenic MZs is shown in the boxes. (E) As in (C), but spleens were used before or after intravenous injection of live S. pneumoniae (Pn14; 1 × 10 , 1 hr) into wild-type mice. (F and G) As in e, but mice were injected intravenously with PBS or 1 × 10 cells of an unencapsulated mutant of serotype 14 S. pneumoniae (mt-Pn14) or Staphylococcus aureus for 1 h, respectively. h As in e, but fractions were immunoblotted for MARCO, SER4/CD169, flotilin-1, and caveolin-1. Scale bars a, b, 20 µm; d, 250 µm Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 7 of 17 Fig. 4 Predominant distribution of complement C1q and C4 in splenic lipid rafts and their upregulation after S. pneumoniae challenge in a SIGN-R1-dependent manner in vivo. a Spleens, livers, and lungs of control mice were fractionated with sucrose gradient ultracentrifugation, and fractions were immunoblotted for C1q, C4, flotilin-1, and caveolin-1. b As in a, but fractions were immunoblotted for IgM, MBL-C, or factor B. c As in a, but wild-type mice were injected intravenously with live S. pneumoniae (Pn14; 1 × 10 , 1 h). d As in c, but fractions were immunoblotted for IgM, MBL- C, or factor B. e As in a, c, but SIGN-R1-KO mice were used, and splenic fractions were analyzed. f, g As in c, but mice were injected intravenously with an unencapsulated mutant of serotype 14 S. pneumoniae (mt-Pn14) or Staphylococcus aureus (1 × 10 , 1 h). All data are representative of five independent experiments presence of S. pneumoniae (Fig. 5g). Furthermore, fluor- Enrichment of SIGN-R1 and C1q enhances the escent microscopy revealed the colocalization of C4 and opsonization, uptake, and decomposition of S. C3 only in SIGN-R1 transfectants (Fig. 5h and Supple- pneumoniae mentary Fig. S5h), indicating that C3 activation was spe- Addition of SIGN-R1 significantly enhanced the fixa- cifically generated at the same location on SIGN-R1 cells tion of C1q on the pneumococcal surface (Fig. 6a). in which the SIGN-R1-mediated classical complement Sequentially, the increased concentration of C1q also pathway was initiated. accelerated the opsonization of iC3b in response to Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 8 of 17 Fig. 5 Dominant C3 activation in splenic lipid rafts in a SIGN-R1-dependent manner following S. pneumoniae challenge in vivo. a Spleens, livers, and lungs of control mice were fractionated with sucrose gradient ultracentrifugation, and fractions were immunoblotted for C3. b As in a,but wild-type mice were injected intravenously with PBS or live S. pneumoniae (Pn14; 1 × 10 ,1 h). c As in a, b, but SIGN-R1-KO mice were used, and splenic fractions were analyzed. d, e As in c, but mice were injected intravenously with an unencapsulated mutant of serotype 14 S. pneumoniae (mt-Pn14) or Staphylococcus aureus (1 × 10 ,1h). f As in c, but CVF-treated mice were used, and splenic fractions were immunoblotted for C3, SIGN-R1, C1q, and C4. g DCEK_WT and DCEK_SIGN-R1 were incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10 , 1 h) without or with 5% NMS in TC buffer for 15 min at 37 °C. Cells were immunostained for C4 or C3 with their respective isotype control IgGs and assessed by FACS. h As in g,but thebinding of C4 (red)orC3 (green) was assessed by fluorescence microscopy after fixing DCEK-SIGN-R1 transfectants with 1% paraformaldehyde. Arrowheads indicate colocalization of C4 and C3. Representative cells are shown in the box. All data are representative of five independent experiments. Scale bars h,10µm Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 9 of 17 Fig. 6 (See legend on next page.) S. pneumoniae and led to the formation of a membrane amount of C3 was fixed on S. pneumoniae under all attack complex on S. pneumoniae (Fig. 6b). Because the conditions. However, C3 activation was dominant only same amount of βC3 was used for fixation, the same with the addition of SIGN-R1, demonstrating the Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 10 of 17 (see figure on previous page) Fig. 6 SIGN-R1-mediated C3 activation and opsonization of S. pneumoniae enhances the uptake and decomposition of the bacteria by SIGN-R1 cells in vitro and in vivo. a Live S. pneumoniae type 14 (Pn14) was incubated with 5% NHS at 37 °C for 30 min in the presence of 10 μg bovine serum albumin, transferrin (Tfe), or purified SIGN-R1, immunostained for C1q, and analyzed by FACS. b As in a, but 10 μg Tfe or C1q was used, and organisms were immunostained for C1q, iC3b, or membrane attack complex (MAC). c As in a, but organisms were immunoblotted for C3. d In total, 1 × 10 mitomycin C-treated and PKH26-labeled S. pneumoniae (PKH26-MitC-Pn14) were incubated with CHO transfectants in the presence of 10 μg C1q or 5% NHS. The binding of organisms to cells was assessed by FACS. e DCEK_SIGN transfectants were incubated with MitC-Pn14 (1 × 10 , 15 h, 37 °C) without or with mouse sera (NMS or C3- or C1q-depleted mouse sera), and cells were immunostained for SIGN-R1 (green) or CPS14 (red), followed by microscopic analysis. Representative cells are shown in the boxes. f CFSE-MitC-Pn14 (1 × 10 , green) were injected intravenously into control, SIGN-R1-KO, or C3-KO mice for 1 h and spleen sections were immunostained for SIGN-R1 (blue) and CPS14 (red). Representative areas in splenic MZs are highlighted in the boxes (middle row). Organisms captured in the red pulp (arrowheads) are highlighted (bottom row). Scale bars e, 20 µm; f,50µm degradation of αC3 and increased fixation of small iC3b Fig. S7a). We then examined whether DC-SIGN bound (43 kDa; Fig. 6c). directly to C4, similar to SIGN-R1 (Supplementary With the addition of C1q or normal human serum Fig. S4a). DC-SIGN transfectants showed clear fixation of (NHS) as a source of C1q, pneumococcal binding was C4 (Fig. 7f). Additionally, DC-SIGN-transfected increased only on SIGN-R1 transfectants (Fig. 6d). HEK293T cells also induced the fixation of C1q or C4 Moreover, pneumococcal uptake and sequential decom- from NHS, confirming again the binding of DC-SIGN to position were observed on SIGN-R1 transfectants only C1q and C4 (Fig. 7g), and their fixation was significantly with normal mouse serum (NMS), but not without serum increased following S. pneumoniae challenge (Fig. 7g). BMT TKO or with C3- or C1q-depleted sera (Fig. 6e and Supple- In splenic fractions from DC-SIGN /SIGN-R1 mentary Fig. S6a). After intravenous injection of S. mice, upregulation of C1q and C4 was obvious following pneumoniae into WT, SIGN-R1-KO, or C3-KO mice, S. pneumoniae challenge (Fig. 7h). Moreover, after intra- BMT pneumococcal uptake and cytoplasmic decomposition venous injection of S. pneumoniae into DC-SIGN / + TKO were obvious only on splenic MZ SIGN-R1 macro- SIGN-R1 mice, systemic C3 activation reappeared in TKO phages from control mice (Fig. 6f and Supplementary Fig. SIGN-R1 mice as in control mice, demonstrating S6b), but completely abolished in splenic MZs from gradual decrease in αC3 and larger iC3b and a rapid SIGN-R1-KO and C3-KO mice and red pulp from all increase and sequential decrease in small iC3b (Fig. 7i). mice (Fig. 6f). The signal specificity of CPS14 on splenic C3 fixation was also completely recovered around splenic + + BMT MZ SIGN-R1 macrophages of control mice in Fig. 6f was MZ DC-SIGN macrophages in DC-SIGN /SIGN- TKO TKO confirmed by immunostaining with its respective isotype R1 mice from SIGN-R1-KO or SIGN-R1 mice as control immunoglobulins (Supplementary Fig. S6c). in control mice (Fig. 7j and Supplementary Fig. S7b), occurring rapidly within 15 min after challenge with S. DC-SIGN may mediate the classical complement pathway pneumoniae and disappearing at 4 h (Supplementary Fig. in LRs from splenic MZ DC-SIGN macrophages following S7c). Immunostaining with isotype control IgG for anti- S. pneumoniae challenge via interactions with C1q C3 antibodies did not yield a C3 signal (Supplementary When DC-SIGN transfectants were treated with Fig. S7d). Moreover, immunoblotting of cadaver spleens S. pneumoniae, DC-SIGN aggregates were dramatically for C1q, C4, and C3 revealed expression in human splenic increased on the cell surface (Fig. 7a). Additionally, when LRs, with predominant expression of C1q and 104-kDa LR fractions of these cells were immunoblotted for DC- αC3′ rather than 113-kDa αC3 (Fig. 7k and Supplemen- SIGN, monomers and dimers of DC-SIGN were obviously tary Fig. S7e). increased in LRs following S. pneumoniae challenge (Fig. 7b). Moreover, splenic MZ DC-SIGN macrophages Discussion BMT TKO from DC-SIGN /SIGN-R1 mice recovered the Although DC-SIGN and SIGN-R1 professionally 35,41 rapid recognition of S. pneumoniae on splenic MZs, even recognize and remove S. pneumonia , the disruption of at 15 min and 1 h after intravenous injection of the LRs on both lectin transfectants resulted in serious organisms (Fig. 7c). In spleens at 1 h, DC-SIGN mono- impairment of the uptake and sequential decomposition mers and multimers were obviously upregulated only in of the organism. Because the disruption of LRs did not splenic LRs following S. pneumoniae challenge (Fig. 7d). alter the expression levels of both lectins on the respective DC-SIGN bound well to purified human C1q or human transfectants, which would lead to normal binding of the C1q from NHS (Fig. 7e). Additionally, DC-SIGN binding organism to the cell surface, the structure of LRs may be to mouse C1q from NMS was confirmed by immuno- more important for the function of both lectins and for blotting and immunostaining (Fig. 7e and Supplementary removal of S. pneumoniae than previously thought. This Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 11 of 17 Fig. 7 (See legend on next page.) Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 12 of 17 (see figure on previous page) Fig. 7 DC-SIGN in splenic lipid rafts may mediate the classical complement pathway in response to S. pneumoniae challenge by interacting with C1q and C4. a DCEK_DC-SIGN transfectants were incubated with mitomycin C-treated S. pneumoniae type 14 (MitC-Pn14; 1 × 10 , 15min, 37°C) and immunostained for DC-SIGN without permeabilization. A representative cell is shown in the box. b After DCEK_DC-SIGN transfectants were incubated with MitC-Pn14 (1 × 10 , 1 h, 37 °C), lysates were fractionated with sucrose gradient ultracentrifugation, and fractions of LRs were BMT TKO immunoblotted for DC-SIGN, flotilin-1, or caveolin-1. c DC-SIGN /SIGN-R1 mice were injected intravenously with CFSE-labeled MitC-Pn14 (green; 8 BMT TKO 1× 10 , 15 min or 1 h), and splenic sections were immunostained for DC-SIGN (red). d As in b, but spleens of DC-SIGN /SIGN-R1 mice were used after intravenous injection of live S. pneumoniae (1 × 10 , 1 h). e (Left) Wild-type or DCEK transfectants were incubated with 5 μg endotoxin-free ovalbumin (efOVA), human C1q, or 3% sera (NMS, NHS, or C1q-depleted human serum) in TC buffer for 1 h at 37 °C, and lysates were immunoblotted for C1q. (Right) Wild-type or DCEK_DC-SIGN transfectants were incubated with 3% NMS for 1 h at 37 °C and immunostained for C1q, followed by microscopic analysis. Representative areas are highlighted from low-power images in Figure S7a. f Wild-type or DCEK transfectants were incubated with 5% NMS for 10 min at 37 °C, immunostained for C4 without permeabilization, and analyzed by FACS. g Empty vector or DC-SIGN-transfected HEK293T cells were incubated without or with MitC-Pn14 (1 × 10 ) in the absence or presence of 5% NHS for 15 min at 37 °C, and cell lysates were BMT TKO immunoblotted for DC-SIGN, C1q, C4, and β-actin. h As in d, but DC-SIGN /SIGN-R1 mice were used, and splenic fractions were immunoblotted TKO BMT TKO 8 for C1q or C4. i Control, SIGN-R1 , and DC-SIGN /SIGN-R1 mice were injected intravenously with Pn14 (1 × 10 ), and their sera were collected TKO BMT TKO at 0, 15, 30, or 60 min and immunoblotted for C3. j As in c, but control, SIGN-R1 KO, SIGN-R1 , and DC-SIGN /SIGN-R1 mice were intravenously injected for 15 min. Splenic sections were immunostained for C3 (red) and SIGN-R1 (green) or C3 (red) and DC-SIGN (green). Representative areas are highlighted from low-power images in Figure S7b. k As in h, but spleens of cadavers were used and immunoblotted for C1q, C4, and C3. Representative data are presented from the results of four cadavers. Scale bars a, e, 20 µm; c, 250 µm; j,50µm speculation was strongly supported by the following could accelerate the generation of a classical pathway C3 results. First, multimers of DC-SIGN or SIGN-R1 were convertase (C4bC2a), and the resulting abundant C4b and preformed in LRs of the respective transfectants, and even C4bC2a could sequentially promote fixation of C3 on the 53–56 SIGN-R1 multimers were preformed in splenic LRs. cell surface . Supporting this possibility, C3, particu- Additionally, DC-SIGN and SIGN-R1 multimers were larly C3α, was most enriched in splenic LRs, despite increased only in LRs following S. pneumoniae challenge having lowest expression in the spleen among examined in vitro or in vivo, but not in non-LRs. Therefore, LRs mouse tissues. Therefore, splenic MZ SIGN-R1 LRs may could provide a spatially confined and optimal platform be optimized to activate C3, although the C3 distribution for DC-SIGN and SIGN-R1 to facilitate clustering, was independent of the existence of SIGN-R1, probably increase their affinity and specificity to pathogens, and due to covalent fixation of C3 fragments on other splenic enhance their clearance, thus affecting their innate pro- cells . Because the complement system should be tightly 7,45 tective functions . controlled under physiological conditions to prevent sig- Raft locations for target antigens favor classical com- nificant damage to self-tissues, enrichment of SIGN-R1, plement activation by concentrating the antigen–antibody C1q, C4, and C3 in splenic MZ SIGN-R1 LRs could also complex into a comparatively small area, thus providing precisely regulate complement activation in splenic MZs; an ideal density for juxtaposed Fc regions to engage indeed, nascent C3b and C4 regulate C1 activation in 46,47 58 C1q . Similarly, both C1q and C4 were exclusively concert with C3 under normal conditions . distributed in splenic LRs among fractions from examined Pneumococcal challenge increased both C1q and C4 and mouse tissues, showing a significant dependence on the activated C3 only in splenic MZ SIGN-R1 LRs without existence of SIGN-R1. SIGN-R1 was exclusively expressed the involvement of other complement pathways. There- 42 + on splenic MZ macrophages and bound directly to C1q fore, splenic MZ SIGN-R1 LRs may provide optimal 39,48 or C4 . Additionally, there is a potential interaction locations for SIGN-R1-mediated C3 activation to accel- 49,50 between C1q and C4 . Therefore, their exclusive dis- erate pneumococcal clearance by sequentially enhancing tribution in splenic LRs is expected, and these proteins pneumococcal iC3b opsonization and rapid phagocytosis + + may be further subdivided into splenic MZ SIGN-R1 primarily in splenic MZ SIGN-R1 macrophages. In par- LRs from heterogeneous LRs of various splenic cells . ticular, S. pneumoniae capsules inhibit its recognition by These findings suggested that splenic MZ SIGN-R1 LRs natural IgM, the binding of the serum proteins to sub- may provide optimal microenvironmental platforms for capsular targets, complement activity, and neutrophil 59,60 reciprocal interactions among SIGN-R1, C1q, and C4, phagocytosis . However, enrichment of SIGN-R1, C1q, thus preforming a trimolecular complex to rapidly acti- C4, and C3 in splenic MZ SIGN-R1 LRs could drama- vate the SIGN-R1-mediated classical complement path- tically enhance the simultaneous recognition of pneumo- way in the spleen. coccal CPSs with SIGN-R1 and C1q, thus overcoming Because SIGN-R1 or C1q directly recognize S. pneu- impairment of the initial pneumococcal recognition . 41,52 moniae , the preformed trimolecular complex of The human spleen and the classical pathway are integral + 39,62–65 SIGN-R1, C1q, and C4 in splenic MZ SIGN-R1 LRs for protection against infection by S. pneumoniae . Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 13 of 17 In particular, the DC-SIGN-C1q complex could also mutant Spn14.H, and Staphylococcus aureus strain mediate a classical complement pathway against S. (RN4220) were used, all of which were kindly provided by pneumoniae in the spleen, representing a potential pro- Professor Vincent A. Fischetti of Rockefeller University tection mechanism in the human spleen . Indeed, the (New York, USA). BMT replaced DC-SIGN on splenic MZs of DC-SIGN / TKO SIGN-R1 mice completely rescued the pneumococcal Cell culture capture, the sequential activation and deposition of C3 DCEK, a mouse L-cell fibroblast line, and human against S. pneumoniae in splenic MZs, which was likely to embryonic kidney (HEK293T) cells were cultured in be related to the DC-SIGN-induced complex of mouse RPMI-1640 medium, and CHO cells were cultured in C1q and C4. Previous studies have shown that recruit- Dulbecco’s modified Eagle’s medium, supplemented with ment of DC-SIGN into LRs with binding to viral glyco- 10% fetal bovine serum, 100 U/mL penicillin G, and 29,31,66 proteins or the gC1qR strengthens the possible 100 mg/mL streptomycin. Stable DCEK transfectants formation of a tetramolecular complex of glycoproteins/ expressing complementary (cDNA) for DC-SIGN or DC-SIGN/C1q/gC1qR and thus increases binding capa- SIGN-R1 (DCEK_DC-SIGNS and DCEK_SIGN-R1 in the 24,67 city through the complex . Therefore, similar to SIGN- figures, respectively) and stable CHO transfectants R1, splenic MZ DC-SIGN LRs may also provide a expressing cDNA for Neo, or SIGN-R1 (CHO_Neo, or favorable microenvironment for the DC-SIGN-mediated CHO_SIGN-R1 in the figures, respectively) were used. classical complement pathway by forming another tetra- molecular complex of DC-SIGN, C1q, gC1qR, and C4, In vivo animal studies finally enhancing sequential C3 activation. Female C57BL/6 mice (6–10 weeks old, weighing Because DC-SIGN is exclusively expressed on human 16–20 g) were purchased (The Jackson Laboratory, Bar splenic perifollicular zone macrophages , the presence of Harbor, ME, USA) and housed under specific pathogen-free DC-SIGN in cadaver splenic LRs indicates that DC-SIGN conditions. SIGN-R1 (CD209b)-KO mice were kindly pro- is expressed in LRs from human splenic perifollicular vided by the Consortium for Functional Glycomics (http:// zone macrophages. Also, similar with the previous www.functionalglycomics.org). DC-SIGN transgenic donor 32,68,69 reports , complements C1q, C4, and C3b were mice were provided by the Rockefeller Gene Targeting enriched in human splenic LRs. Therefore, these cadaver Resource Center and identified by PCR using DC-SIGN splenic perifollicular zone DC-SIGN LRs may also be gene primers forward (5′-CgggATCCgAgTggggTgACA TgAgTgACT-3′) and reverse (5′-ACgCgTCgACAAA important for the DC-SIGN-mediated classical comple- ment system against S. pneumoniae, explaining why the AgggggTgAAgTTCTgCTACg-3′). For in vivo experiments spleen and the classical pathway are integral for protec- using animal models, all studies were approved by the 63–65 tion against infections to S. pneumoniae in humans . Institutional Animal Care and Use Committee of Konkuk The importance of LRs has been restricted to their roles University (permit number: KU11107) and performed in in receptor-mediated intracellular events from the plasma strict accordance with the approved guidelines for animal membrane, so far. However, splenic MZ LRs provide care and animal experimentation. Animal welfare was bidirectional platforms not only for usual events associated overseen by local committees. Mice were housed in a with the intracellular milieu, for example, recognition and temperature-controlled room with an automated phagocytosis of pathogens in vivo, but also unusual events darkness–light cycle system and had ad libitum access to associated with the extracellular milieu, such as the com- food and water. Mice were challenged with 1 × 10 colony- plement system, orchestrating early and complicated host forming unit of S. pneumoniae suspended in 100 mL of PBS responses to microbial infection. Thus, the spleen could be in PBS intravenously. Prior to tissue dissection, the mice equipped with the most sensitive system to target various were sacrificed via euthanasia using an overdose of CO 35,36 pathogens, such as S. pneumoniae and human with a flow rate 20% of the cage volume per minute, fol- 37,38 immunodeficiency virus-1 . These findings can explain lowing AVMA Guidelines for the Euthanasia of Animals. how such a small portion of splenic SIGN-R1 macro- phages (<0.05%) helps the spleen to efficiently protect Bacterial growth conditions and fluorescent labeling hosts against S. pneumoniae, providing insights into the Streptococcus pneumoniae capsular serotype 14 involvement of LRs in the innate immune system and the (DCC1490), the unencapsulated S. pneumoniae Tn916 roles of DC-SIGN in the human spleen. mutant Spn14.H (mt-Pn14), and Staphylococcus aureus were grown in brain heart infusion broth (DIFCO) to Materials and methods mid-logarithmic phase (18 h, 37 °C). Bacteria were inac- Bacterial strains tivated with 50 μg/mL mitomycin C (Sigma) for 1 h and Streptococcus pneumoniae capsular serotype 14 suspensions of 10 bacteria were labeled with PKH26 (DCC1490), the unencapsulated S. pneumoniae Tn916 following the manufacturer’s instructions or with 5 mM Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 14 of 17 CFSE (Sigma-Aldrich) for 30 min at 37 °C. The fluor- of pneumococcal-bound cells was calculated in five areas escent bacteria were injected intravenously into mice or for each sample from five independent experiments. incubated with cells. Cell fractionation and purification of LRs with sucrose Cellular and tissue immunofluorescence microscopy gradients Cells on coverslips or 10 μm OCT (Tissue-Tek) frozen Tissues from mice or cells were solubilized in 2 mL of spleen sections were fixed with 100% acetone (10 min, 1% Triton X-100 (Junsei Chemical Co., Ltd.) in MES- room temperature) and stained, where indicated, with buffered saline (MBS, 25 mM MES, pH 6.5, 150 mM DAPI (4′,6-diamidino-2-phenylindole) (blue) or FITC-, NaCl). After homogenizing with 10 up-and-down strokes PE-, AMCA-, or Alexa Fluor-conjugated donkey anti- of a tight-fitting Dounce homogenizer, the tissue or cel- chicken IgY, goat anti-hamster IgG, donkey anti-rabbit lular extracts were adjusted to 4 mL with sucrose con- IgG, goat anti-rat IgG, and streptavidin as secondary centrations of 45% or 40%, respectively, and overlaid with reagents (purchased from Abcam, Jackson ImmunoR- 4 mL of 30% sucrose and 4 mL of 5% sucrose in MBS. The esearch Laboratories, or Molecular Probes). Cells and sucrose gradient was formed by centrifugation at spleen sections were examined for fluorescence with a 200,000 × g for 18–20 h at 4 °C using a Beckman SW41ti deconvolution fluorescence BX61-32FDIC microscope rotor. After centrifugation, the sucrose gradients were (Olympus Corp., Tokyo, Japan). Images were acquired fractionated into 12 fractions without pelleting, and an EZ with a Coolsnap system (Roper Scientific, Inc., opaque buoyant band corresponding to the LRs was col- AZ, USA). lected at the interface between the 30% and 5% sucrose gradients. The same quantity of proteins from each frac- Flow cytometry tion was used for immunoblotting analysis. Cells used in FACS analysis were detached with 1 mM EDTA in PBS for 10 min and pre-incubated 10 min with Plasmids and transfection 2.4G2 monoclonal antibody at 4 °C to block Fc receptors. Clone pMX DC-SIGN was a gift from Dr. Chae Gyu Cells or S. pneumoniae were incubated with 3–5% mouse Park (Rockefeller University). This clone c DC-SIGN or human serum in 200 μL TC buffer (10 mM Tris-HCl, expression was monitored by immunoblotting analysis 140 mM NaCl, 2 mM CaCl , 2 mM MgCl , and 1% bovine using anti-DC-SIGN antibodies. DCEK transfectants were 2 2 serum albumin [BSA]), and complement binding to cells transiently transfected with plasmids encoding dynamin II or organisms was detected by immunostaining with K44A (dominant-negative dynamin with a point mutation respective antibodies for 30 min at 4 °C. Cytometric ana- in the nucleotide-binding site, a gift from Professor lysis was performed using a FACScan (Becton Dickinson, Seung-Jae Lee, Seoul National University College of San Jose, CA, USA) and CellQuestPro (BD Biosciences). Medicine, Seoul, Korea) or with an empty pcDNA3 vector Subsequent data analysis was performed with CellQuest- for 48 h, using Lipofectamine Plus reagent (Invitrogen Life Pro (BD Biosciences). Technologies, Carlsbad, CA, USA) according to the manufacturer’s specifications. Streptococcus pneumoniae uptake and decomposition analysis in vitro and in vivo SIGN-R1 TKO mouse generation Cells (1 × 10 ) were incubated with mitomycin SIGN-R1 TKO or isotype control mice were generated C-treated and CFSE-labeled S. pneumoniae (1 × 10 ) for by intravenous injection of 100 μg 22D1 antibody or iso- the indicated times at 37 °C, and bacterial binding to cells type hamster IgG for 48 h. The 22D1 antibody selectively and cytoplasmic decomposition of capsular poly- and transiently depleted the surface SIGN-R1 molecule, saccharides of S. pneumoniae were examined with a but not SIGN-R1 macrophages in the splenic MZ, per- fluorescence microscope. Mice were intravenously injec- mitting analysis of its function in vivo, as in a previous ted with mitomycin C-treated and CFSE-labeled report . 8 39,41 S. pneumoniae (1 × 10 ) for the indicated times , and bacterial binding and decomposition were examined on DC-SIGN transgenic mouse generation − + BMT SIGN-R1 or SIGN-R1 cells of spleen tissues. DC-SIGN transgenic (DC-SIGN /WT) mice were generated from the reconstitution of lethally irradiated Quantification of bacterial decomposition ratios C57BL/6 mice with bone marrow cells of DC-SIGN To quantify the pneumococcal decomposition ratio, the transgenic donor mice. SIGN-R1-depleted DC-SIGN BMT TKO number of pneumococcal-bound or -decomposing cells transgenic (DC-SIGN /SIGN-R1 ) mice were gen- was counted, and the average percentage of erated from the intravenous injection of 22D1 for 48 h BMT pneumococcal-decomposing cells from the total number into DC-SIGN /WT mice. Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 15 of 17 Complement C3-depleted mice polyvinylidene difluoride membranes, followed by incu- Control mice were obtained by intraperitoneally bation with antibodies. Antibody-reactive bands on the injecting 60 U/kg of cobra venom factor one day prior to blots were visualized with peroxidase-labeled secondary the experimental challenge. C3 depletion was confirmed antibodies, followed by treatment with West-ZOL plus by Western blot analysis for C3 by using sera. (Intron) or Immobilon (Millipore). Mouse infection studies Human cadaver spleen studies Mitomycin C-treated fluorescent bacteria (1 × 10 ) were Human cadaveric spleens were donated from the intravenously administered to mice for the indicated Department of Anatomy, School of Medicine, Konkuk times. Mice were sacrificed, and spleen sections were University (Seoul, Korea). examined by deconvolution fluorescence microscopy. Software used in this study Assay for in vivo and in vitro C3 processing Fluorescent images were analyzed with the MetaMorph To quantify C3 processing in tissues in vivo, 1 × 10 software (Universal Imaging). Immunoblotting signals S. pneumoniae were injected intravenously, and tissue were detected using LAS-4200 (Fuji Film). lysates were collected at the indicated times, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis Statistical analyses (SDS-PAGE), and immunoblotted with polyclonal anti- The number of cells binding to bacteria and the number mouse C3 antibodies. For in vitro C3 processing, 1 × 10 of cells decomposing bacteria were counted. The average S. pneumoniae were incubated with 5% NHS in TC buffer percentage of pneumococcal decomposition of the total (140 mM NaCl, 2 mM CaCl , 2 mM MgCl , 10 mM Tris, pneumococcal binding to cells was calculated in five areas 2 2 pH 7.5, supplemented with 1% BSA) for 30 min at 37 °C. of each sample from three independent experiments and The bacteria were washed, mixed, and boiled with 20 µL shown in the indicated graphs. Data are presented as of 2× SDS sample buffer. Bacterial lysates were separated mean ± SD. Statistical significance between groups were by SDS-PAGE and immunoblotted with polyclonal anti- determined by two-way analysis of variance, followed by mouse C3 antibodies. These antibodies detected the Tukey’s post hoc tests and unpaired Student’s t tests with components of native C3, αC3, and βC3, as well as the a two-tailed test. p Value < 0.05 was taken to indicate fragments of αC3 (molecular weights in humans: 113 kDa statistical significance (not significant, ns; *p < 0.05; for αC3, 104 kDa of αC3′, 63 kDa and 41 kDa for iC3b **p < 0.01; ***p < 0.001) fragments, and 75 kDa for βC3; molecular weights in rats: 120 kDa for αC3, 70 kDa and 43 kDa for iC3b fragments, Acknowledgements and 65 kDa for βC3) that are generated during C3 pro- This work was supported by the Basic Science Research Program through the cessing by C3 convertases by immunoblotting analysis. In National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (S201803S00049); Health Technology R & D the steady state, αC3 and βC3 are predominant, with a few Project through the Korea Health Industry Development Institute (KHIDI), Ministry larger iC3b. With activation, αC3 and larger iC3b are of Health & Welfare, Republic of Korea (HI17C1713); and Basic Science Research rapidly processed into smaller fragments, including Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2017R1C1B2010487). 43 kDa iC3b, but not βC3, which serves as a loading control for the immunoblotting analysis, resulting in loss of most of the detectable αC3 as well as larger iC3b, but Author details accumulation of the smaller iC3b in C3 activation in Department of Biomedical Science and Technology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea. immunoblotting analysis . Department of Obstetrics and Gynecology, Division of Maternal and Fetal Medicine, Research Institute of Medical Science, Konkuk University School of Western blot analysis and immunodetection Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea. Department of Anatomy, Research Institute of Medical Science, Konkuk University School of Tissues, cells, and bacteria were lysed in RIPA buffer Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea. Department (150 mM NaCl, 50 mM Tris-HCl, pH 8.0/1% Nonidet P- of Oncology, Georgetown University Medical Center, Washington, DC, USA. 40/0.5% sodium deoxycholate/0.1% SDS) supplemented Department of Pathology, New York University School of Medicine, New York, NY 10016, USA. Laboratory Animal Center, KBIO Health, Osongsarngmyon-ro with 0.2% protease inhibitor cocktail (Sigma), and lysed 123, Ghungju-si, Chungbuk, Korea. Department of Veterinary Pharmacology samples were mixed with an equal volume of 2× SDS and Toxicology, Veterinary Science Research Institute, College of Veterinary sample buffer containing 2-mercaptoethanol and boiled at Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea. Department of Infectious Diseases, College of Veterinary Medicine, 95 °C for 5 min (100 °C for 10 min). Diluted sera (1:50) or Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea fractions of LRs were mixed with 2× or 5× SDS sample buffer with 2-mercaptoethanol and boiled at 95 °C for 10 min (100 °C for 10 min). The samples were separated Conflict of interest by SDS-PAGE on 4–15% gradient gels and transferred to The authors declare that they have no conflict of interest. Official journal of the Cell Death Differentiation Association Yang et al. Cell Death Discovery (2019) 5:133 Page 16 of 17 Publisher’s note 25. Triantafilou, M., Morath, S., Mackie, A., Hartung, T. & Triantafilou, K. Lateral Springer Nature remains neutral with regard to jurisdictional claims in diffusion of Toll-like receptors reveals that they are transiently confined within published maps and institutional affiliations. lipid rafts on the plasma membrane. J. Cell Sci. 117,4007–4014 (2004). 26. 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