Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021 is online at: average * The Journal of Immunology published online 20 April 2012 from submission to initial decision 4 weeks from acceptance to publication http://www.jimmunol.org/content/early/2012/04/20/jimmun ol.1103760 Complement Protein C1q Directs Macrophage Polarization and Limits Inflammasome Activity during the Uptake of Apoptotic Cells Marie E. Benoit, Elizabeth V. Clarke, Pedro Morgado, Deborah A. Fraser and Andrea J. Tenner J Immunol Submit online. Every submission reviewed by practicing scientists ? is published twice each month by http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://www.jimmunol.org/content/suppl/2012/04/20/jimmunol.110376 0.DC1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of October 2, 2021. Published April 20, 2012, doi:10.4049/jimmunol.1103760 The Journal of Immunology Complement Protein C1q Directs Macrophage Polarization and Limits Inflammasome Activity during the Uptake of Apoptotic Cells Marie E. Benoit, Elizabeth V. Clarke, Pedro Morgado, Deborah A. Fraser, and Andrea J. Tenner Deficiency in C1q, the recognition component of the classical complement cascade and a pattern recognition receptor involved in apoptotic cell clearance, leads to lupus-like autoimmune diseases characterized by auto-antibodies to self proteins and aberrant innate immune cell activation likely due to impaired clearance of apoptotic cells. In this study, we developed an autologous system using primary human lymphocytes and human monocyte-derived macrophages (HMDMs) to characterize the effect of C1q on macrophage gene expression profiles during the uptake of apoptotic cells. C1q bound to autologous apoptotic lymphocytes mod- ulated expression of genes associated with JAK/STAT signaling, chemotaxis, immunoregulation, and NLRP3 inflammasome acti- Downloaded from vation in LPS-stimulated HMDMs. Specifically, C1q sequentially induced type I IFNs, IL-27, and IL-10 in LPS-stimulated HMDMs and IL-27 in HMDMs when incubated with apoptotic lymphocyte conditioned media. Coincubation with C1q tails prevented the induction of type I IFNs and IL-27 in a dose-dependent manner, and neutralization of type I IFNs partially prevented IL-27 induction by C1q. Finally, C1q decreased procaspase-1 cleavage and caspase-1–dependent cleavage of IL-1b suggesting a potent inhibitory effect of C1q on inflammasome activation. These results identify specific molecular pathways induced by C1q to suppress macrophage inflammation and provide potential therapeutic targets to control macrophage polarization and thus http://www.jimmunol.org/ inflammation and autoimmunity. The Journal of Immunology, 2012, 188: 000–000. he complement system, a powerful effector of the innate moral defense system to sense danger by recognizing pathogen- immune system, consists of a group of proteins circulating associated molecular patterns (PAMPs) but is also activated by T as inactive precursors in the blood and in extracellular damage-associated molecular patterns (DAMPs) or altered self fluids. Upon activation through the classical, lectin, or alternative tissues. Dysregulated complement activation has been associated pathway, a cascade of proteolytic cleavages and formation of with the development of various diseases including rheumatoid central enzymatic complexes (C3 and C5 convertases) leads to arthritis and Alzheimer’s disease (2, 3). A causal link between by guest on October 2, 2021 the generation of active fragments resulting in the opsonization complement deficiency and systemic lupus erythematosus (SLE) of invading pathogens (C1q, C3b, and iC3b), release of proin- involves in part the role of complement in physiological waste flammatory chemotactic factors (C3a and C5a), which recruit disposal mechanisms, in particular clearance of dying cells (4). leukocytes to the site of infection or injury, and finally formation Although activation by all three complement pathways can con- of the membrane attack complex (C5b-9) and subsequent lysis tribute to enhanced uptake of apoptotic cells by phagocytic cells of the pathogen (1, 2). Complement functions as an important hu- (5–8), homozygous deficiency of any of the early complement components of the classical pathway (C1q, C1r, C1s, C4, and C2) . Department of Molecular Biology and Biochemistry, Institute for Immunology, Uni- predisposes to the development of SLE with 90% of individuals versity of California, Irvine, Irvine, CA 92697 with genetic deficiency of C1q developing severe SLE (9). Received for publication December 22, 2011. Accepted for publication March 20, C1q is known to play a prominent nonredundant tissue-specific 2012. role in the clearance of apoptotic cells in vitro and in vivo (10– This work was supported by Grant UL1 RR031985 from the National Center for 14). C1q binds to apoptotic cells and cellular debris through its Research Resources (a component of the National Institutes of Health and the Na- globular heads (10, 15) and to phagocytic receptors through its tional Institutes of Health Roadmap for Medical Research), National Institutes of Health Grants AI 41090 and AG 00538, and the Cypress College Science, Technol- collagen tails (1, 16). Although at first thought to be primarily of ogy, Engineering, and Math Summer Bridge Program. liver origin, C1q is predominantly synthesized in vivo by peripheral The gene expression data presented in this article have been submitted to the Gene tissue macrophages and dendritic cells (17, 18) and by myeloid cells Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number in vitro (8, 19–21). Although C1q is most often bound to C1r and GSE30177. C1s in the circulation (22), this local synthesis of C1q is hypothe- Address correspondence and reprint requests to Dr. Andrea J. Tenner, Department of Molecular Biology and Biochemistry, 3205 McGaugh Hall, University of California, sized to be the major source of C1q for the rapid opsonization of Irvine, Irvine, CA 92697. E-mail address: [email protected] dying cells in tissue before recruitment of plasma-derived compo- The online version of this article contains supplemental material. nents such as C1r and C1s and subsequent activation of the com- Abbreviations used in this article: AL, apoptotic lymphocyte; ASC, apoptosis-asso- plement cascade. In addition, induced synthesis of C1q has been ciated speck-like protein containing a CARD domain; DAMP, damage-associated mo- detected in several injury models in vivo and in vitro[(23, 24); lecular pattern; EAL, early apoptotic lymphocyte; GO, gene ontology; HMDM, human monocyte-derived macrophage; HMGB1, high mobility group box 1; HSP, heat shock reviewed in Ref. 3], suggesting that the induction of C1q synthesis protein; LAL, late apoptotic lymphocyte; PAMP, pathogen-associated molecular pat- in tissue may be a response to injury that promotes rapid clearance tern; pDC, plasmacytoid dendritic cell; PI, propidium iodide; qRT-PCR, quantitative of apoptotic cells and concomitant suppression of inflammation. For real-time PCR; SLE, systemic lupus erythematosus. example, interaction of C1q with human monocytes or dendritic Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 cells results in the downregulation of proinflammatory cytokines www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103760 2 C1q LIMITS MACROPHAGE AND INFLAMMASOME ACTIVATION upon TLR4 stimulation by LPS (25, 26). Recently, we showed that C1q binding assay C1q enhances uptake of apoptotic Jurkat T cells by human mono- EALs and LALs were incubated with 150 mg/ml purified human C1q for cytes but has no effect on the basal clearance level of these apoptotic 1 h in PBS/1% human serum albumin at 37˚C. Binding of C1q was as- cells by human monocyte-derived macrophages (HMDMs) and sessed for every experiment by flow cytometry using an mAb against C1q dendritic cells (8). In addition, although C1q influences the induc- (Quidel) and FITC–anti-mouse IgG (Jackson ImmunoResearch Laborato- . tion of cytokines in all myeloid cell types tested in this study, both ries). For each experiment, C1q binding was 50% for EALs or LALs. the degree and direction of modulation depend on the state of dif- Uptake assay ferentiation of the phagocytic cell (8). However, because several C1q receptors have been identified and none has been shown spe- PKH26-labeled or unlabeled EALs and LALs, precoated or not with C1q, were incubated with HMDMs at a 5:1 ratio for 1 h (optimal ratio and time cifically to mediate C1q-enhancement of phagocytosis of apoptotic determined in preliminary experiments, see Supplemental Fig. 2B) in cells (1, 12, 27), the intracellular signaling pathways engaged upon phagocytosis buffer (RPMI 1640, 25 mM HEPES, and 5 mM MgCl2). For interaction of C1q with phagocytic cells remain to be fully eluci- uptake quantification, cells were washed, harvested with trypsin/EDTA, dated. In addition, because characterization of macrophage acti- and stained