Complement protein C1q directs macrophage polarization and limits inflammasome activity during the uptake of apoptotic cells

Abstract

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 modulated expression of genes associated with JAK/STAT signaling, chemotaxis, immunoregulation, and NLRP3 inflammasome activation 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-1β 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 inflammation and autoimmunity.

Figure 1. C1q binding to EAL and LAL and effect on the uptake by HMDMs

(A and B) EAL and LAL were incubated without (gray peak) or with 150 µg/ml C1q (black lane) for 1 h, washed and stained for C1q. Representative FACS plot of multiple experiments are shown. (B) Percentage of C1q binding and anti-C1q MFI determined by flow cytometry. Results represent means ± s.d. (n = 5). (C–D) HMDMs were incubated with PKH26-labeled EAL and LAL, pre-incubated or not with C1q, at a 5:1 ratio for 1 h, washed and fixed. Cells were stained with FITC-phalloidin and analyzed by confocal microscopy (Fig. S2) to determine the percentage of phagocytosis and the number of targets per HMDMs (C) or stained with CD11c-FITC antibodies and analyzed by flow cytometry (D).Results represent means ± s.d. (n = 3 different donors), two-way ANOVA, * p < 0.05 and ** p < 0.01. (E–F) PKH26-prelabeled EAL and LAL (red) were incubated with C1q for 1 h, washed and then added to HMDMs at a 5:1 ratio for 1 h. Cells were fixed and stained with anti-C1q antibodies (blue) and FITC-phalloidin (green) and analyzed by confocal microscopy. Representative micrographs of 3 independent experiments are shown in E. Scale bar = 10 µm. (F) Percentage of HMDM-bound/ingested EAL or LAL bound or not to C1q. Results represent means ± s.d. (n = 3), two-way ANOVA, * p < 0.05 and ** p < 0.01.

Figure 2. Gene expression and main biological processes modulated by C1q in HMDMs during the uptake of AL

(A) Pearson correlation coefficient-based heat map (complete linkage method) representation of the Log2 fold-change (all conditions performed in triplicate) of HMDMs incubated with EAL, C1q-EAL, LAL and C1q-LAL and then stimulated with LPS for 3 h over unstimulated HMDMs. (B) GO-based functional annotation of genes modulated by C1q in HMDMs. Major biological processes are shown as the percentage of differentially expressed annotated genes (redundancy is due to the involvement of individual genes in multiple biological processes). (C–F) Network diagrams of chemotaxis (C), inflammation/cytokines (D), JAK/STAT signalling (E) and NLRP3 inflammasome activation (F) pathways modulated by C1q in HMDMs. Node colors represent changes in gene expression in C1q-EAL vs. EAL or C1q-LAL vs. LAL, shown using a color gradient (blue = down-regulated, white = not modulated and yellow = up-regulated by C1q).

Figure 3. Increase of type I IFNs, IL-27 and IL-10 expression by C1q in LPS-stimulated HMDMs during the uptake of AL

HMDMs were incubated with EAL (left panels) or LAL (right panels), pre-incubated or not with C1q, at a 5:1 ratio for 1 h and then stimulated with 10 ng/ml LPS (A–H) or 10–1000 ng/ml (I) in HL-1 for up to 18 h. Changes in mRNA levels for IFNα (A and B), IFNβ (C and D), IL-27 (E and F) and IL-10 (G and H) were determined by qRT-PCR. Protein levels of IFNα (A and B, insets) were detected 6 h after LPS stimulation and IL-27 (E and F, insets and I) and IL-10 (G and H, insets) after 18 h of LPS stimulation. (A–H) LPS data (black squares) are identical between left and right panels (single LPS control experiment performed for both EAL and LAL simultaneously). Results represent means ± s.d. (n = 2–3 different donors), two-way ANOVA, * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 4. Increased IL-27 expression by C1q in resting HMDMs during the uptake of AL

HMDMs were incubated with EAL or LAL, pre-incubated or not with C1q, at a 5:1 ratio for 1 h. HMDMs were then cultured in HL-1 for 3 h in absence of LPS (A) or in presence of EAL or LAL conditioned media (B). Changes in type I IFNs, IL-27 and IL-10 were determined by qRT-PCR. Results represent means ± s.d. (n = 3 different donors), two-way ANOVA, ** p < 0.01 and *** p < 0.001.

Figure 5. Co-incubation with C1q tails or inhibition of type I IFNs reduces the induction of IL-27 by C1q in LPS-stimulated HMDMs

HMDMs were incubated with EAL, C1q-EAL, LAL or C1q-LAL at a 5:1 ratio for 1 h (A–B) in presence of C1q tails and then stimulated with 10 ng/ml LPS for 3 h or (C–E) stimulated with LPS for 18 h in presence of 1 µg/ml control mouse IgG1 or neutralizing antibodies against IFNα and/or IFNβ (C–D) or goat IgG or neutralizing antibodies against IL-27 (E). mRNA levels were determined by qRT-PCR and proteins levels by ELISAs. Results represent means ± s.d. (n = 2–3 different donors, performed in duplicates), two-way ANOVA, * p < 0.05, ** p < 0.01 and ***, p < 0.001.

Figure 6. C1q decreased procaspase-1 and pro-IL-1β cleavage in LPS-stimulated HMDMs

HMDMs were incubated with PKH26-labeled EAL and LAL, pre-incubated with C1q, at a 5:1 ratio for 1 h and then stimulated with 10 ng/ml LPS. (A–B) HMDMs were stimulated with LPS for 6 h. ATP (1 mM) was added 90 min before the end of the stimulation. Cleaved caspase-1 was detected by FITC fluorescent caspase-1 probes and HMDMs were stained with a blue cell tracker. Representative merged micrographs of 3 independent experiments (from 3 different donors) are shown. Scale bar = 50 µm. Areas in white boxes were enlarged to show PKH26-AL (top, red), cleaved caspase-1 (middle, green) and the merge (bottom). (B) Quantification of cleaved caspase-1 in HMDMs. (C) NLRP3, procaspase-1, ASC and actin expression in HMDM cell extracts. Representative blots of 2 independent experiments are shown. (D–E) Levels of pro-IL-1β relative to β-actin levels (D, cell extracts) and mIL-1β relative to pro-IL-1β (E, supernatants) in HMDMs stimulated with LPS for 18 h with ATP added during the last 3 h of stimulation. Representative blots of 3 independent experiments are shown. All results represent means ± s.d. (n = 3 different donors), two-way ANOVA, * p < 0.05, ** p < 0.01 and ***, p < 0.001.

Figure 7. Main biological pathways modulated by C1q in HMDMs

C1q increases the expression of type I IFNs and IL-27, known to act sequentially to stimulate expression of IL-10 (also up-regulated by C1q), IL-33, known to promote alternative activation of macrophages, IL-37, a potent natural suppressor of innate inflammatory responses and JAK3 that may be involved in IL-27 up-regulation. C1q also suppresses procaspase-1 and pro-IL-1β cleavage and subsequent mIL-1β release through possibly increase expression of negative regulators of inflammasome activity and indirectly (at later times) through increase IL-10 expression, which is known to decrease pro-IL-1β mRNA levels. C1q may thus prevent excessive and dysregulated inflammasome activation induced by the release of DAMPs (HMGB1, HSPs, ATP) from apoptotic cells during secondary necrosis that can activate TLR4 and the NLRP3 inflammasome. Orange arrows represent up-regulated genes and blue lines represent down-regulated genes by C1q.