Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis

Abstract

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

Keywords: atherosclerosis; cell-autonomous complement; complement factor H; complement protein C3; efferocytosis; inflammation; local complement production; macrophages; plaque necrosis.

Figure 1.. Global complement factor H deficiency protects from necrotic core formation in a C3-dependent manner

C3^+/+Cfh^+/+Ldlr^−/−, C3^+/+Cfh^−/−Ldlr^−/−, C3^−/−Cfh^+/+Ldlr^−/− and C3^−/−Cfh^−/−Ldlr^−/− mice were fed an atherogenic diet for 10 weeks (n=12, 14, 14 and 18, respectively; pooled from two independent experiments). (A) Plasma cholesterol and triglyceride levels measured by an automated enzymatic method. (B) Total plasma C3 levels quantified by ELISA. (C) Quantitative analysis of atherosclerosis of the whole aorta. Data are expressed as percentage of Sudan IV-stained area of the entire aorta. Representative images are shown. (D) Quantification of aortic root plaque size. Values represent the average μm² of nine sections throughout the entire aortic origin. Representative images of Masson’s trichrome-stained sections are shown. Original magnification, 50X; scale bars, 200 μm. (E) Assessment of necrotic core formation in cross sections at the aortic origin. Bars indicate the percentage of necrotic area per total lesion area throughout the entire aortic origin. Representative images are shown. Original magnification, 50X; scale bars, 200 μm. Each symbol represents individual mice. Mean±s.e.m., statistical significance was evaluated by one-way ANOVA followed by Tukey’s multiple comparison test (*P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 2.. Complement factor H controls cell-autonomous C3 activation in monocyte-derived macrophages and delays the resolution of inflammation

(A) C3 and C5 mRNA and protein levels of monocyte-derived macrophages (Mo-Macs) from thioglycolate-injected mice measured by qRT-PCR and ELISA. (B) C3 mean fluorescence unit (MFI) of peritoneal Ly6C^hi and Ly6C^lo monocytes of thioglycolate-injected mice determined by intracellular flow cytometry. (C) Intracellular C3 levels normalized to cellular protein content in cell lysates of sorted circulating Ly6C^hi monocytes and resident peritoneal macrophages (Res-Macs) from unchallenged mice as well as Ly6C^hi monocytes and Mo-Macs from thioglycolate-injected mice, quantified by ELISA. (D) Intracellular C3 levels of peritoneal Ly6C^hi monocytes of thioglycolate-injected Cfh^+/+ and Cfh^−/− mice quantified by ELISA. Representative histograms show C3 levels within peritoneal Ly6C^hi monocytes assessed by intracellular flow cytometry. (E-G) Characterization of Mo-Macs in the peritoneal lavage fluid of thioglycolate-injected Cfh^+/+ vs Cfh^−/− mice. (E) Intracellular C3 levels of adherent Mo-Macs by ELISA. (F) Bar graphs and representative cytometry plots show surface and intracellular C3 staining of Mo-Macs measured by flow cytometry. (G) C3a/C3 ratio, a measure of complement activation, as judged by total intracellular C3a and C3 levels in lysates of adherent Mo-Macs quantified by ELISA. (H-L) Characterization of the resolution of inflammation in the peritoneal lavage fluid of Cfh^+/+ vs Cfh^−/− mice 2, 24, 72 and 168 hours after thioglycolate injection. Absolute numbers of (H) Mo-Macs and (I) neutrophils, quantified by flow cytometry. (J) Resolution curves and (K) the corresponding resolution interval calculated by the resolution indices extrapolated from the curves are shown. (L) Absolute numbers of early (AnnV⁺7-AAD⁻) and late (AnnV⁺7-AAD⁺) apoptotic cells, assessed by flow cytometry. Each symbol represents individual mice. Data are representative of at least three independent experiments. Mean±s.e.m., two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 3.. Complement factor H-deficient macrophages display heightened efferocytotic capacity

(A) Ex vivo efferocytosis assay using adherent Mo-Macs isolated from the peritoneal cavity of thioglycolate-injected Cfh^+/+ vs Cfh^−/− mice. Percentages of CMFDA⁺ efferocytotic Mo-Macs were quantified by flow cytometry. Representative histograms are shown. (B) Assessment of opsonization as judged by the percentage of C3⁺ and 7-aminoactinomycin D⁺ (7-AAD) double positive Mo-Macs in the peritoneal cavity of Cfh^+/+ and Cfh^−/− mice determined by flow cytometry. (C) Ex vivo efferocytosis assay showing percentages of CMFDA⁺ efferocytotic Mo-Macs, quantified by flow cytometry. (D-I) Characterization of Mo-Macs in the peritoneal lavage fluid of thioglycolate-injected Cfh^+/+ vs Cfh^−/− mice. (D) Volcano plot of genes with expression change exceeding a factor of 1.5 (q < 0.1) from a genome-wide transcriptome profiling by RNA-sequencing. (E-F) Enrichr analysis. The eight most overrepresented (E) biological pathways as well as (F) biological processes of all differentially expressed genes between wild-type and CFH-deficient Mo-Macs are shown. Color coding of (E) represents the number of genes featured in each pathway. (G) Heat map of LAP-associated genes upregulated in CFH-deficient macrophages. (H) Fold change in mean fluorescence intensity (MFI) of the surface levels of CD16/32, TIM4, VSIG4, CD11b and MHCII on CFH-deficient Mo-Macs compared to controls, measured by flow cytometry. (I) Western blot analysis of intracellular LC3-I and LC3-II levels in lysates of Mo-Macs isolated from the peritoneal cavity of thioglycolate-injected Cfh^+/+ vs Cfh^−/− mice. A representative blot is shown. (J) qRT-PCR analysis of Itgam, Timd4 and Vsig4 transcript levels of ex vivo peritoneal Mo-Macs of thioglycolate-injected mice supplemented with increasing concentrations of purified mouse C3. (K) Western blot analysis of intracellular LC3-I and LC3-II levels in lysates of Mo-Macs incubated with or without purified C3 (1ug). (L) pHrodo positivity and MFI of CFH-deficient Mo-Macs following Bafilomycin A1 (BafA1; 200 nM) treatment and pHRodo+ apoptotic cell loading, measured by flow cytometry. (M) Schematic representation of BafA1 treatment of Cfh^−/− mice. (N) Absolute numbers of Mo-Macs with corresponding flow cytometry plots and (O) total counts of 7-AAD⁺ Mo-Macs in the peritoneal cavity of Cfh^−/− mice injected with or without BafA1 after thioglycolate injection, quantified by flow cytometry. Pool of two independent experiments. Each symbol represents individual mice. Mean±s.e.m., two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 4.. Monocyte/macrophage-derived complement factor H is sufficient to delay the resolution of inflammation

(A) Single-cell RNA-seq data from wild-type spleen, represented by t-distributed stochastic neighbor embedding (t-SNE). Left panel demonstrates the clustering and annotation of splenic immune cell subsets. Gene expression profile of Cfh is shown. (B) Relative gene expression of Cfh in FACS-sorted splenic immune cell subsets of unchallenged mice measured by qRT-PCR. Data are shown relative to Cfh expression in Ly6C^hi monocytes. (C) qRT-PCR analysis of C3 and multiple complement regulatory proteins (Cfh, C4bp, Fhr-b, Cfi, Cfd, Cd55, Cd55b, Cr1l) in circulating Ly6C^hi monocytes sorted from the blood of thioglycolate-injected vs control mice. (D) Total secreted CFH levels in the supernatant of Mo-Macs treated with 100 ng/ml IFNγ. (E-L) Lethally irradiated wild-type mice were reconstituted with bone marrow from Cfh^+/+ vs Cfh^−/− mice and were subjected to thioglycolate-induced sterile peritonitis, 10 weeks after transplantation (n=5 vs n=6, n=3 vs n=6 of two independent experiments). (E) Schematic representation of chimeric models of CFH deficiency. (F) C3 concentration in the serum, peritoneal lavage fluid as well as cell-associated C3 levels normalized to cellular protein content in lysates of adherent Mo-Macs of bone marrow chimeric mice measured by ELISA. (G) Bar graphs and representative flow cytometry plots show intracellular C3 levels of Mo-Macs measured by flow cytometry. (H) C3a/C3 ratio, a measure of complement activation, as judged by total intracellular C3a and C3 levels in lysates of adherent Mo-Macs quantified by ELISA. (I) Fold change in mean fluorescence intensity (MFI) of the surface levels of CD11b, TIM4, VSIG4 and MHCII on Mo-Macs of bone marrow-chimeric mice, measured by flow cytometry. (J-K) (J) Mo-Macs absolute numbers and (K) frequency of late apoptotic (AnnV⁺ 7-AAD⁺) Mo-Macs in the peritoneal lavage fluid of bone marrow-chimeric mice 72 hours after thioglycolate injection, quantified by flow cytometry. Representative plots are shown. Each symbol represents individual mice. Mean±s.e.m., two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 5.. Complement factor H dominates local complement expression in aortic macrophages

(A-D) Ldlr^−/− mice were fed a standard (SD) or Western-type diet (WTD) for 12 weeks (n=8 vs n=10). qRT-PCR analysis of Cfh and C3 transcript levels in (A&C) circulating and splenic Ly6C^hi monocytes and in (B&D) total liver samples. Data are expressed relative to gene expression in SD-fed mice. (E-F) Relative expression of (E) fluid-phase complement regulatory proteins (Cfh, Cfhr2, C4bp, Cpn1, Serping1, Clu and Vtn) as well as (F) complement cascade components in CD45⁺CD11b⁺CD64⁺ macrophages sorted from plaques of WTD-fed Ldlr^−/− mice. Data are expressed as transcripts per million (TPM) relative to Cd68 gene. (G) Expression of complement repressors in aortic leukocyte subsets from a meta-analysis of 9 single-cell RNA-sequencing studies. Average expression level (log2 scale) indicated by saturation of blue (dark blue is highest, with minimum scaling to 0). Dot size represents relative percentage of cells that expressed the corresponding genes within each cluster. (H) Schematic representation of mixed 1:1 bone marrow transplantation experiment from control (WT, CD45.1) and CFH-deficient (CFH KO UBI-GFP, CD45.2) into Ldlr-deficient (Ldlr^−/− CD45.2) mice, following a 12-week WTD feeding. (I) Percent of chimerism among blood Ly6C^hi monocytes at harvest with a representative dot plot, quantified by flow cytometry. (J) Mean fluorescence intensity (MFI) of surface levels of CD11b, MHCII, TIM4 and VSIG4 and (K) percent of chimerism among aortic macrophages, measured by flow cytometry. Each symbol represents individual mice. Mean±s.e.m., two-tailed Student’s t-tests (*P < 0.05, ****P < 0.0001).

Figure 6.. Hematopoietic deletion of complement factor H attenuates atherosclerosis by promoting lesional efferocytosis

(A) Schematic representation of chimeric models of hematopoietic CFH deficiency. (A-L) Lethally irradiated Ldlr^−/− mice were reconstituted with bone marrow from Cfh^+/+ vs Cfh^−/− mice and were fed an atherogenic diet for 12 weeks, starting 5 weeks after transplantation (n=14 vs n=12). (B-D) Total plasma (B) CFH, (C) C3 as well as (D) C3a and C5a levels quantified by ELISA. (E) Quantification of lesional intact C3 content. Representative images of C3-stained sections are shown. Original magnification, 50X; scale bars, 200 μm. (F) Quantification of aortic root plaque size. Representative images of Masson’s trichrome-stained sections are shown. Original magnification, 50X; scale bars, 200 μm. (G) Measurement of lesion volume. The dot plots represent the average μm² of nine sections throughout the entire aortic origin, bar graphs show total lesion volume (mm³). (H) Assessment of necrotic core formation in cross sections at the aortic origin. Percentages of necrotic area of total lesion area are shown. Representative images are shown. (I) Measurement of MAC-3⁺ lesional macrophage content per total cellular area by immunohistochemistry. (J) Quantification of dying F4/80⁺ macrophages per cellular area (mm²) by TUNEL staining using fluorescence microscopy. (K) Evaluation of lesional efferocytosis. Efferocytotic capacity was determined as the ratio of free vs macrophage-associated apoptotic cells using fluorescence microscopy. Representative images of sections stained with DAPI, TUNEL and F4/80 are shown. Yellow hashes (#) show free, while pink asterisks (*) indicate macrophage-associated apoptotic cells. Scale bars 100 μm. (L) Quantification of ATG5⁺ lesional area per total cellular area by immunohistochemistry. Each symbol represents individual mice. Representative images are shown. Mean±s.e.m., two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 7.. Complement C3- and CFH-producing inflammatory aortic macrophages populate human coronary arteries

(A) CFH secretion by untreated vs interferon-gamma (IFNγ)-stimulated THP-1 monocytes evaluated by ELISA. (B-C) Representative flow cytometry histograms and bar graphs show intracellular (B) CFH and (C) C3 levels of untreated vs IFNγ-stimulated THP-1 Mo-Macs evaluated by flow cytometry. Data are representative of three independent experiments. (D) Enrichr analysis of coronary artery disease (CAD) candidate genes from the CAD1000G Gene list, retrieved from Zhao et al 2016. The ten most overrepresented biological pathways are shown. (E) Uniform manifold approximation and projection (UMAP) visualization of aortic myeloid cell populations from human coronary artery plaques re-clustered from the raw data published by Wirka et al. 2019. Red circle highlights a distinct population of C3^high inflammatory macrophages. Right: C3 gene expression profile is shown in human aortic myeloid cell populations, average expression level (log2 scale) indicated by saturation of blue (dark blue is highest, with minimum scaling to 0). (F) Enrichr analysis of the top differentially expressed genes enriched in C3^high inflammatory macrophages. The most overrepresented biological pathways and molecular processes are shown. (G) Expression of C3 and CFH in single cells from human aortic myeloid cell subsets. Average expression level (log2 scale) indicated by saturation of blue (see above). Dot size represents relative percentage of cells within the respective cluster that expressed the corresponding genes. Mean±s.e.m., two-tailed Student’s t-tests (***P < 0.001, ****P < 0.0001).