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Intracellular C3 regulates the immune response to infection via NF-κB signaling

ABSTRACT: Complement factor C3 is one of the most abundant proteins in the bloodstream and a central part of the complement system. Upon activation, C3 facilitates bacterial recognition and clearance of bacteria in the extracellular environment. Initially regarded as a serum effector component, C3 is also emerging as an intracellular protein regulating basic cellular processes. Previously, we reported that intracellular, cytosolic C3 can opsonize bacteria and impact their virulence. In this study, we show that cells lacking C3 exhibit altered gene expression that influences immune responses to infection and inflammation. We observed decreased cytokine secretion in C3-deficient cells, which was rescued by expression of non-canonical, cytosolic C3. Further investigation revealed that C3 deficiency impairs signal transduction within the NF-κB signaling pathway which is attributed to decreased expression of Toll-like receptors. This effect is reversed in cells expressing cytosolic C3, where receptor expression and pathway activation are restored. Therefore, we propose a novel role of intracellular, cytosolic C3 in shaping immune responses by modulating the expression of receptors critical to pathogen recognition.

ORGANISM(S): Homo sapiens

PROVIDER: GSE301708 | GEO | 2025/12/31

REPOSITORIES: GEO

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Project description:Purpose: Production of broad-spectrum antibodies against T cell-dependent antigens requires marginal zone (MZ) B cells to present antigenic peptides complexed to MHC II. Here we describe the trogocytic acquisition of these complexes from dendritic cells (DCs). Complement C3 (C3) binds to MHC II on murine and human DCs. Recognition of C3 by MZ B cell complement receptor 2 triggers trogocytosis of DC plasma membrane and its receptors, which become exposed on the MZ B cells. Excessive trogocytosis is deleterious but prevented by membrane ubiquitin ligase MARCH1, which limits the number of MHC II–C3 complexes displayed on the DC surface. MHC II molecules are thus receptors for constitutively activated and potentially harmful C3, an activity that enables acquisition of DC-like functions by trogocytic MZ B cells. Methods: mRNA profiles of splenic cDC1s, B cells and CD11cintCD24+CD8int cells of wild type (WT) and March1-/- mice mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. All 100-base-pair single-end reads were mapped to the reference mouse genome (GRCm38/mm10) using STAR aligner, and gene-wise counts were obtained using Subread package. Differential expression analysis of RNA sequencingdata was carried out using the edgeR and limma-voom packages in Bioconductor. Results: Using an optimized data analysis workflow, we mapped about 14 million sequence reads per sample to the mouse genome (build mm10) and identified 16,182 genes in the different cell types from WT and March1−/− mice.Transcriptomic profiling revealed over 6500 genes differentially expressed between cDC1s and B cells. Principle component analyses revealed very few differences between WT and March1−/− cDC1s, and between WT and March1−/− B cells. Notably, the transcriptome of CD11cintCD24+CD8int cells was similar to those of WT or March1−/− B cells, indicating they belonged to the B cell lineage. Conclusions: In our study we have described that MHC II ubiquitination by MARCH1 plays two important roles: first, to enhance the removal of surface MHC II–C3 complexes on all APCs; and second, to limit MZ B cell trogocytosis and elimination of cDCs. It is conceivable these two functions, more than the regulation of MHC II antigen presentation, have been the major drivers for the conservation of MHC II ubiquitination through evolution.

Project description:Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption the GAPAID consortium examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n=211), with other systemic autoimmune diseases (n=65) and non-autoimmune control subjects (n=149) in two rheumatology tertiary care centers. Standard clinical and laboratory data were collected from all subjects and serum complement levels were determined in SLE patients. The genotype of SNP rs1143679 in the ITGAM gene was also determined. On-chip formation of immune complexes was examined using a functional immunoassay on autoantigen microarray. The amount of antigen-bound IgM, IgG and complement C4 and C3 was quantified on autoantigens comprising nucleic acids, proteins and lipids. Our results show that the relatively high complement consumption of nucleic acids is further increased upon binding of IgM and IgG. This is true even when serum complement levels are decreased due to complement consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, most protein and lipid autoantigens show positive correlation with C4 and C3 levels. Genetic analysis reveals that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) have lower levels of dsDNA specific IgM among SLE patients. Regarding organ involvement we find that besides anti-C1q IgG, low levels of dsDNA specific IgM and low complement C4 binding to C1q are also associated with renal injury. In summary, nucleic acids maintain a skewed complement deposition balance when bound by IgG and IgM, depleting the early classical complement pathway from other physiological processes. Dysfunction of the receptor responsible for complement-mediated apoptotic debris removal promotes the development of autoantibodies targeting nucleic acids. These observations provide serological and genetic evidence for complement-mediated clearance deficiency of apoptotic debris in lupus.

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Intracellular C3 regulates the immune response to infection via NF-κB signaling

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