Guang Sheng Ling

Integrin CD11b positively regulates TLR4-induced signalling pathways in dendritic cells but not in macrophages

Tuned and distinct responses of macrophages and dendritic cells to Toll-like receptor 4 (TLR4) activation induced by lipopolysaccharide (LPS) underpin the balance between innate and adaptive immunity. However, the molecule(s) that confer these cell-type-specific LPS-induced effects remain poorly understood. Here we report that the integrin αM (CD11b) positively regulates LPS-induced signalling pathways selectively in myeloid dendritic cells but not in macrophages. In dendritic cells, which express lower levels of CD14 and TLR4 than macrophages, CD11b promotes MyD88-dependent and MyD88-independent signalling pathways. In particular, in dendritic cells CD11b facilitates LPS-induced TLR4 endocytosis and is required for the subsequent signalling in the endosomes. Consistent with this, CD11b deficiency dampens dendritic cell-mediated TLR4-triggered responses in vivo leading to impaired T-cell activation. Thus, by modulating the trafficking and signalling functions of TLR4 in a cell-type-specific manner CD11b fine tunes the balance between adaptive and innate immune responses initiated by LPS.

C1q as a unique player in angiogenesis with therapeutic implication in wound healing

Significance C1q is a well-known initiator of the complement classical pathway and interacts with several immune and nonimmune cells inducing complement activation-independent functions. Endothelial cells represent one of the potential targets of C1q that binds to cell surface-expressed receptors and stimulates inflammation. Here we report a unique and hitherto unrecognized function of C1q to promote angiogenesis acting through the globular heads. The angiogenic activity of C1q was supported by its ability to induce new vessel formation in in vitro and in vivo models of wound healing. These findings have important implications for the treatment of clinical diseases associated with impaired angiogenesis such as chronic skin ulcers in diabetic patients. We have previously shown that C1q is expressed on endothelial cells (ECs) of newly formed decidual tissue. Here we demonstrate that C1q is deposited in wound-healing skin in the absence of C4 and C3 and that C1q mRNA is locally expressed as revealed by real-time PCR and in situ hybridization. C1q was found to induce permeability of the EC monolayer, to stimulate EC proliferation and migration, and to promote tube formation and sprouting of new vessels in a rat aortic ring assay. Using a murine model of wound healing we observed that vessel formation was defective in C1qa−/− mice and was restored to normal after local application of C1q. The mean vessel density of wound-healing tissue and the healed wound area were significantly increased in C1q-treated rats. On the basis of these results we suggest that C1q may represent a valuable therapeutic agent that can be used to treat chronic ulcers or other pathological conditions in which angiogenesis is impaired, such as myocardial ischemia.

Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

The CD11b/CD18 integrin (complement receptor 3, CR3) is a surface receptor on monocytes, neutrophils, macrophages and dendritic cells that plays a crucial role in several immunological processes including leukocyte extravasation and phagocytosis. The minor allele of a non-synonymous CR3 polymorphism (rs1143679, conversation of arginine to histidine at position 77: R77H) represents one of the strongest genetic risk factor in human systemic lupus erythematosus, with heterozygosity (77R/H) being the most common disease associated genotype. Homozygosity for the 77H allele has been reported to reduce adhesion and phagocytosis in human monocytes and monocyte-derived macrophages, respectively, without affecting surface expression of CD11b. Herein we comprehensively assessed the influence of R77H on different CR3-mediated activities in monocytes, neutrophils, macrophages and dendritic cells. R77H did not alter surface expression of CD11b including its active form in any of these cell types. Using two different iC3b-coated targets we found that the uptake by heterozygous 77R/H macrophages, monocytes and neutrophils was significantly reduced compared to 77R/R cells. Allele-specific transduced immortalized macrophage cell lines demonstrated that the minor allele, 77H, was responsible for the impaired phagocytosis. R77H did not affect neutrophil adhesion, neutrophil transmigration in vivo or Toll-like receptor 7/8-mediated cytokine release by monocytes or dendritic cells with or without CR3 pre-engagement by iC3b-coated targets. Our findings demonstrate that the reduction in CR3-mediated phagocytosis associated with the 77H CD11b variant is not macrophage-restricted but demonstrable in other CR3-expressing professional phagocytic cells. The association between 77H and susceptibility to systemic lupus erythematosus most likely relates to impaired waste disposal, a key component of lupus pathogenesis.

C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation.

Complement C1q is the activator of the classical pathway. However, it is now recognized that C1q can exert functions unrelated to complement activation. Here we show that C1q, but not C4, is expressed in the stroma and vascular endothelium of several human malignant tumours. Compared with wild-type (WT) or C3- or C5-deficient mice, C1q-deficient (C1qa−/−) mice bearing a syngeneic B16 melanoma exhibit a slower tumour growth and prolonged survival. This effect is not attributable to differences in the tumour-infiltrating immune cells. Tumours developing in WT mice display early deposition of C1q, higher vascular density and an increase in the number of lung metastases compared with C1qa−/− mice. Bone marrow (BM) chimeras between C1qa−/− and WT mice identify non-BM-derived cells as the main local source of C1q that can promote cancer cell adhesion, migration and proliferation. Together these findings support a role for locally synthesized C1q in promoting tumour growth.

In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils

Mature neutrophils are notoriously short‐lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time‐consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional‐Hoxb8 immortalized precursor‐derived neutrophils. In vitro‐derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single‐cell analysis to define functional attributes. We have validated this approach using CD11b‐deficient neutrophils and replicated the key findings observed in gene‐targeted animals and in naturally CD11b‐deficient humans. Furthermore, we show that by retroviral trans‐duction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher‐throughput genetic modification and in vivo functional analysis to the neutrophil‐lineage.—McDonald, J. U., Cortini, A, Rosas, M., Fossati‐Jimack, L., Ling, G. S., Lewis, K. J., Dewitt, S., Liddiard, K., Brown, G. D., Jones, S. A, Hallett, M. B., Botto, M., Taylor, P. R. In vivo functional analysis and genetic modification of in vitro‐derived mouse neutrophils. FASEB J. 25, 1972‐1982 (2011). www.fasebj.org

C1q Modulates the Response to TLR7 Stimulation by Pristane-Primed Macrophages: Implications for Pristane-Induced Lupus.

The complement component C1q is known to play a controversial role in the pathogenesis of systemic lupus erythematosus, but the underlying mechanisms remain poorly understood. Intraperitoneal injection of pristane induces a lupus-like syndrome whose pathogenesis implicates the secretion of type I IFN by CD11b+ Ly6Chigh inflammatory monocytes in a TLR7-dependent fashion. C1q was also shown to influence the secretion of IFN-α. In this study, we explored whether C1q deficiency could affect pristane-induced lupus. Surprisingly, C1qa−/− mice developed lower titers of circulating Abs and milder arthritis compared with the controls. In keeping with the clinical scores, 2 wk after pristane injection the peritoneal recruitment of CD11b+ Ly6Chigh inflammatory monocytes in C1qa−/− mice was impaired. Furthermore, C1q-deficient pristane-primed resident peritoneal macrophages secreted significantly less CCL3, CCL2, CXCL1, and IL-6 when stimulated in vitro with TLR7 ligand. Replenishing C1q in vivo during the pristane-priming phase rectified this defect. Conversely, pristane-primed macrophages from C3-deficient mice did not show impaired cytokine production. These findings demonstrate that C1q deficiency impairs the TLR7-dependent chemokine production by pristane-primed peritoneal macrophages and suggest that C1q, and not C3, is involved in the handling of pristane by phagocytic cells, which is required to trigger disease in this model.

C1q restrains autoimmunity and viral infection by regulating CD8+ T cell metabolism

Complement is a CD8+ T cell metabolic rheostat Systemic lupus erythematosus (SLE) is associated with deficiencies in the complement protein C1q. Although C1q plays a role in the clearance of apoptotic cells, there are several redundant clearance pathways. Disruption of one pathway does not lead to an autoimmune defect. In a chronic graft-versus-host disease model of SLE, Ling et al. show that C1q dampens CD8+ T cell responses to self-antigens. C1q modulates metabolism through the mitochondrial cell-surface protein p32/gC1qR. The lack of C1q during a viral infection also enhances CD8+ T cell responses. Thus, C1q plays a role as a “metabolic rheostat” for effector CD8+ T cells. Science, this issue p. 558 The initiator of the complement cascade, which protects against lupus, controls CD8+ T cell responses via mitochondrial metabolism. Deficiency of C1q, the initiator of the complement classical pathway, is associated with the development of systemic lupus erythematosus (SLE). Explaining this association in terms of abnormalities in the classical pathway alone remains problematic because C3 deficiency does not predispose to SLE. Here, using a mouse model of SLE, we demonstrate that C1q, but not C3, restrains the response to self-antigens by modulating the mitochondrial metabolism of CD8+ T cells, which can themselves propagate autoimmunity. C1q deficiency also triggers an exuberant effector CD8+ T cell response to chronic viral infection leading to lethal immunopathology. These data establish a link between C1q and CD8+ T cell metabolism and may explain how C1q protects against lupus, with implications for the role of viral infections in the perpetuation of autoimmunity.

Complement receptor 3 mediates renal protection in experimental C3 glomerulopathy

C3 glomerulopathy is a complement-mediated renal disease that is frequently associated with abnormalities in regulation of the complement alternative pathway. Mice with deficiency of factor H (Cfh–/–), a negative alternative pathway regulator, are an established experimental model of C3 glomerulopathy in which complement C3 fragments including iC3b accumulate along the glomerular basement membrane. Here we show that deficiency of complement receptor 3 (CR3), the main receptor for iC3b, enhances the severity of spontaneous renal disease in Cfh–/– mice. This effect was found to be dependent on CR3 expression on bone marrow–derived cells. CR3 also mediated renal protection outside the setting of factor H deficiency, as shown by the development of enhanced renal injury in CR3-deficient mice during accelerated nephrotoxic nephritis. The iC3b–CR3 interaction downregulated the proinflammatory cytokine response of both murine and human macrophages to lipopolysaccharide stimulation in vitro, suggesting that the protective effect of CR3 on glomerular injury was mediated via modulation of macrophage-derived proinflammatory cytokines. Thus, CR3 has a protective role in glomerulonephritis and suggests that pharmacologic potentiation of the macrophage CR3 interaction with iC3b could be therapeutically beneficial.

Intranasal peptide‐induced tolerance and linked suppression: consequences of complement deficiency

A role for complement, particularly the classical pathway, in the regulation of immune responses is well documented. Deficiencies in C1q or C4 predispose to autoimmunity, while deficiency in C3 affects the suppression of contact sensitization and generation of oral tolerance. Complement components including C3 have been shown to be required for both B‐cell and T‐cell priming. The mechanisms whereby complement can mediate these diverse regulatory effects are poorly understood. Our previous work, using the mouse minor histocompatibility (HY) model of skin graft rejection, showed that both C1q and C3 were required for the induction of tolerance following intranasal peptide administration. By comparing tolerance induction in wild‐type C57BL/6 and C1q‐, C3‐, C4‐ and C5‐deficient C57BL/6 female mice, we show here that the classical pathway components including C3 are required for tolerance induction, whereas C5 plays no role. C3‐deficient mice failed to generate a functional regulatory T (Treg) –dendritic cell (DC) tolerogenic loop required for tolerance induction. This was related to the inability of C3‐deficient DC to up‐regulate the arginine‐consuming enzyme, inducible nitric oxide synthase (Nos‐2), in the presence of antigen‐specific Treg cells and peptide, leading to reduced Treg cell generation. Our findings demonstrate that the classical pathway and C3 play a critical role in the peptide‐mediated induction of tolerance to HY by modulating DC function.

Complement C3 Exacerbates Imiquimod-Induced Skin Inflammation and Psoriasiform Dermatitis

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