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This shows the efficiency of immuodepletion experiments

The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3−/− mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ+/TNFα+/IL10+ CD4+ and CD8+ T cells. Immunodepletion of CD4+ but not CD8+ T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5–dependent pathway.Significance: This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. Cancer Res; 78(1); 143–56. ©2017 AACR.

These figures show experiments of tumor growth in Factor B-/- mice. They also show gating strategy for flow cytometry and changes in inflammatory cells. Data showing loss of CIITA in cancer cells alters the response to complement depletion.

Complement activation at a particular location is determined by the balance of activating and inhibitory proteins. Factor H is a key regulator of the alternative pathway of complement, and genetic or acquired impairments in Factor H are associated with glomerular injury. The human Factor H-related proteins (FHRs) comprise a family of five proteins that are structurally related to Factor H. Variations in the genes or expression levels of the FHRs are also associated with glomerular disease, although the mechanisms of glomerular protection/injury are incompletely understood. To explore the role of the FHRs on complement regulation/ dysregulation in the kidney, we expressed and purified recombinant murine FHRs (FHRs A, B, C and E). These four distinct FHRs contain binding regions with high amino acid sequence homology to binding regions within Factor H, but we observed different interactions of the FHRs with Factor H binding ligands, including heparin and C3d. There was differential binding of the FHRs to the resident kidney cell types (mesangial, glomerular endothelial, podocytes, and tubular epithelial). All four FHRs caused complement dysregulation on kidney cell surfaces in vitro, although the magnitude of the effect differed among the FHRs and also varied among the different kidney cells. However, only FHR E caused glomerular complement dysregulation when injected in vivo but did not exacerbate injury when injected into mice with ischemic acute kidney injury, an alternative pathway-mediated model. Thus, our experiments demonstrate that the FHRs have unique, and likely context-dependent, effects on the different cell types within the kidney.

Experiments in mouse models have shown that the complement cascade is activated within the kidney after ischemia-reperfusion and that complement activation contributes to tubular injury in this setting. Less is known, however, about complement activation in human kidneys after ischemia or whether complement activation in the tubulointerstitium can be detected by measurement of complement fragments in the urine. We hypothesized that urine biomarkers of complement activation would rapidly increase in patients who develop ischemic acute kidney injury, signaling complement activation within the kidney. We confirmed that the alternative pathway of complement is activated in the kidneys of mice after ischemia-reperfusion, and we found that levels of factor B fragments (generated during alternative pathway activation) rapidly increase in the urine. We next performed a case-control study in which we measured complement fragments in human urine samples from patients undergoing cardiac surgery using ELISAs. The level of Ba increased after cardiac surgery and was significantly higher in patients who developed acute kidney injury. The increase in Ba also correlated with magnitude of the subsequent rise in serum creatinine and with the need for hemodialysis during the hospitalization. These findings demonstrate that the alternative pathway of complement is activated in patients who develop acute kidney injury after cardiac surgery and that increases in the level of urine Ba may be a predictive and functional biomarker of severe kidney injury.

Focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) are common forms of idiopathic nephrotic syndrome. The causes of these diseases are incompletely understood, but the response of patients to immunosuppressive therapies suggests that their pathogenesis is at least in part immune mediated. Preclinical and clinical research indicates that activation of the classical pathway of complement contributes to glomerular injury in FSGS. Glomerular IgM deposits are also prominent in some patients, raising the possibility that IgM is a trigger of classical pathway activation. In the present study, we examined the pattern of complement activation in the glomeruli and plasma of patients with nephrotic syndrome. We also tested whether patients with FSGS and MCD have elevated levels of natural IgM reactive with epitopes on glomerular endothelial cells and cardiolipin. We found evidence of classical pathway activation in patients with idiopathic nephrotic syndrome compared with healthy control subjects. We also detected higher levels of self-reactive IgM to both targets. Based on these results, IgM and classical pathway activation may contribute to disease pathogenesis in some patients with FSGS and MCD. NEW & NOTEWORTHY IgM is detected in biopsies from some patients with nephrotic syndrome, although this has been attributed to passive trapping of the protein. We found, however, that IgM colocalizes with complement activation fragments in some glomeruli. We also found that affected patients had higher levels of IgM reactive to glomerular endothelial cell epitopes. Thus, IgM activates the complement system in the glomeruli of some patients with nephrotic syndrome and may contribute to injury.

Although it was long believed that the complement system helps the body to identify and remove transformed cells, it is now clear that complement activation contributes to carcinogenesis and can also help tumors to escape immune-elimination. Complement is activated by several different mechanisms in various types of cancer, and complement activation fragments have multiple different downstream effects on cancer cells and throughout the tumor microenvironment. Thus, the role of complement activation in tumor biology may vary among different types of cancer and over time within a single tumor. In multiple different pre-clinical models, however, complement activation has been shown to recruit immunosuppressive myeloid cells into the tumor microenvironment. These cells, in turn, suppress anti-tumor T cell immunity, enabling the tumor to grow. Based on extensive pre-clinical work, therapeutic complement inhibitors hold great promise as a new class of immunotherapy. A greater understanding of the role of complement in tumor biology will improve our ability to identify those patients most likely to benefit from this treatment and to rationally combine complement inhibitors with other cancer therapies.

Hepatocellular carcinoma (HCC) is difficult to detect, carries a poor prognosis, and is one of few cancers with an increasing yearly incidence. Molecular defects in complement factor H (CFH), a critical regulatory protein of the complement alternative pathway (AP), are typically associated with inflammatory diseases of the eye and kidney. Little is known regarding the role of CFH in controlling complement activation within the liver. While studying aging CFH-deficient (fH(–/–)) mice, we observed spontaneous hepatic tumor formation in more than 50% of aged fH(–/–) males. Examination of fH(–/–) livers (3–24 months) for evidence of complement-mediated inflammation revealed widespread deposition of complement-activation fragments throughout the sinusoids, elevated transaminase levels, increased hepatic CD8(+) and F4/80(+) cells, overexpression of hepatic mRNA associated with inflammatory signaling pathways, steatosis, and increased collagen deposition. Immunostaining of human HCC biopsies revealed extensive deposition of complement fragments within the tumors. Investigating the Cancer Genome Atlas also revealed that increased CFH mRNA expression is associated with improved survival in patients with HCC, whereas mutations are associated with worse survival. These results indicate that CFH is critical for controlling complement activation in the liver, and in its absence, AP activation leads to chronic inflammation and promotes hepatic carcinogenesis.

The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3−/− mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ+/TNFα+/IL10+ CD4+ and CD8+ T cells. Immunodepletion of CD4+ but not CD8+ T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5–dependent pathway. Significance: This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. Cancer Res; 78(1); 143–56. ©2017 AACR.

Complement activation probably contributes to glomerular inflammation and damage in IgA nephropathy. In this issue, 2 groups report that levels of factor H–related protein 1 are elevated in patients with IgA nephropathy and correlate with disease progression. These studies provide new evidence that the complement cascade is important to the pathogenesis of this disease. These results also suggest that factor H–related protein 1 levels may be useful for identifying those patients at high risk of disease progression.

International audience; Antibody-mediated rejection (AbMR) adversely affects long-term graft survival in kidney transplantation. Currently, the diagnosis of AbMR requires a kidney biopsy, and detection of complement C4d deposition in the allograft is one of the diagnostic criteria. Complement activation also generates several soluble fragments which could potentially provide non-invasive biomarkers of the process. Furthermore, microvesicles released into the plasma from injured cells can serve as biomarkers of vascular injury. To explore whether soluble complement fragments or complement fragments bound to endothelial microvesicles can be used to non-invasively detect AbMR, we developed an in vitro model in which human endothelial cells were exposed to anti-HLA antibodies and complement sufficient serum. We found that complement fragments C4a and sC5b-9 were increased in the supernatants of cells exposed to complement-sufficient serum compared to cells treated complement-deficient serum. Furthermore, complement activation on the cell surface was associated with the release of microvesicles bearing C4 and C3 fragments. We next measured these analytes in plasma from kidney transplant recipients with biopsy-proven acute AbMR (n = 9) and compared the results with those from transplant recipients who also had impaired allograft function but who did not have AbMR (n = 30). Consistent with the in vitro results, complement fragments C4a and Ba were increased in plasma from patients with AbMR compared to control subjects (P

Humoral autoimmunity is central to the development of systemic lupus erythematosus (SLE). Complement receptor type 2 (CR2)/CD21 plays a key role in the development of high-affinity Abs and long-lasting memory to foreign Ags. When CR2 is bound by its primary C3 activation fragment–derived ligand, designated C3d, it coassociates with CD19 on B cells to amplify BCR signaling. C3d and CR2 also mediate immune complex binding to follicular dendritic cells. As the development of SLE involves subversion of normal B cell tolerance checkpoints, one might expect that CR2 ligation by C3d-bound immune complexes would promote development of SLE. However, prior studies in murine models of SLE using gene-targeted Cr2−/− mice, which lack both CR2 and complement receptor 1 (CR1), have demonstrated contradictory results. As a new approach, we developed a highly specific mouse anti-mouse C3d mAb that blocks its interaction with CR2. With this novel tool, we show that disruption of the critical C3d–CR2 ligand-receptor binding step alone substantially ameliorates autoimmunity and renal disease in the MRL/lpr model of SLE.

Background Endothelial microparticles are associated with chronic kidney disease ( CKD ) and complement activation. We hypothesized that the complement pathway is activated in patients with CKD via endothelial microparticles and that complement activation correlates with endothelial dysfunction in CKD . Methods and Results We analyzed complement data of 30 healthy subjects, 30 patients with stage III / IV CKD , and 30 renal transplant recipients with stage III / IV CKD , evaluating the potential correlation of complement fragments with brachial artery flow–mediated dilation, Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and urinary albumin/creatinine ratio. Endothelial microparticles were characterized via proteomic analysis and compared between study groups. Complement fragment Ba was significantly increased in CKD and post–kidney transplant CKD . Plasma Ba levels correlated significantly with lower brachial artery flow–mediated dilation, lower Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and higher urinary albumin/creatinine ratio. Factor D levels were significantly higher in the plasma microparticles of patients with CKD versus healthy controls. Plasma microparticles isolated from patients with CKD and containing factor D activated the alternative pathway in vitro. Conclusion The alternative complement pathway is activated in CKD and correlates with endothelial dysfunction and markers of CKD . Future studies are needed to evaluate whether endothelial microparticles with increased factor D play a pathologic role in CKD ‐associated vascular disease. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 02230202.

Factor H is a circulating protein that regulates activation of the alternative pathway (AP) of complement. Mutations and genetic variations of factor H are associated with several AP-mediated diseases, highlighting the critical role of factor H in AP regulation. AP-mediated inflammation is typically triggered by illness or tissue injury, however, and tissue injury can trigger AP activation in individuals with fully functional factor H. This suggests that factor H function is affected by local conditions within tissues. We hypothesized that inducible proteins impair the ability of factor H to locally control the AP, thereby increasing AP activation. We used purified murine factor H to immunoprecipitate binding partners from mouse kidneys. Using immunoaffinity liquid chromatography–mass spectrometry, we identified annexin A2 as a factor H binding partner. Further experiments showed that annexin A2 reduces the binding of factor H to cell surfaces. Recombinant annexin A2 impaired complement regulation by factor H and increased complement activation on renal cell surfaces in vitro and in vivo. In a murine model of acute pneumococcal otitis media, the administration of annexin A2 increased AP-mediated bacterial opsonization and clearance. In conclusion, the local production of annexin A2 within tissues suppresses regulation of the AP by factor H. Annexin A2 can contribute to AP-mediated tissue inflammation by locally impairing factor H function, but it can also improve complement-mediated bacterial clearance.

Natural IgM binds to glomerular epitopes in several progressive kidney diseases. Previous work has shown that IgM also binds within the glomerulus after ischemia/reperfusion (I/R) but does not fully activate the complement system. Factor H is a circulating complement regulatory protein, and congenital or acquired deficiency of factor H is a strong risk factor for several types of kidney disease. We hypothesized that factor H controls complement activation by IgM in the kidney after I/R, and that heterozygous factor H deficiency would permit IgM-mediated complement activation and injury at this location. We found that mice with targeted heterozygous deletion of the gene for factor H developed more severe kidney injury after I/R than wild-type controls, as expected, but that complement activation within the glomeruli remained well controlled. Furthermore, mice that are unable to generate soluble IgM were not protected from renal I/R, even in the setting of heterozygous factor H deficiency. These results demonstrate that factor H is important for limiting injury in the kidney after I/R, but it is not critical for controlling complement activation by immunoglobulin within the glomerulus in this setting. IgM binds to glomerular epitopes after I/R, but it is not a significant source of injury.

International audience; Mutations in the complement regulatory proteins areassociated with several different diseases. Although these mutations cause dysregulated alternative pathway activation throughout the body, the kidneys are the most common site of injury. The susceptibility of the kidney to alternative pathway-mediated injury may be due to limited expression of complement regulatory proteins on several tissue surfaces within the kidney. To examine the roles of the complement regulatory proteins factor H and Crry inprotecting distinct renal surfaces from alternative pathway mediated injury, we generated mice with targeted deletions of the genes for both proteins. Surprisingly, mice with combined genetic deletions of factor H and Crry developed significantly milder renal injury than mice deficient in only factor H. Deficiency of both factor H and Crry was associated with C3 deposition at multiple locations within the kidney, but glomerular C3 deposition was lower than that in factor H alone deficient mice. Thus, factor H and Crry are critical for regulating complement activation at distinct anatomic sites within the kidney.However, widespread activation of the alternative pathway reduces injury by depleting the pool of C3 available at any1 location