Liver regeneration stimulated by a loss of liver mass leads to hepatocyte and nonparenchymal cell proliferation and rapid restoration of liver parenchyma. Mice with targeted disruption of the interleukin-6 (IL-6) gene had impaired liver regeneration characterized by liver necrosis and failure. There was a blunted DNA synthetic response in hepatocytes of these mice but not in nonparenchymal liver cells. Furthermore, there were discrete G1 phase (prereplicative stage in the cell cycle) abnormalities including absence of STAT3 (signal transducer and activator of transcription protein 3) activation and depressed AP-1, Myc, and cyclin D1 expression. Treatment of IL-6-deficient mice with a single preoperative dose of IL-6 returned STAT3 binding, gene expression, and hepatocyte proliferation to near normal and prevented liver damage, establishing that IL-6 is a critical component of the regenerative response.

The involvement of complement activation products in promoting tumor growth has not yet been recognized. Here we show that generation of complement C5a in the tumor microenvironment enhanced tumor growth by suppressing the anti-tumor CD8+ T cell-mediated response. This suppression was associated with the recruitment of myeloid-derived suppressor cells (MDSCs) into tumors and augmentation of their T cell-directed suppressive capabilities. Amplification of MDSC suppressive capacity by C5a occurred through regulation of the production of reactive oxygen and nitrogen species. Pharmacological blockade of C5a receptor significantly impaired tumor growth to a degree comparable to the effect produced by the anti-cancer drug Taxol. Thus, this study demonstrates a therapeutic role for complement inhibition in the treatment of cancer.

Interleukin‐6 null (IL‐6−/−) mice have impaired liver regeneration and increased liver necrosis following partial hepatectomy that is corrected with IL‐6 treatment. Following acute carbon tetrachloride (CCl4 ) treatment, we found that IL‐6−/− mice developed increased hepatocellular injury and defective regeneration with significant blunting of signal transducer‐and‐activator of transcription protein 3 (STAT3) and nuclear factor‐κB (NF‐κB) activation and reduced hepatocyte DNA synthetic and mitotic responses. After CCl4 treatment, unlike partial hepatectomy, increased hepatocyte apoptosis was noted in IL‐6−/− livers. Pretreatment with IL‐6 before CCl4 reduced acute CCl4 injury and apoptosis and accelerated regeneration in both IL‐6+/+ and −/− livers. Repetitive doses of CCl4 in the presence or absence of phenobarbital resulted in increased injury and fibrosis in IL‐6 −/− compared with +/+ livers. After acute and chronic injury, IL‐6−/− livers showed the protracted presence of α‐smooth muscle actin associated with activated stellate cells, indicating a disturbed response in wound healing that progressed to fibrosis. These data provide evidence for an important role for IL‐6 in reducing CCl4 –induced acute and chronic liver injury and fibrosis.

Therapeutic modulation of the complement system has become increasingly important in line with the growing recognition of the role of complement in numerous diseases. Compstatin, a peptidic inhibitor that acts at the central level of the complement cascade, is currently in clinical evaluation but routes to improve its efficacy have not yet been fully explored. Here, we report improvements in both the inhibitory potency and pharmacokinetic parameters of compstatin that broaden its clinical applications. Selective modification of the compstatin N-terminus with non-proteinogenic amino acids resulted in the first analogue with subnanomolar binding affinity (KD = 0.5 nM) and other similarly potent derivatives with improved solubility in clinically relevant solvents. Detailed structure-activity relationship studies based on biophysical and computational methods revealed key structural determinants for the observed improvements. Importantly, pharmacokinetic evaluation in non-human primates revealed target-driven elimination kinetics with plasma half-life values exceeding expectations for peptidic drugs (close to 12 hours). This successful optimization strategy is expected to pave the way for systemic administration of compstatin in a range of clinical conditions.

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Chronic periodontitis is induced by a dysbiotic microbiota and leads to inflammatory destruction of tooth-supporting connective tissue and bone. The third component of complement, C3, is a point of convergence of distinct complement activation mechanisms but its involvement in periodontitis was not previously addressed. We investigated this question using two animal species models, namely, C3-deficient or wild-type mice and non-human primates (NHP) locally treated with a potent C3 inhibitor (the compstatin analog Cp40) or an inactive peptide control. In mice, C3 was required for maximal periodontal inflammation and bone loss and for the sustenance of the dysbiotic microbiota. The effect of C3 on the microbiota was therefore different from that reported for the C5a receptor, which is required for the initial induction of dysbiosis. C3-dependent bone loss was demonstrated in distinct models, including Porphyromonas gingivalis-induced periodontitis, ligature-induced periodontitis, and aging-associated periodontitis. Importantly, local treatment of NHP with Cp40 inhibited ligature-induced periodontal inflammation and bone loss, which correlated with lower gingival crevicular fluid levels of proinflammatory mediators (e.g., IL-17 and RANKL) and decreased osteoclastogenesis in bone biopsy specimens, as compared to control treatment. This is the first time, for any disease, that complement inhibition in NHP was shown to inhibit inflammatory processes that lead to osteoclastogenesis and bone loss. These data strongly support the feasibility of C3-targeted intervention for the treatment of human periodontitis.

Beyond its role in immunity, complement mediates a wide range of functions in the context of morphogenetic or tissue remodeling processes. Angiogenesis is crucial during tissue remodeling in multiple pathologies; however, the knowledge about the regulation of neovascularization by the complement components is scarce. Here we studied the involvement of complement in pathological angiogenesis. Strikingly, we found that mice deficient in the central complement component C3 displayed increased neovascularization in the model of retinopathy of prematurity (ROP) and in the in vivo Matrigel plug assay. In addition, antibody-mediated blockade of C5, treatment with C5aR antagonist, or C5aR deficiency in mice resulted in enhanced pathological retina angiogenesis. While complement did not directly affect angiogenesis-related endothelial cell functions, we found that macrophages mediated the antiangiogenic activity of complement. In particular, C5a-stimulated macrophages were polarized toward an angiogenesis-inhibitory phenotype, including the up-regulated secretion of the antiangiogenic soluble vascular endothelial growth factor receptor-1. Consistently, macrophage depletion in vivo reversed the increased neovascularization associated with C3- or C5aR deficiency. Taken together, complement and in particular the C5a-C5aR axes are potent inhibitors of angiogenesis.

Although the complement system has been implicated in liver regeneration after toxic injury and partial hepatectomy, the mechanism or mechanisms through which it participates in these processes remains ill-defined. In this study, we demonstrate that complement activation products (C3a, C3b/iC3b) are generated in the serum of experimental mice after CCl4 injection and that complement activation is required for normal liver regeneration. Decomplementation by cobra venom factor resulted in impaired entry of hepatocytes into S phase of the cell cycle. In addition, livers from C3-deficient (C3−/−) mice showed similarly impaired proliferation of hepatocytes, along with delayed kinetics of both hepatocyte hyperplasia and removal of injured liver parenchyma. Restoration of hepatocyte proliferative capabilities of C3−/− mice through C3a reconstitution, as well as the impaired regeneration of C3a receptor-deficient mice, demonstrated that C3a promotes liver cell proliferation via the C3a receptor. These findings, together with data showing two waves of complement activation, indicate that C3 activation is a pivotal mechanism for liver regeneration after CCl4 injury, which fulfills multiple roles; C3a generated early after toxin injection is relevant during the priming of hepatocytes, whereas C3 activation at later times after CCl4 treatment contributes to the clearance of injured tissue.