Wen-Chao Song

Tissue-specific deletion of Crry from mouse proximal tubular epithelial cells increases susceptibility to renal ischemia–reperfusion injury

The murine cell surface protein Crry is a key complement regulator with similar activities to human membrane cofactor protein (MCP) and decay-accelerating factor. MCP plays a critical role in preventing complement-mediated tissue injury and its mutation has been implicated in several human kidney diseases. Study of Crry in mice has relevance to understanding MCP activity in human diseases but such efforts have been hampered by the embryonic lethality phenotype of Crry gene knockout. Here we used a conditional gene targeting approach and deleted Crry from the mouse proximal tubular epithelial cells where Crry is prominently expressed. Absence of Crry from proximal tubular epithelial cells resulted in spontaneous C3 deposition on the basolateral surface but no apparent renal disease in unchallenged mice. However, mice deficient in Crry on proximal tubular epithelial cells developed exacerbated renal injury when subjected to renal ischemia reperfusion showing increased blood urea nitrogen levels, higher tubular injury scores, more tubular epithelial cell apoptosis, and inflammatory infiltrates. Renal ischemia reperfusion injury in the Crry conditional knockout mice was prevented by blocking C3 and C5 activation using an anti-properdin or anti-C5 mAb, respectively. Thus, Crry has a critical role in protecting proximal tubular epithelial cells during ischemia reperfusion challenge. Our results highlight the latent risk for inflammatory kidney injury associated with defects in membrane complement regulators.

Decay-Accelerating Factor 1 Deficiency Exacerbates Leptospiral-Induced Murine Chronic Nephritis and Renal Fibrosis

Leptospirosis is a global zoonosis caused by pathogenic Leptospira, which can colonize the proximal renal tubules and persist for long periods in the kidneys of infected hosts. Here, we characterized the infection of C57BL/6J wild-type and Daf1−/− mice, which have an enhanced host response, with a virulent Leptospira interrogans strain at 14 days post-infection, its persistence in the kidney, and its link to kidney fibrosis at 90 days post-infection. We found that Leptospira interrogans can induce acute moderate nephritis in wild-type mice and is able to persist in some animals, inducing fibrosis in the absence of mortality. In contrast, Daf1−/− mice showed acute mortality, with a higher bacterial burden. At the chronic stage, Daf1−/− mice showed greater inflammation and fibrosis than at 14 days post-infection and higher levels at all times than the wild-type counterpart. Compared with uninfected mice, infected wild-type mice showed higher levels of IL-4, IL-10 and IL-13, with similar levels of α-smooth muscle actin, galectin-3, TGF-β1, IL-17, IFN-γ, and lower IL-12 levels at 90 days post-infection. In contrast, fibrosis in Daf1−/− mice was accompanied by high expression of α-smooth muscle actin, galectin-3, IL-10, IL-13, and IFN-γ, similar levels of TGF-β1, IL-12, and IL-17 and lower IL-4 levels. This study demonstrates the link between Leptospira-induced murine chronic nephritis with renal fibrosis and shows a protective role of Daf1.

Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, and tumor control during t-cell immunotherapy

ABSTRACT Cancer immunotherapy relies upon the ability of T cells to infiltrate tumors. The endothelium constitutes a barrier between the tumor and effector T cells, and the ability to manipulate local vascular permeability could be translated into effective immunotherapy. Here, we show that in the context of adoptive T cell therapy, antitumor T cells, delivered at high enough doses, can overcome the endothelial barrier and infiltrate tumors, a process that requires local production of C3, complement activation on tumor endothelium and release of C5a. C5a, in turn, acts on endothelial cells promoting the upregulation of adhesion molecules and T-cell homing. Genetic deletion of C3 or the C5a receptor 1 (C5aR1), and pharmacological blockade of C5aR1, impaired the ability of T cells to overcome the endothelial barrier, infiltrate tumors, and control tumor progression in vivo, while genetic chimera mice demonstrated that C3 and C5aR1 expression by tumor stroma, and not leukocytes, governs T cell homing, acting on the local endothelium. In vitro, endothelial C3 and C5a expressions were required for endothelial activation by type 1 cytokines. Our data indicate that effective immunotherapy is a consequence of successful homing of T cells in response to local complement activation, which disrupts the tumor endothelial barrier.

Properdin Contributes to Allergic Airway Inflammation through Local C3a Generation

Complement is implicated in asthma pathogenesis, but its mechanism of action in this disease remains incompletely understood. In this study, we investigated the role of properdin (P), a positive alternative pathway complement regulator, in allergen-induced airway inflammation. Allergen challenge stimulated P release into the airways of asthmatic patients, and P levels positively correlated with proinflammatory cytokines in human bronchoalveolar lavage (BAL). High levels of P were also detected in the BAL of OVA-sensitized and challenged but not naive mice. Compared with wild-type (WT) mice, P-deficient (P−/−) mice had markedly reduced total and eosinophil cell counts in BAL and significantly attenuated airway hyperresponsiveness to methacholine. Ab blocking of P at both sensitization and challenge phases or at challenge phase alone, but not at sensitization phase alone, reduced airway inflammation. Conversely, intranasal reconstitution of P to P−/− mice at the challenge phase restored airway inflammation to wild-type levels. Notably, C3a levels in the BAL of OVA-challenged P−/− mice were significantly lower than in wild-type mice, and intranasal coadministration of an anti-C3a mAb with P to P−/− mice prevented restoration of airway inflammation. These results show that P plays a key role in allergen-induced airway inflammation and represents a potential therapeutic target for human asthma.

C5 inhibition prevents renal failure in a mouse model of lethal C3 glomerulopathy

C3 glomerulopathy is a potentially life-threatening disease of the kidney caused by dysregulated alternative pathway complement activation. The specific complement mediator(s) responsible for kidney injury in C3 glomerulopathy are yet to be defined and no specific therapy is currently available. We previously developed a mouse model of lethal C3 glomerulopathy with factor H and properdin gene double mutations. Therefore, we used this model to examine the role of C5 and C5a receptor (C5aR) in the pathogenesis of the disease. Disease severity in these factor H/properdin double-mutant mice was found to be correlated with plasma C5 levels, and prophylactic anti-C5 mAb therapy was effective in preventing lethal C3 glomerulopathy. When given to these double-mutant mice that had already developed active disease with severe proteinuria, anti-C5 mAb treatment also prevented death in half of the mice. Deficiency of C5aR significantly reduced disease severity, suggesting that C5aR-mediated inflammation contributed to C3 glomerulopathy. Thus, C5 and C5aR have a critical role in C3 glomerulopathy. Hence, early intervention targeting these pathways may be an effective therapeutic strategy for patients with C3 glomerulopathy.

Overactivation of Complement Alternative Pathway in Postpartum Atypical Hemolytic Uremic Syndrome Patients with Renal Involvement.

Postpartum atypical hemolytic uremic syndrome (aHUS) is a life‐threatening syndrome with unclear pathogenesis. The current study aimed to investigate the clinical and pathological features, complement activation status, and the genetic variations in a Chinese cohort of patients with renal biopsy‐proven postpartum aHUS.

Murine systemic thrombophilia and hemolytic uremic syndrome from a factor H point mutation

Complement plays a key role in host defense, but its dysregulation can cause autologous tissue injury. Complement activation is normally controlled by regulatory proteins, including factor H (FH) in plasma and membrane cofactor protein (MCP) on the cell surface. Mutations in FH and MCP are linked to atypical hemolytic uremic syndrome, a type of thrombotic microangiopathy (TMA) that causes renal failure. We describe here that disruption of FH function on the cell surface can also lead to disseminated complement-dependent macrovascular thrombosis. By gene targeting, we introduced a point mutation (W1206R) into murine FH that impaired its interaction with host cells but did not affect its plasma complement-regulating activity. Homozygous mutant mice carrying this mutation developed renal TMA as well as systemic thrombophilia involving large blood vessels in multiple organs, including liver, lung, spleen, and kidney. Approximately 30% of mutant mice displayed symptoms of stroke and ischemic retinopathy, and 48% died prematurely. Genetic deficiency of complement C3 and factor D prevented both the systemic thrombophilia and renal TMA phenotypes. These results demonstrate a causal relationship between complement dysregulation and systemic angiopathy and suggest that complement activation may contribute to various human thrombotic disorders involving both the micro- and macrovasculature.

Modeling complement-driven diseases in transgenic mice: Values and limitations.

Remarkable advances have been made over past decades in understanding the pathogenesis of complement-mediated diseases. This has led to development of new therapies for, and in some cases re-classification of, complement-driven diseases. This success is due to not only insight from human patients but also studies using transgenic animal models. Animal models that mimic human diseases are useful tools to understand the mechanism of disease and develop new therapies but there are also limitations due to species differences in their complement systems. This review provides a summary of transgenic animal models for three human diseases that are at the forefront of anti-complement therapy, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). They are discussed here as examples to highlight the values and limitations of animal modeling in complement-driven diseases.

Direct Binding of the pH-Regulated Protein 1 (Pra1) from Candida albicans Inhibits Cytokine Secretion by Mouse CD4+ T Cells

Opportunistic infections with the saprophytic yeast Candida albicans are a major cause of morbidity in immunocompromised patients. While the interaction of cells and molecules of innate immunity with C. albicans has been studied to great depth, comparatively little is known about the modulation of adaptive immunity by C. albicans. In particular, direct interaction of proteins secreted by C. albicans with CD4+ T cells has not been studied in detail. In a first screening approach, we identified the pH-regulated antigen 1 (Pra1) as a molecule capable of directly binding to mouse CD4+ T cells in vitro. Binding of Pra1 to the T cell surface was enhanced by extracellular Zn2+ ions which Pra1 is known to scavenge from the host in order to supply the fungus with Zn2+. In vitro stimulation assays using highly purified mouse CD4+ T cells showed that Pra1 increased proliferation of CD4+ T cells in the presence of plate-bound anti-CD3 monoclonal antibody. In contrast, secretion of effector cytokines such as IFNγ and TNF by CD4+ T cells upon anti-CD3/ anti-CD28 mAb as well as cognate antigen stimulation was reduced in the presence of Pra1. By secreting Pra1 C. albicans, thus, directly modulates and partially controls CD4+ T cell responses as shown in our in vitro assays.

Retinal Pre-Conditioning by CD59a Knockout Protects against Light-Induced Photoreceptor Degeneration.

Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments. Retinal degeneration and function were compared in WT versus KO mice following light damage. Gene expression changes, endoplasmic reticulum (ER) stress, and glial cell activation were also compared. At baseline, the ERG responses and rhodopsin levels were lower in CD59aKO compared to wild-type (WT) mice. Following LD, the ERG responses were better preserved in CD59aKO compared to WT mice. Correspondingly, the number of photoreceptors was higher in CD59aKO retinas than WT controls after LD. Under normal light conditions, CD59aKO mice had higher levels than WT for GFAP immunostaining in Müller cells, mRNA and protein levels of two ER-stress markers, and neurotrophic factors. The reduction in photon capture, together with the neurotrophic factor upregulation, may explain the structural and functional protection against LD in the CD59aKO.