Alexandru Tatomir

The role of complement system in adipose tissue-related inflammation

Abstract As the common factor linking adipose tissue to the metabolic context of obesity, insulin resistance and atherosclerosis are associated with a low-grade chronic inflammatory status, to which the complement system is an important contributor. Adipose tissue synthesizes complement proteins and is a target of complement activation. C3a-desArg/acylation-stimulating protein stimulates lipogenesis and affects lipid metabolism. The C3a receptor and C5aR are involved in the development of adipocytes’ insulin resistance through macrophage infiltration and the activation of adipose tissue. The terminal complement pathway has been found to be instrumental in promoting hyperglycemia-associated tissue damage, which is characteristic of the major vascular complications of diabetes mellitus and diabetic ketoacidosis. As a mediator of the effects of the terminal complement complex C5b-9, RGC-32 has an impact on energy expenditure as well as lipid and glucose metabolic homeostasis. All of this evidence, taken together, indicates an important role for complement activation in metabolic diseases.

The role of complement activation in atherogenesis: the first 40 years.

The pathogenesis of atherosclerotic inflammation is a multi-step process defined by the interweaving of excess modified lipid particles, monocyte-macrophages populations, and innate immune and adaptive immunity effectors. A part of innate immunity, the complement system, is an important player in the induction and progression of atherosclerosis. The accumulation of either oxidized or enzymatically modified LDL—bound to C-reactive protein or not—prompts complement activation leading to the assembly of the terminal complement C5b-9 complex in the atherosclerotic lesion. The sublytic C5b-9 assembly leads to the activation and proliferation of smooth muscle and endothelial cells, accompanied by the release of various chemotactic, pro-adhesion, and procoagulant cytokines from these cells. Response gene to complement (RGC)-32, an essential effector of the terminal complement complex C5b-9, also affects atherogenesis, propelling vascular smooth muscle cell proliferation and migration, stimulating endothelial proliferation, and promoting vascular lesion formation. A substantial amount of experimental work has suggested a role for the complement system activation during atherosclerotic plaque formation, with the proximal classical complement pathway seemingly having a protective effect and terminal complement contributing to accelerated atherogenesis. All these data suggest that complement plays an important role in atherogenesis.

RGC-32 is expressed in the human atherosclerotic arterial wall: Role in C5b-9-induced cell proliferation and migration.

The complement system is an important player in the development of atherosclerosis. Previously reported as a cell cycle regulator, RGC-32 is an essential effector of the terminal complement complex, C5b-9. In this study, our aims were to determine the expression of RGC-32 in the human atherosclerotic arterial wall and to delineate the mechanisms through which RGC-32 affects C5b-9-induced endothelial cell proliferation and migration. We now demonstrate that RGC-32 is expressed in human aortic atherosclerotic wall and that RGC-32 expression increases with the progression of atherosclerosis. Furthermore, silencing of RGC-32 expression abolished C5b-9-induced human aortic endothelial cell (HAEC) proliferation and migration. Of the 279 genes differentially expressed in HAECs after RGC-32 silencing, the genes involved in cell adhesion and cell cycle activation were significantly regulated by RGC-32. RGC-32 silencing caused a significant reduction in the expression of cyclin D1, cyclin D3, Akt, ROCK1, Rho GDP dissociation inhibitor alpha and profilin. These data suggest that RGC-32 mediates HAEC migration through the regulation of RhoA and ROCK1 expression and is involved in actin cytoskeletal organization. Thus, RGC-32 has promising therapeutic potential with regard to angiogenesis and atherosclerosis.

RGC-32 Promotes Th17 Cell Differentiation and Enhances Experimental Autoimmune Encephalomyelitis

Th17 cells play a critical role in autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Response gene to complement (RGC)-32 is a cell cycle regulator and a downstream target of TGF-β that mediates its profibrotic activity. In this study, we report that RGC-32 is preferentially upregulated during Th17 cell differentiation. RGC-32−/− mice have normal Th1, Th2, and regulatory T cell differentiation but show defective Th17 differentiation in vitro. The impaired Th17 differentiation is associated with defects in IFN regulatory factor 4, B cell–activating transcription factor, retinoic acid–related orphan receptor γt, and SMAD2 activation. In vivo, RGC-32−/− mice display an attenuated experimental autoimmune encephalomyelitis phenotype accompanied by decreased CNS inflammation and reduced frequency of IL-17– and GM-CSF–producing CD4+ T cells. Collectively, our results identify RGC-32 as a novel regulator of Th17 cell differentiation in vitro and in vivo and suggest that RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases.

Phosphorylated SIRT1 as a biomarker of relapse and response to treatment with glatiramer acetate in multiple sclerosis

We have previously shown that SIRT1 mRNA expression was significantly lower in relapsing MS patients compared to those in remission. Our goal was to longitudinally investigate the role of active, phosphorylated SIRT1 (p-SIRT1) as a potential biomarker of relapse and predictor for response to glatiramer acetate (GA) treatment in patients with relapsing remitting multiple sclerosis (MS). We also want to investigate the downstream effects of SIRT1 activation by measuring the trimethylation of histone 3 at lysine 9 (H3K9me3). A cohort of 15 GA-treated patients was clinically monitored using the Expanded Disability Status Scale (EDSS) and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 6, and 12 months after initiation of the therapy. P-SIRT1 and H3K9me3 levels were assayed by Western blotting using specific antibodies. Statistically significant lower levels of p-SIRT1 protein (p < 0.0001) and H3K9me3 (p = 0.001) were found during relapses when compared to stable MS patients. Non-responders to GA treatment were defined as patients who exhibited at least two relapses following initiation of GA treatment. Statistically significant lower levels of p-SIRT1 protein (p = 0.02) and H3K9me3 (p = 0.004) were found in GA non-responders compared to responders. Using receiver operating characteristic analysis, area under the curve (AUC) for p-SIRT1 was 77% (p = 0.007) and for H3K9me3 was 81% (p = 0.002) for prediction of relapse. For predicting responsiveness to GA treatment, AUC was 75% (P = 0.01) for H3K9me3. Our data suggest that p-SIRT1 and H3K9me3 could serve as potential biomarkers for MS relapse. In addition, H3K9me3 could serve as possible biomarker to predict response to GA treatment.

The complement system as a biomarker of disease activity and response to treatment in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system. The complement system has an established role in the pathogenesis of MS, and evidence suggests that its components can be used as biomarkers of disease-state activity and response to treatment in MS. Plasma C4a levels have been found to be significantly elevated in patients with active relapsing-remitting MS (RRMS), as compared to both controls and patients with stable RRMS. C3 levels are also significantly elevated in the cerebrospinal fluid (CSF) of patients with RRMS, and C3 levels are correlated with clinical disability. Furthermore, increased levels of factor H can predict the transition from relapsing to progressive disease, since factor H levels have been found to increase progressively with disease progression over a 2-year period in patients transitioning from RRMS to secondary progressive (SP) MS. In addition, elevations in C3 are seen in primary progressive (PP) MS. Complement components can also differentiate RRMS from neuromyelitis optica. Response gene to complement (RGC)-32, a novel molecule induced by complement activation, is a possible biomarker of relapse and response to glatiramer acetate (GA) therapy, since RGC-32 mRNA expression is significantly decreased during relapse and increased in responders to GA treatment. The predictive accuracy of RGC-32 as a potential biomarker (by ROC analysis) is 90% for detecting relapses and 85% for detecting a response to GA treatment. Thus, complement components can serve as biomarkers of disease activity to differentiate MS subtypes and to measure response to therapy.

Markers of immune-mediated inflammation in the brains of young adults and adolescents with type 1 diabetes and fatal diabetic ketoacidosis. Is there a difference?

Due to the limited data on diabetic ketoacidosis and brain edema (DKA/BE) in children/adolescents and the lack of recent data on adults with type 1 diabetes (T1D), we addressed the question of whether neuroinflammation was present in the fatal DKA of adults. We performed immunohistochemistry (IHC) studies on the brains of two young adults with T1D and fatal DKA and compared them with two teenagers with poorly controlled diabetes and fatal DKA. C5b-9, the membrane attack complex (MAC) had significantly greater deposits in the grey and white matter of the teenagers than the young adults (p=0.03). CD59, a MAC assembly inhibitory protein was absent, possibly suppressed by the hyperglycemia in the teenagers but was expressed in the young adults despite comparable average levels of hyperglycemia. The receptor for advanced glycation end products (RAGE) had an average expression in the young adults significantly greater than in the teenagers (p=0.02). The autophagy marker Light Chain 3 (LC3) A/B was the predominant form of programmed cell death (PCD) in the teenage brains. The young adults had high expressions of both LC3A/B and TUNEL, an apoptotic cell marker for DNA fragmentation. BE was present in the newly diagnosed young adult with hyperglycemic hyperosmolar DKA and also in the two teenagers. Our data indicate that significant differences in neuroinflammatory components, initiated by the dysregulation of DKA and interrelated metabolic and immunologic milieu, are likely present in the brains of fatal DKA of teenagers when compared with young adults.

RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis

Extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-β downstream effects, but its role in gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we investigated its involvement in TGF-β-induced ECM expression and the upregulation of the reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin. In cultured neonatal rat astrocytes, collagens I, IV, and V, fibronectin, α-SMA, and nestin were significantly induced by TGF-β stimulation, and RGC-32 silencing resulted in a significant reduction in their expression. Using astrocytes isolated from RGC-32 knock-out (KO) mice, we found that the expression of TGF-β-induced collagens I, IV, and V, fibronectin, and α-SMA was significantly reduced in RGC-32 KO mice when compared with wild-type (WT) mice. SIS3 inhibition of Smad3 phosphorylation was also associated with a significant reduction in RGC-32 nuclear translocation and TGF-β-induced collagen I expression. In addition, during experimental autoimmune encephalomyelitis (EAE), RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype. Taken together, our data demonstrate that RGC-32 plays an important role in mediating TGF-β-induced reactive astrogliosis in EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.

Letter by Rus et al Regarding Article, “RGC-32 (Response Gene to Complement 32) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis”

The recently published article by Cui et al1 sheds new light on the role of RGC-32 (response gene to complement 32) in atherogenesis. This interesting article describes a new mechanism of action of RGC-32 in inducing the expression of the endothelial cell adhesion molecules ICAM-1 and VCAM-1, which are necessary for monocyte recruitment into the atherogenic lesion core. We would like to add a few comments on several findings reported in this article. We have also examined the expression of RGC-32 in the human aortic atherosclerotic wall.2 We found that RGC-32 was expressed in human aortic atherosclerotic lesions: by endothelial cells, by inflammatory cells (CD4+ and CD68+ cells) in intimal thickenings and fibrous …

AI-05 Response gene to complement-32 promotes plasma cell differentiation and enhances lupus-like chronic graft versus host disease

Background Response Gene to Complement (RGC)−32 is an intracellular protein that plays a role in cell growth and promotes cell cycle activation and Akt phosphorylation. RGC-32 is also a downstream target of TGF-β in fibroblasts and renal proximal tubular cells and plays a role in renal fibrogenesis. In immune cells, RGC-32 is expressed by both T and B lymphocytes. Our prior studies showed that RGC-32 promotes Th17 differentiation of mouse CD4 T cells and is highly expressed in human IL-17 CD4 cells. Whether RGC-32 plays a role in the activation and differentiation of B cells and the development of autoimmunity is not known. We used WT and RGC-32 KO mice to determine whether lack of RGC-32 impairs B cell differentiation and activation and alters autoimmune parameters in the chronic graft versus host disease (cGVHD) model of lupus. Materials and methods B cells were cultured with lps, anti-CD40 mAb, IL-21 and IL-6, IL-4 or TGFβ and RGC-32 mRNA and protein expression was determined. TLR-dependent and T dependent B cell differentiation to plasma cells (PC) was induced with lps and with CD40mAb plus IL-4. cGVHD was induced with 100×106 Bm12 splenocytes injected into WT or RGC-32 KO recipients. Host B cell number and activation, anti-dsDNA Ab production, germinal centre (GC) B cell number and proliferation, PC number, expression of transcription factors IRF4 and Blimp1 were assessed at 2 and 4 weeks. Results RGC-32 mRNA was upregulated in B cells by lps, anti-CD40 mAb, IL-21 and IL-6. RGC-32 KO B cells failed to differentiate normally to PC as demonstrated by a 2-fold reduction in PC numbers generated after lps and anti-CD40+ IL-4 stimulation and impaired upregulation of Prdm1 and IRF4 mRNA. RGC-32 transcripts were upregulated in spleen cells from cGVHD mice and protein expression was detected in B cells and GC cells. RGC-32KO hosts displayed an attenuated autoimmune phenotype as demonstrated by: 1) decreased production of anti-dsDNA autoAb. 2) decreased number and proliferation of GC B cells. 3) decreased number of IgG anti-dsDNA secreting PC and 4) decreased IRF4 and Prdm1 mRNA expression. Conclusions These results suggest that expression of RGC-32 in B cells is critical for optimal GC proliferation, PC differentiation and autoantibody production in a murine model of lupus. These data support the idea that RGC-32 blockade has the potential to attenuate autoimmune parameters of cGVHD and possibly reverse abnormalities in the T and B cell pathways that contribute to lupus pathogenesis.