C5a/C5aR1 mediates IMQ-induced psoriasiform skin inflammation by promoting IL-17A production from γδ-T cells.

Psoriasis is a chronic relapsing inflammatory skin disease, affecting up to 3% of the global population. Accumulating evidence suggests that the complement system is involved in its pathogenesis. Our previous study revealed that the C5a/C5aR1 pathway is crucial for disease development. However, the underlying mechanisms remain largely unknown. To explore potential mechanisms, psoriatic skin lesions and histological changes were assessed following imiquimod (IMQ) cream treatment. Inflammatory cytokine expression was tested by real-time RT-PCR. Immunohistochemistry and flow cytometry were used to identify inflammatory cell infiltration and interleukin (IL-17A) IL-17A expression. A C5aR1 antagonist (C5aR1a) and PI3K inhibitor (wortmannin) were used for blocking experiments (both in vivo and in vitro) to explore the mechanism. C5a/C5aR1-pathway inhibition significantly attenuated psoriasis-like skin lesions with decreased epidermal hyperplasia, downregulated type 17-related inflammatory gene expression, and reduced IL-17A-producing γδ-T cell responses. Mechanistically, C5a/C5aR1 promoted the latter phenotype via PI3K-Akt signaling. Consistently, C5aR1 deficiency clearly ameliorated IMQ-induced chronic psoriasiform dermatitis, with a significant decrease in IL-17A expression. Finally, blocking C5aR1 signaling further decreased psoriasiform skin inflammation in IL-17-deficient mice. Results suggest that C5a/C5aR1 mediates experimental psoriasis and skin inflammation by upregulating IL-17A expression from γδ-T cells. Blocking C5a/C5aR1/IL-17A axis is expected to be a promising strategy for psoriasis treatment.

LIGHT aggravates sepsis-associated acute kidney injury via TLR4-MyD88-NF-κB pathway.

Sepsis-associated acute kidney injury (SA-AKI) is a common clinical critical care syndrome. It has received increasing attention due to its high morbidity and mortality; however, its pathophysiological mechanisms remain elusive. LIGHT, the 14th member of the tumour necrosis factor (TNF) superfamily and a bidirectional immunoregulatory molecule that regulates inflammation, plays a pivotal role in disease pathogenesis. In this study, mice with an intraperitoneal injection of LPS and HK-2 cells challenged with LPS were employed as a model of SA-AKI in vivo and in vitro, respectively. LIGHT deficiency notably attenuated kidney injury in pathological damage and renal function and markedly mitigated the inflammatory reaction by decreasing inflammatory mediator production and inflammatory cell infiltration in vivo. The TLR4-Myd88-NF-κB signalling pathway in the kidney of LIGHT knockout mice was dramatically down-regulated compared to the controls. Recombinant human LIGHT aggravated LPS-treated HK-2 cell injury by up-regulating the expression of the TLR4-Myd88-NF-κB signalling pathway and inflammation levels. TAK 242 (a selective TLR4 inhibitor) reduced this trend to some extent. In addition, blocking LIGHT with soluble receptor fusion proteins HVEM-Fc or LTßR-Fc in mice attenuated renal dysfunction and pathological damage in SA-AKI. Our findings indicate that LIGHT aggravates inflammation and promotes kidney damage in LPS-induced SA-AKI via the TLR4-Myd88-NF-κB signalling pathway, which provide potential strategies for the treatment of SA-AKI.

Use of etanercept to treat rheumatoid arthritis in an HIV-positive patient: a case-based review.

Rheumatoid arthritis (RA) is a relatively common autoimmune disease that is associated with progressive disability and systemic complications, with a relatively high socioeconomic burden. The treatment of RA has been revolutionized by the use of biological drugs, such as anti-tumor necrosis factor (TNF) agents. A wide spectrum of RA disease severity has been reported among patients with human immunodeficiency virus (HIV) infection. Yet, only a few cases using anti-TNF therapy have been described in this clinical population. Therefore, the aim of our case-based review was to describe the successful use of etanercept in a 38-year-old female patient with RA concomitant with HIV infection, who had been resistant to the first-line anti-rheumatic therapies. As per routine care guidelines, the patient was screened for hepatitis virus infection, latent tuberculosis, and other infectious conditions, prior to the initiation of etanercept treatment. CD4 cell count, HIV viral load, and adverse effects were closely monitored during the treatment. The HIV infection remained stable with etanercept treatment, without the need for anti-retrovirus agents. No adverse effects and serious infections were identified during the treatment. Therefore, anti-TNF therapy is a viable alternative for the treatment of RA in patients with HIV, who do not respond to conventional anti-rheumatic therapies. The relationship between TNF-α and HIV infection, as well as cautionary guidelines regarding the utilization of anti-TNF therapy in this clinical population, is discussed.

LIGHT/IFN-γ triggers ß cells apoptosis via NF-κB/Bcl2-dependent mitochondrial pathway.

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN-γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ-induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ - induced pancreatic beta cell destruction. LIGHT and IFN-γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V(+) cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF-κB activation, the combination of LIGHT and IFN-γ caused an obvious decrease in expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, but an increase in expression of the pro-apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF-κB activation and Bak expression, and peri-insulitis in non-obese diabetes mice. Inhibition of NF-κB activation with the specific NF-κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl-xL down-regulation and Bax up-regulation, and led to a significant increase in LIGHT- and IFN-γ-treated cell viability. Moreover, cleaved caspase-9, -3, and PARP (poly (ADP-ribose) polymerase) were observed after LIGHT and IFN-γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT- and IFNγ-induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN-γ induces beta cells apoptosis via an NF-κB/Bcl2-dependent mitochondrial pathway.

Proteomics reveals defective peroxisomal fatty acid oxidation during the progression of acute kidney injury and repair.

Acute kidney injury (AKI) is characterized by a rapid decrease in renal function with high mortality and risk of progression to chronic kidney disease (CKD). Ischemia and reperfusion injury (IRI) is one of the major causes of AKI. However, the cellular and molecular responses of the kidney to IRI are complex and not fully understood. Herein, we conducted unbiased proteomics and bioinformatics analyses in an IRI mouse model on days 3, 7, and 21, and validated the results using IRI, unilateral ureteral obstruction (UUO), and biopsies from patients with AKI or CKD. The results indicated an obvious temporal expression profile of differentially expressed proteins and highlighted impaired lipid metabolism during the progression of AKI to CKD. Acyl-coenzyme A oxidase 1 (Acox1), the first rate-limiting enzyme of peroxisomal fatty acid beta-oxidation, was then selected, and its disturbed expression in the two murine models validated the proteomic findings. Accordingly, Acox1 expression was significantly downregulated in renal biopsies from patients with AKI or CKD, and its expression was negatively correlated with kidney injury score. Furthermore, in contrast to the decreased Acox1 expression, lipid droplet accumulation was remarkably increased in these renal tissues, suggesting dysregulation of fatty acid oxidation. In conclusion, our results suggest that defective peroxisomal fatty acid oxidation might be a common pathological feature in the transition from AKI to CKD, and that Acox1 is a promising intervention target for kidney injury and repair.

Decoy receptor 2 mediates the apoptosis-resistant phenotype of senescent renal tubular cells and accelerates renal fibrosis in diabetic nephropathy.

Apoptotic resistance leads to persistent accumulation of senescent cells and sustained expression of a senescence-associated secretory phenotype, playing an essential role in the progression of tissue fibrosis. However, whether senescent renal tubular epithelial cells (RTECs) exhibit an apoptosis-resistant phenotype, and the role of this phenotype in diabetic nephropathy (DN) remain unclear. Our previous study was the first to demonstrate that decoy receptor 2 (DcR2) is associated with apoptotic resistance in senescent RTECs and renal fibrosis. In this study, we aimed to further explore the mechanism of DcR2 in apoptosis-resistant RTECs and renal fibrosis in DN. DcR2 was co-localized with fibrotic markers (α-SMA, collagen IV, fibronectin), senescent marker p16, and antiapoptotic proteins FLIP and Bcl2 but rarely co-localized with caspase 3 or TUNEL. DcR2 overexpression promoted renal fibrosis in mice with streptozotocin (STZ)-induced DN, as evidenced by augmented Masson staining and upregulated expression of fibrotic markers. DcR2 overexpression also enhanced FLIP expression while reducing the expression of pro-apoptotic proteins (caspases 8 and 3) in senescent RTECs, resulting in apoptotic resistance. In contrast, DcR2 knockdown produced the opposite effects in vitro and in vivo. Moreover, quantitative proteomics and co-immunoprecipitation experiments demonstrated that DcR2 interacted with glucose-related protein 78 kDa (GRP78), which has been shown to promote apoptotic resistance in cancer. GRP78 exhibited co-localization with senescent and antiapoptotic markers but was rarely co-expressed with caspase 3 or TUNEL. Additionally, GRP78 knockdown decreased the apoptosis resistance of HG-induced senescent RTECs with upregulated cleaved caspase 3 and increased the percentage of apoptotic RTECs. Mechanistically, DcR2 mediated apoptotic resistance in senescent RTECs by enhancing GRP78-caspase 7 interactions and promoting Akt phosphorylation. Thus, DcR2 mediated the apoptotic resistance of senescent RTECs and renal fibrosis by interacting with GRP78, indicating that targeting the DcR2-GRP78 axis represents a promising therapeutic strategy for DN.

Response to belimumab in thrombotic thrombocytopenic purpura associated with systemic lupus erythematosus: a case-based review.

Thrombotic thrombocytopenic purpura (TTP), a life-threatening syndrome characterized by acute microangiopathic hemolytic anemia, thrombocytopenia, and visceral ischemia, can be classified as congenital TTP (inherited due to a mutation in ADAMTS13) and acquired TTP. The acquired TTP is further classified as idiopathic and secondary TTP. Systemic lupus erythematosus (SLE) is regarded as one of the most common causes of secondary TTP (SLE-TTP). In contrast to patients with idiopathic TTP, some patients with SLE-TTP, especially those diagnosed with refractory TTP, are resistant to plasma exchange and high-dose corticosteroids and usually require second-line drugs, including newly developed biologicals. Belimumab, a B-lymphocyte stimulator-specific inhibitor, was the first approved new therapy for SLE in the past 50 years. Only two cases of SLE-TTP using belimumab have been reported; however, detailed information has not been made available. Herein, we describe a 28-year-old female patient who presented with palm petechiae, strong tawny urine, and yellow stained skin and sclera, and was diagnosed with SLE-TTP supported by high anti-ANA titers; positive anti-SSA/SM; pleural effusion; decreased platelet count, hemoglobin, and complement C3/C4 counts; increased lactate dehydrogenase level, along with increased schistocytes; and a significant deficiency of ADAMTS13 activity. Belimumab (10 mg/kg) was administered after six plasma exchanges. Good efficiency and outcomes without any adverse events, SLE, or TTP relapse were observed during 12 months of follow-up. Therefore, belimumab is a promising choice for SLE-TTP management. In addition, we provide a focused review of the existing literature on the pathogenesis, diagnosis, and therapeutic strategies for SLE-TTP.

LIGHT deficiency attenuates acute kidney disease development in an in vivo experimental renal ischemia and reperfusion injury model.

Ischemia-reperfusion (I/R), a leading risk factor of acute kidney injury (AKI), is associated with high mortality and risk of progression to chronic kidney disease. However, the molecular mechanism of I/R-AKI remains not fully understood, which hinders its efficient clinical treatment. In this study, we observed that LIGHT deficiency remarkably attenuated I/R-AKI, as evidenced by rescued renal function, ameliorated tubular cell apoptosis, and alleviated inflammatory responses. Consistently, blocking LIGHT signaling with its soluble receptor fusion proteins (HVEM-IgG-Fc or LTßR-IgG-Fc) improved I/R renal dysfunction. RNA-sequencing and corresponding results indicated that LIGHT promoted oxidative stress and inflammation triggered by ischemic injury. Moreover, LIGHT signaling augmented ischemic stress-induced mitochondrial dysfunction characterized by an imbalance in mitochondrial fission and fusion, decreased mtDNA copies, impaired mitophagy, and increased mitochondrial membrane potential (ΔΨm). Mechanistically, LIGHT promoted mitochondrial fission by enhancing Drp1 phosphorylation (Ser616) and its translocation to the mitochondria. In conclusion, these results suggest that LIGHT-HVEM/LTßR signaling is critical for the I/R-AKI pathogenesis and it is further confirmed to be related to the increase in I/R-induced oxidative stress and mitochondria dysfunction, which may be the underlying mechanism of LIGHT signaling-mediated I/R-AKI.

Complement component 3 prevents imiquimod-induced psoriatic skin inflammation by inhibiting apoptosis in mice.

Complement component 3 (C3), a pivotal molecule in the complement system, is an essential immune mediator in various diseases, including psoriasis. However, the mechanistic role of C3 in psoriasis pathology and development remains elusive. Here, we showed that C3 deficiency dramatically augmented imiquimod-induced psoriasis-like skin inflammation, characterized by greater epidermal hyperplasia, inflammatory cell infiltration, and inflammatory gene expression than those in wild-type counterparts. In addition, C3 deficiency promoted imiquimod-induced skin cell apoptosis and supported greater proportions of IFN-γ+ T cells in the inflamed tissues. Accordingly, C3 supplement in the C3 deficient mice reduced skin inflammation and cells apoptosis. Moreover, blocking apoptosis with Z-VAD-FMK, a broad caspase inhibitor, markedly attenuated imiquimod-induced psoriasis-like skin inflammation and IFN-γ+ T cell responses in C3-deficient mice. Collectively, our results suggest that C3 prevents imiquimod-induced psoriasis-like skin inflammation by inhibiting apoptosis.

Fibrinogen-like protein 2 deficiency aggravates renal fibrosis by facilitating macrophage polarization.

Renal fibrosis has no effective target for its prevention or reversal. Fibinogen-like protein 2 (Fgl2) is a novel prothrombinase exhibiting coagulation activity and immunomodulatory effects. Although Fgl2 is known to play a vital role in the development of liver and interstitial fibrosis, its function in renal fibrosis remains unclear. In this study, Fgl2 expression was found to be markedly increased in kidney tissues from mice with unilateral ureteral obstruction (UUO)-induced renal fibrosis and patients with chronic kidney disease. However, Fgl2 deficiency aggravated UUO-induced renal fibrosis, as evidenced by the significantly increasing collagen I, fibronectin, and α-SMA expression, extracellular matrix deposition, and profibrotic factor (TGF-ß1) secretion. Administration of rmFgl2 (recombinant mouse Fgl2) significantly alleviated UUO-induced renal fibrosis in mice, suggesting that the increased fibrosis can be reversed by supplementing rmFgl2. Although there was no difference in the percentages of total macrophages between Fgl2+/+ and Fgl2-/- mice, Fgl2 deficiency remarkably facilitated M2 macrophage polarization and accelerated M1 macrophage polarization to a low degree, during UUO-induced renal fibrosis development in mice. Similar results were observed when Fgl2+/+ and Fgl2-/- mice bone marrow-derived macrophages were treated for M1 or M2 polarization. Moreover, Fgl2 deficiency significantly increased the phosphorylation of STAT6, a critical mediator of M2 polarization, in both UUO-induced fibrotic kidney tissues and bone marrow-derived M2 macrophages. In conclusion, the aggravation of renal fibrosis by Fgl2 deficiency is facilitated by the p-STAT6-dependent upregulation of macrophage polarization, especially of M2.

C5aR deficiency attenuates the breast cancer development via the p38/p21 axis.

Emerging evidence has shown activation of the complement component C5 to C5a in cancer tissues and C5aR expression in breast cancer cells relates to the tumor development and poor prognosis, suggesting the involvement of complement C5a/C5aR pathway in the breast cancer pathogenesis. In this study, we found that as compared to the non-tumoral tissues, both C5aR and MAPK/p38 showed an elevated expression, but p21/p-p21 showed lower expression, in the tumoral tissues of breast cancer patients. Mice deficient in C5aR or mice treated with the C5aR antagonist exhibited attenuation of breast cancer growth and reduction in the p38/p-p38 expression, but increase in p21/p-p21 expression, in the tumor tissues. Pre-treatment of the breast cancer cells with recombinant C5a resulted in reduced p21 expression, and MAPK/p38 inhibitors prevented C5a-induced reduction in p21 expression, suggesting the involvement of the MAPK/p38 signaling pathway in the C5a/C5aR-mediated suppression of p21/p-p21 expression. These results provide evidence that breast cancer development may rely on C5a/C5aR interaction, for which MAPK/p38 pathway participate in down-regulating the p21 expression. Inhibition of C5a/C5aR pathway is expected to be helpful for the treatment of patients with breast cancer.

Tumor necrosis factor superfamily 14 is critical for the development of renal fibrosis.

OBJECTIVE: Tumor necrosis factor superfamily protein 14 (TNFSF14) was recently identified as a risk factor in some fibrosis diseases. However, the role of TNFSF14 in renal fibrosis pathogenesis remains unknown. RESULTS: It was found that TNFSF14 levels were significantly increased both in UUO-induced renal fibrotic mice and in patients with fibrotic nephropathy, compared with those in controls. Accordingly, Tnfsf14 deficiency led to a marked reduction in renal fibrosis lesions and inflammatory cytokines expression in the UUO mice. Furthermore, the levels of Sphk1, a critical molecule that causes fibrotic nephropathy, were remarkably reduced in Tnfsf14 KO mice with UUO surgery. In vitro recombinant TNFSF14 administration markedly up-regulated the expression of Sphk1 of primary mouse renal tubular epithelial cells (mTECs). CONCLUSION: TNFSF14 is a novel pro-fibrotic factor of renal fibrosis, for which TNFSF14 up-regulates Sphk1 expression, which may be the underlying mechanism of TNFSF14-mediated renal fibrosis. METHODS: We investigated the effect of TNFSF14 on renal fibrosis and the relationship between TNFSF14 and pro-fibrotic factor sphingosine kinase 1 (Sphk1) by using the unilateral urethral obstruction (UUO)-induced mice renal fibrosis as a model and the specimen of patients with fibrosis nephropathy, by Masson trichrome staining, immunohistochemistry, qRT-PCR, and western blot analysis.

C5a/C5aR1 Pathway Is Critical for the Pathogenesis of Psoriasis.

Psoriasis is one of the most common chronic inflammatory skin diseases, affecting ~2% of the population. The lack of characterization of the pathogenesis of psoriasis has hindered efficient clinical treatment of the disease. In our study, we observed that expression of complement component 5a receptor 1(C5aR1) was significantly increased in skin lesions of both imiquimod (IMQ) and IL23-induced psoriatic mice and patients with psoriasis. C5aR1 deficiency or treatment with C5a receptor 1 antagonist (C5aR1a) in mice significantly attenuated psoriasis-like skin lesions and expression of inflammatory cytokines and chemokines. Moreover, C5aR1 deficiency significantly decreased IMQ-induced infiltration of plasmacytoid dendritic cells (pDCs), monocytes and neutrophils in psoriatic skin lesions and functions of pDCs, evidenced by the remarkable reduction in the IMQ-induced production of interferon-α (IFN-α) and tumor necrosis factor α (TNF-α), and FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent pDCs differentiation. Accordingly, in vitro treatment with recombinant C5a accelerated pDCs migration and the differentiation of bone marrow cells into pDCs. Furthermore, biopsies of psoriatic patients showed a dramatic increase of C5aR1+ pDCs infiltration in psoriatic skin lesions, compared to healthy subjects. Our results provide direct evidence that C5a/C5aR1 signaling plays a critical role in the pathogenesis of psoriasis. Inhibition of C5a/C5aR1 pathway is expected to be beneficial in the treatment of patients with psoriasis.

Mesenchymal stem cells alleviate acute kidney injury by down-regulating C5a/C5aR pathway activation.

BACKGROUND: Acute kidney injury (AKI) leads to serious renal damage, and early inhibition of inflammation is necessary for its treatment. C5a/C5aR signaling activation promotes inflammatory response in tissue injury. Anti-inflammatory activity of mesenchymal stem cells (MSCs) makes it possible to alleviate AKI by controlling the C5a/C5aR signaling activation. METHODS: Ischemia reperfusion (I/R)-induced AKI models in wild-type and C5aR KO mice were used. In addition, human bone marrow MSCs (hBM-MSCs) or C5aR antagonist were injected in this model. All animals were killed at 72 h after reperfusion. In vitro, the LPS-activated macrophage line RAW264.7 cells were co-cultured with or without hBM-MSCs in the presence of recombinant C5a or not for indicated time points. After that, C5aR expression, the inflammatory factor production, and NF-κB translocation in RAW264.7 cells were measured. RESULTS: hBM-MSC treatment and C5a/C5aR signaling blockade or C5aR-deficiency exhibited similar attenuated effects on I/R-induced AKI, macrophages infiltration, and the pro-inflammatory cytokines TNF-α and IL-1ß expression in renal tissues in mice. Moreover, hBM-MSC administration led to a significant reduction in C5a levels in serum and C5aR expression in the kidney tissues in mice after I/R. In vitro, upon co-culture with hBM-MSCs, both C5aR expression and the secretion of pro-inflammatory factors TNF-α, IL-6, and nitric oxide in LPS-activated macrophages were markedly reduced. Accordingly, recombinant complement C5a accelerated LPS-induced NF-κB translocation and pro-inflammatory factors expression in macrophages, but the addition of hBM-MSCs reversed these C5a-induced effects. CONCLUSIONS: The present study indicates that hBM-MSCs alleviate AKI via suppressing C5a/C5aR-NF-κB pathway activation.

Celastrol alleviates renal fibrosis by upregulating cannabinoid receptor 2 expression.

Renal fibrosis is the final manifestation of various chronic kidney diseases, and no effective therapy is available to prevent or reverse it. Celastrol, a triterpene that derived from traditional Chinese medicine, is a known potent anti-fibrotic agent. However, the underlying mechanisms of action of celastrol on renal fibrosis remain unknown. In this study, we found that celastrol treatment remarkably attenuated unilateral ureteral obstruction (UUO)-induced mouse renal fibrosis. This was evidenced by the significant reduction in tubular injury; collagen deposition; accumulation of fibronectin, collagen I, and α-smooth muscle actin; and the expression levels of pro-fibrotic factors Vim, Cola1, and TGF-ß1 mRNA, as well as inflammatory responses. Celastrol showed similar effects in a folic acid-induced mouse renal fibrosis model. Furthermore, celastrol potentiated the expression of the anti-fibrotic factor cannabinoid receptor 2 (CB2R) in established mouse fibrotic kidney tissues and transforming growth factor ß1 (TGF-ß1)-stimulated human kidney 2 (HK-2) cells. In addition, the CB2R antagonist (SR144528) abolished celastrol-mediated beneficial effects on renal fibrosis. Moreover, UUO- or TGF-ß1-induced activation of the pro-fibrotic factor SMAD family member 3 (Smad3) was markedly inhibited by celastrol. Inhibition of Smad3 activation by an inhibitor (SIS3) markedly reduced TGF-ß1-induced downregulation of CB2R expression. In conclusion, our study provides the first direct evidence that celastrol significantly alleviated renal fibrosis, by contributing to the upregulation of CB2R expression through inhibiting Smad3 signaling pathway activation. Therefore, celastrol could be a potential drug for treating patients with renal fibrosis.

C5a/C5aR pathway accelerates renal ischemia-reperfusion injury by downregulating PGRN expression.

Recent reports indicate that the complement C5a/C5aR pathway and progranulin (PGRN) deficiency both contribute to ischemia-reperfusion (IR)-induced acute kidney injury. However, the underlying relationship between the C5a/C5aR signaling pathway and PGRN expression during acute kidney injury is poorly understood. In this study, we showed that C5aR expression was significantly upregulated after renal IR, and that C5aR deficiency led to a marked increase in PGRN expression and a significant reduction in tubular damage and production of inflammatory cytokines. In accordance with these results, recombinant C5a caused downregulation of PGRN protein and mRNA levels in renal tubular epithelial cells (HK-2 cells), which could be negated by disruption of C5a/C5aR signaling by the C5aR antagonist, as confirmed by immunofluorescence, western blotting, and quantitative real-time PCR. Moreover, C5aR deficiency resulted in attenuated NF-κB expression 24h after IR, and recombinant C5a potentiated TNFα-induced NF-κB activation in HK-2 cells. Inhibition of NF-κB activation reversed C5a-induced downregulation of PGRN expression. Our results show for the first time that the complement C5a/C5aR pathway aggravates IR-induced acute kidney injury by suppressing PGRN expression and confirm that suppression of PGRN expression is associated with increased NF-κB activation induced by C5a.

Increased expression of Fibrinogen-Like Protein 2 is associated with poor prognosis in patients with clear cell renal cell carcinoma.

Fibrinogen-like protein 2 (FGL2) is highly expressed in various tumour tissues and plays a vital role in tumour initiation and progression. This study evaluated the clinical significance of FGL2 in patients with clear cell renal cell carcinoma (ccRCC). FGL2 expression in fresh and 170 archived paraffin-embedded ccRCC tissues was measured by quantitative RT-PCR, western blotting, and immunohistochemitry. FGL2 expression was significantly upregulated in ccRCC. Statistical analyses by using Kaplan-Meier method showed that high FGL2 expression was associated with poor overall survival (OS) and recurrence-free survival (RFS) of patients with ccRCC. Multivariate analyses indicated that FGL2 was as an independent prognostic factor of survivaland that tumoural FGL2 levels could significantly predict the prognosis of patients with early-stage ccRCC. Nomogram systems, which integrated FGL2 expression and other clinical parameters, were established and were found to be better than TNM staging in predicting the OS and RFS of patients with ccRCC. FGL2 silencing led to a significant reduction in cells viability and increase in cells apoptosis, accompanied with a reduced ERK1/2 and p38 MAPK activation, in ccRCC cells. Thus, our results suggest that high FGL2 expression is a novel, independent, and an adverse prognostic factor of clinical outcomes in patients with ccRCC.

Complement component 3 deficiency prolongs MHC-II disparate skin allograft survival by increasing the CD4(+) CD25(+) regulatory T cells population.

Recent reports suggest that complement system contributes to allograft rejection. However, its underlying mechanism is poorly understood. Herein, we investigate the role of complement component 3 (C3) in a single MHC-II molecule mismatched murine model of allograft rejection using C3 deficient mice (C3(-/-)) as skin graft donors or recipients. Compared with C3(+/+) B6 allografts, C3(-/-) B6 grafts dramatically prolonged survival in MHC-II molecule mismatched H-2(bm12) B6 recipients, indicating that C3 plays a critical role in allograft rejection. Compared with C3(+/+) allografts, both Th17 cell infiltration and Th1/Th17 associated cytokine mRNA levels were clearly reduced in C3(-/-) allografts. Moreover, C3(-/-) allografts caused attenuated Th1/Th17 responses, but increased CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cell expression markedly in local intragraft and H-2(bm12) recipients. Depletion of Treg cells by anti-CD25 monoclonal antibody (mAb) negated the survival advantages conferred by C3 deficiency. Our results indicate for the first time that C3 deficiency can prolong MHC-II molecule mismatched skin allograft survival, which is further confirmed to be associated with increased CD4(+) CD25(+) Treg cell population expansion and attenuated Th1/Th17 response.

STAT1-mediated down-regulation of Bcl-2 expression is involved in IFN-γ/TNF-α-induced apoptosis in NIT-1 cells.

Tumor necrosis factor (TNF)-α and interferon (IFN)-γ are the major pro-inflammatory cytokines involved in beta-cell destruction. The fate of islet beta-cells in the cytokine-induced intrinsic mitochondrial apoptotic pathway is determined by the interaction between members of the Bcl-2 family. However, the mechanism through which beta-cell apoptosis is regulated remains unclear. In this study, we treated the murine beta-cell line NIT-1 with TNF-α and IFN-γ and then investigated the regulation of signal transducer and activator of transcription-1 (STAT-1) and expression of the members of the Bcl-2 family in this apoptotic pathway. Results showed that TNF-α and IFN-γ synergistically reduced NIT-1 cell viability. In addition, the decrease in cell growth was due to apoptosis as shown by apoptotic body formation, detected by confocal laser microscope, and a significant increase in Annexin-Vup(+) cell percentage, detected by flow cytometry. Combination treatment with TNF-α and IFN-γ caused a remarkable increase in the release of cytochrome c, and in the activation of caspase-9 and caspase-3, as well as, an obvious enhancement in STAT-1 phosphorylation; the treatment, however, resulted in the down-regulation in Bcl-2 expression. The enhancement in STAT-1 activity and a down-regulation in Bcl-2 expression was also observed in MIN6 cells, another murine beta-cell derived line, after cells exposure to the combination of TNF-α and IFN-γ treatment. Knockdown of STAT-1 gene expression by siRNA or inhibition of STAT-1 activation with fludarabine reversed Bcl-2 down-expression and led to a significant decrease in apoptosis in TNF-α- and IFN-γ-treated NIT-1 cells. Taken together, our results suggest that STAT1-mediated down-regulation of Bcl-2 is involved in NIT-1 cell apoptosis induced by combination treatment with TNF-α and IFN-γ.

Caspase-3 is involved in IFN-γ- and TNF-α-mediated MIN6 cells apoptosis via NF-κB/Bcl-2 pathway.

TNF-α and IFN-γ are the major pro-inflammatory cytokines in the ß-cell destruction. However, the underlying mechanism remains unclear. The present study used a murine insulinoma cell line MIN6 for further investigation of the effect of Caspase-3 on the cytokines-induced pancreatic ß-cell apoptosis and analyzed the mechanisms involved in the activation of Caspase-3. It was showed that the combination of IFN-γ and TNF-α significantly reduced the viability of MIN6 cells and the observed cells growth inhibition was due to cell apoptosis as judged by the morphological changes under a confocal laser scanning microscopy and FACS assay of Annexin-V/7-AAD double staining. Accompanying with NF-κB activation and Bcl-2 downregulation, both the cleaved Caspase-3 and PARP, a known substrate of Caspase-3 in vivo, were observed at 24 and 12 h, respectively, after cells exposure to IFN-γ and TNF-α treatment. Pretreatment of Caspase-3 inhibitors remarkably attenuated IFN-γ- and TNF-α-induced cells apoptosis. Inhibition of NF-κB activation led to the increase in Bcl-2 expression, a significant attenuation in Caspase-3 activity, and an obvious amelioration in cells viability in IFN-γ- and TNF-α-treated MIN6 cells. Taken together, our results indicate that Caspase-3 is critical for the induction of MIN6 cells apoptosis and it's activation is further confirmed to be related to the NF-κB-mediated Bcl-2 downregulation, which may be the underlying mechanism of IFN-γ- and TNF-α-mediated MIN6 cells apoptosis.