TLR7 activation in epilepsy of tuberous sclerosis complex Abstract Background Neuroinflammation and toll-like receptors (TLR) of the innate immune system have been implicated in epilepsy. We previously reported high levels of microRNAs miR-142-3p and miR-223-3p in epileptogenic brain tissue resected for the treatment of intractable epilepsy in children with tuberous sclerosis complex (TSC). As miR-142-3p has recently been reported to be a ligand and activator of TLR7, a detector of exogenous and endogenous single-stranded RNA, we evaluated TLR7 expression and downstream IL23A activation in surgically resected TSC brain tissue. Methods Gene expression analysis was performed on cortical tissue obtained from surgery of TSC children with pharmacoresistent epilepsy. Expression of TLRs 2, 4 and 7 was measured using NanoString nCounter assays. Real-time quantitative PCR was used to confirm TLR7 expression and compare TLR7 activation, indicated by IL-23A levels, to levels of miR-142-3p. Protein markers characteristic for TLR7 activation were assessed using data from our existing quantitative proteomics dataset of TSC tissue. Capillary electrophoresis Western blots were used to confirm TLR7 protein expression in a subset of samples. Results TLR7 transcript expression was present in all TSC specimens. The signaling competent form of TLR7 protein was detected in the membrane fraction of each sample tested. Downstream activation of TLR7 was found in epileptogenic lesions having elevated neuroinflammation indicated by clinical neuroimaging. TLR7 activity was significantly associated with tissue levels of miR-142-3p. Conclusion TLR7 activation by microRNAs may contribute to the neuroinflammatory cascade in epilepsy in TSC. Further characterization of this mechanism may enable the combined of use of neuroimaging and TLR7 inhibitors in a personalized approach towards the treatment of intractable epilepsy.

Upregulated CD200 in pre-retinal proliferative fibrovascular membranes of proliferative diabetic retinopathy patients and its correlation with vascular endothelial growth factor Abstract Objective and design The objective was to determine the expression of CD200 in the pre-retinal proliferative fibrovascular membranes (PFVM) of patients with proliferative diabetic retinopathy (PDR) and to clarify its correlation with vascular endothelial growth factor (VEGF) and corresponding receptors. Methods PFVM samples were collected by vitrectomy from 14 patients with PDR, and 11 non-diabetic patients who accepted vitrectomy for idiopathic epiretinal membranes removal. The expression of CD200, VEGF,VEGF-R1 and VEGF-R2 was measured via qPCR and immunofluorescent staining. Results The mRNA level of CD200 was significantly higher in PDR patients than that in control patients. Meanwhile, CD200 and CD31 were found co-located and statistically associated in PFVM of PDR patients. The mRNA levels of VEGF, VEGF-R1 and VEGF-R2 were also significantly higher in PDR patients. Moreover, statistical association was found between CD200 and VEGF, VEGF-R1 in mRNA levels. But there was no significant correlationship between CD200 and VEGF-R2. Conclusions These results suggest a significantly increased expression of CD200 in PFVM of patients with PDR and present a crucial association between CD200 and VEGF-involved pathway. It represents a potential therapy that interfering with CD200 may inhibit the VEFG expression and neovascular formation in PDR patients.

Tumor necrosis factor alpha (TNF-α) and its soluble receptors are associated with disability, disability progression and clinical forms of multiple sclerosis Abstract Background The association between tumor necrosis factor (TNF)-α, soluble TNF receptor (sTNFR)1 and sTNFR2 with clinical characteristics of multiple sclerosis (MS) remains unclear. Objective To examine whether TNF-α, sTNFR1 and sTNFR2 are associated with MS diagnosis, disability, disability progression and clinical forms of MS. Materials and subjects The study included 147 patients with relapsing–remitting MS (RRMS), 21 with progressive clinical forms (ProgMS) and 70 controls. Expanded Disability Status Scale (EDSS) evaluated disability as mild (EDSS < 3.0) or moderate/high (EDSS ≥ 3.0). Multiple Sclerosis Severity Score (MSSS) evaluated disability progression as no progression (MSSS < 5) and progression (MSSS ≥ 5). Baseline data of subjects and plasma levels of TNF-α, sTNFR1, sTNFR2 were obtained. Results The MS diagnosis explained 44.6% and 12.3% of TNF-α and sTNFR2 levels, respectively. Moderate/high disability and disability progression were best predicted by sTNFR1 and age (positively) and ProgMS were best predicted by sTNFR1 (positively) and sTNFR2 (negatively), coupled with age and sex. A composite score reflecting the sTNFR1/sTNFR2 ratio showed a positive association with ProgMS after adjusting for age and sex. Conclusion Increased sTNFR1 and age were positively associated with disability and disability progression, whereas increased sTNFR1 (positively) and sTNFR2 (negatively) were associated with ProgMS, suggesting a distinct role of them in the immunopathological mechanisms of MS.

Regulation of oxidized LDL-induced inflammatory process through NLRP3 inflammasome activation by the deubiquitinating enzyme BRCC36 Abstract Objective Oxidized Low-Density Lipoprotein (oxLDL) is a well-established pro-inflammatory marker that activates the NLRP3 inflammasome. Ubiquitination plays an important role in modulating the stability and functions of various proteins. BRCC36 is a ubiquitin-modifying enzyme that plays a crucial role in protein stabilization and activation in the cytosol, but its role in OxLDL-induced NLRP3 inflammasome activation is not known. Here, we have investigated the role of deubiquitinating enzyme BRCC36 in regulating NLRP3 inflammasome during oxLDL stimulation. Methods Raw 264.7 murine macrophages were stimulated with oxLDL and effect of BRCC36 deubiquitination activity was assessed by fluorometric assay, and protein expression was assessed by Western blotting. The level of IL-1β measured by ELISA and LDH activity as pyroptotic cell death marker was assessed by fluorometric assay. Results The results showed that oxLDL increased the level of NLRP3 in macrophages and also the level of active caspase-1 and IL-1β. It also modulated the expression of deubiquitinating enzymes and caused pyroptotic cell death as indicated by LDH release. Inhibiting the proteasomal activity by MG132 and siRNA-mediated silencing of BRCC36 in macrophages potentially suppressed oxLDL-induced NLRP3 inflammasome activation and IL-1β secretion. Furthermore, the inhibition of proteasomal deubiquitinating activity with specific BRCC36 inhibitor G5 also reduced the inflammatory cell death. Conclusion Taken together, our study suggests that deubiquitinating enzyme BRCC36 inhibition could potentially suppress oxLDL-induced inflammatory process by inhibiting NLRP3 activation and resultant IL-1β secretion.

Saikosaponin C exerts anti-HBV effects by attenuating HNF1α and HNF4α expression to suppress HBV pgRNA synthesis Abstract Objective Saikosaponin c (SSc), a compound purified from the traditional Chinese herb of Radix Bupleuri was previously identified to exhibit anti-HBV replication activity. However, the mechanism through which SSc acts against HBV remains unknown. In this study, we investigated the mechanism of SSc mediated anti-HBV activity. Methods HepG2.2.15 cells were cultured at 37 ℃ in the presence of 1–40 μg/mL of SSc or DMSO as a control. The expression profile of HBV markers, cytokines, HNF1α and HNF4α were investigated by real-time quantitative PCR, Elisa, Western blot and Dot blotting. Knockdown of HNF1α or HNF4α in HepG2.2.15 cells was mediated by two small siRNAs specifically targeting HNF1α or HNF4α. Results We found that SSc stimulates IL-6 expression, leading to attenuated HNF1α and HNF4α expression, which further mediates suppression of HBV pgRNA synthesis. Knockdown of HNF1α or HNF4α in HepG2.2.15 cells by RNA interference abrogates SSc’s anti-HBV role. Moreover, SSc is effective to both wild-type and drug-resistant HBV mutants. Conclusion SSc inhibits pgRNA synthesis by targeting HNF1α and HNF4α. These results indicate that SSc acts as a promising compound for modulating pgRNA transcription in the therapeutic strategies against HBV infection.

MHTP, a synthetic tetratetrahydroisoquinoline alkaloid, attenuates lipopolysaccharide-induced acute lung injury via p38MAPK/p65NF-κB signaling pathway-TLR4 dependent Abstract Introduction This study investigated the mechanism of action of a synthetic tetrahydroisoquinoline alkaloid, MHTP, in an experimental model of acute lung injury (ALI) in two distinct moments: 72 h and 10 days. Methodology To realize this study, 2.5 mg/kg of lipopolysaccharide (LPS) was intranasally administered in BALB/c mice, and nasal instillation of MHTP (1.25; 2.5; 5.0; 10 or 20 mg/kg) was administrated at 1, 24, and 48 h after LPS challenge. The data were statistically analyzed and p < 0.05 was considered statistically significant. Results MHTP treatment (2.5, 5.0, 10 or 20 mg/kg) significantly decreased neutrophil migration into the bronchoalveolar lavage fluid (BALF), tissue inflammatory cell infiltration, edema, and hemorrhage as well as collagen fiber deposition on the perialveolar regions at both moments. TNF-α and IL-6 levels were significantly diminished in the MHTP-treated animals at 72 h and maintained them, at a basal level, at 10-day observation. These effects of MHTP are due to downregulating p38MAPkinese/p65NFκB signaling pathway-TLR4 dependent. Also, the MHTP treatment promoted a survival rate at 100% and improved their body weights during the 10-day observation. Unlike, the LPS group (non-treated LPS challenged animals) presented less than 50% of surviving rate at 72 h and the animals that survived did not improve their physiological state at 10-day observation. Conclusions These data showed for the first time the beneficial and effective activity of a nasal treatment with a synthetic tetrahydroisoquinoline alkaloid on an experimental model of ALI and pointed out the molecular mechanism related to it.

Tannic acid prevents macrophage-induced pro-fibrotic response in lung epithelial cells via suppressing TLR4-mediated macrophage polarization Abstract Background Polarized macrophages induce fibrosis through multiple mechanisms, including a process termed epithelial-to-mesenchymal transition (EMT). Mesenchymal cells contribute to the excessive accumulation of fibrous connective tissues, leading to organ failure. This study was aimed to investigate the effect of tannic acid (TA), a natural dietary polyphenol on M1 macrophage-induced EMT and its underlying mechanisms. Materials First, we induced M1 polarization in macrophage cell lines (RAW 264.7 and THP-1). Then, the conditioned-medium (CM) from these polarized macrophages was used to induce EMT in the human adenocarcinomic alveolar epithelial (A549) cells. We also analysed the role of TA on macrophage polarization. Results We found that TA pre-treated CM did not induce EMT in epithelial cells. Further, TA pre-treated CM showed diminished activation of MAPK in epithelial cells. Subsequently, TA was shown to inhibit LPS-induced M1 polarization in macrophages by directly targeting toll-like receptor 4 (TLR4), thereby repressing LPS binding to TLR4/MD2 complex and subsequent signal transduction. Conclusion It was concluded that TA prevented M1 macrophage-induced EMT by suppressing the macrophage polarization possibly through inhibiting the formation of LPS-TLR4/MD2 complex and blockage of subsequent downstream signal activation. Further, our findings may provide beneficial information to develop new therapeutic strategies against chronic inflammatory diseases.

Aquaporin-1 attenuates macrophage-mediated inflammatory responses by inhibiting p38 mitogen-activated protein kinase activation in lipopolysaccharide-induced acute kidney injury Abstract Objective This study was designed to investigate the role of AQP1 in the development of LPS-induced AKI and its potential regulatory mechanisms in the inflammatory responses of macrophages. Methods Male Wistar rats were injected intraperitoneally with LPS, and biochemical and histological renal damage was assessed. The levels of inflammatory mediators, macrophage markers and AQP1 in blood and kidney tissues were assessed by ELISA. RTPCR was used to assess changes in the relative levels of AQP1 mRNA induced by LPS. Western blot and immunofluorescence analyses were performed to assay the activation of the p38 MAPK and NF-κB pathways, respectively. The same detection methods were used in vitro to determine the regulatory mechanisms underlying AQP1 function. Results AQP1 mRNA levels were dramatically decreased in AKI rats following the increased expression of inflammatory factors. In vitro experiments demonstrated that silencing the AQP1 gene increased inflammatory mediator secretion, altered the classical activation of macrophages, greatly enhanced the phosphorylation of p38 and accelerated the translocation of NF-κB. Furthermore, these results were blocked by doramapimod, a p38 inhibitor. Therefore, these effects were mediated by the increased phosphorylation of p38 MAPK. Conclusion Our results suggest that altered AQP1 expression may be associated with the development of inflammation in AKI. AQP1 plays a protective role in modulating acute renal injury and can attenuate macrophage-mediated inflammatory responses by downregulating p38 MAPK activity in LPS-induced RAW264.7 cells. The pharmacological targeting of AQP1-mediated p38 MAPK signalling may provide a novel treatment approach for AKI.

Familial hypogammaglobulinemia with high RTE and naïve T lymphocytes Abstract Most of primary immunodeficiencies with hypogammaglobulinemia are associated with reduced memory B cells. T cell development may be interesting as well, but increased recent thymic emigrants are rarely reported in these patients. We report the case of a family (mother and her two sons) diagnosed with common variable immunodeficiency 10 due to a mutation in the NFKB2 gene. Laboratory findings showed that all three patients presented hypogammaglobulinemia, reduced memory B cells and elevated naïve T lymphocytes and recent thymic emigrants. This feature, in the absence of glucocorticoid deficiency, may suggest a primary thymic dysfunction. Interestingly, the mother presented the worst immune phenotype, as regards both antibody production and NK function, indicating that immune function may deteriorate in the course of time. We conclude that close monitoring of immune functions may widen the knowledge on the CVID10 and improve the patients’ care.

Vamorolone, a dissociative steroidal compound, reduces collagen antibody-induced joint damage and inflammation when administered after disease onset Abstract Objective and design The objective of this study was to assess the effect of vamorolone, a first-in-class dissociative steroidal compound, to inhibit inflammation when administered after disease onset in the murine collagen antibody-induced arthritis model of arthritis. Animals 84 DBA1/J mice were used in this study (n = 12 per treatment group). Treatment Vamorolone or prednisolone was administered orally after disease onset for a duration of 7 days. Methods Disease score and bone erosion were assessed using previously described scoring systems. Cytokines were measured in joints via immunoassay, and joint cathepsin B activity (marker of inflammation) was assessed using optical imaging of joints on live mice. Results We found that vamorolone treatment led to a reduction of several disease parameters including disease score, joint inflammation, and the presence of pro-inflammatory mediators to a degree similar of that observed with prednisolone treatment. More importantly, histopathological analysis of affected joints showed that vamorolone treatment significantly reduced the degree of bone erosion while this bone-sparing property was not observed with prednisolone treatment at any of the tested doses. Conclusions While many intervention regimens in other studies are administered prior to disease onset in animal models, the current study involves delivery of the potential therapeutic after disease onset. Based on the findings, vamorolone may offer an efficacious, yet safer alternative to conventional steroidal compounds in the treatment of rheumatoid arthritis and other inflammatory diseases.