NLRP12 negatively modulates inducible nitric oxide synthase (iNOS) expression and tumor necrosis factor-α production in Porphyromonas gingivalis LPS-treated mouse macrophage cell line (RAW264.7) Abstract Objective The aim of the present study is to investigate the participation of NLRP12 in Porphyromonas gingivalis LPS-activated mouse macrophages. Methods NLRP12-depleted mouse macrophages were stimulated with P. gingivalis LPS (1 μg/ml.). At indicated time points, the treated cells were lysed and the supernatant from treated cells was collected. Gene and protein expression of NLRP12 and iNOS were determined by RT-PCR and immunoblotting, respectively. The level of TNF-α production in the supernatant of the activated cells was determined by ELISA. Results and conclusion NLRP12 was upregulated in response to stimulation with P. gingivalis LPS. In addition, when NLRP12 was depleted in P. gingivalis LPS-treated macrophages, an increase in TNF-α production and iNOS expression were observed when compared to those of the control cells, indicating that NLRP12 downregulates the inflammatory cytokine and antimicrobial molecule production in the macrophages.

Interactions between carboxypeptidase M and kinin B1 receptor in endothelial cells Abstract Introduction Carboxypeptidase M (CPM) is a glycosylphosphatidylinositol anchored enzyme that plays an important role in the kallikrein–kinin system (KKS). CPM catalytic domain hydrolyzes Arg from C-terminal peptides (i.e., bradykinin and kallidin), generating des-Arg-kinins, the agonists of B1 receptor (B1R). It is known that CPM and kinin B1R are co-localized in the plasma membrane microdomains, where they interact with each other, facilitating receptor signaling. Aims We hypothesized here that this CPM-B1R interaction could also affect the activity of the enzyme. Methods Thus, in this work, we evaluated the impact of B1R presence or absence on CPM activity and expression, using primary culture of microvascular endothelial cells from wild-type, kinin B1R knockout mice (B 1 −/− ), and transgenic rats overexpressing B1 receptor exclusively in the endothelium. In addition, HEK293T cells, as wells as B 1 −/− primary culture of endothelial cells, both transfected with B1R, were also used. Results CPM expression and activity were downregulated in cells of knockout mice compared to control and this reduction was rescued after B1R transfection. Cells overexpressing B1R presented higher levels of CPM mRNA, protein, and activity. This profile was reverted by pre-incubation with the B1R antagonist, R715, in highly expressing receptor cells. Conclusions Our data show that kinin B1R positively modulates both CPM expression and activity, suggesting that CPM-B1R interaction in membrane microdomains might affect enzyme activity, beyond interfering in receptors signaling. This work highlights the interactions among different components of KKS and contributes to a better understanding of its patho-physiological role.

Organ crosstalk: the potent roles of inflammation and fibrotic changes in the course of organ interactions Abstract Background Organ crosstalk can be defined as the complex and mutual biological communication between distant organs mediated by signaling factors. Normally, crosstalk helps to coordinate and maintain homeostasis, but sudden or chronic dysfunction in any organ causes dysregulation in another organ. Many signal molecules, including cytokines and growth factors, are involved in the metabolic dysregulation, and excessive or inappropriate release of these molecules leads to organ dysfunction or disease (e.g., obesity, type 2 diabetes). Aim and method The aim of this review is to reveal the impact of organ crosstalk on the pathogenesis of diseases associated with organ interactions and the role of inflammatory and fibrotic changes in the organ dysfunction. After searching in MEDLINE, PubMed and Google Scholar databases using ‘organ crosstalk’ as a keyword, studies related to organ crosstalk and organ interaction were compiled and examined. Conclusion The organ crosstalk and the functional integration of organ systems are exceedingly complex processes. Organ crosstalk contributes to metabolic homeostasis and affects the inflammatory response, related pathways and fibrotic changes. As in the case of interactions between adipose tissue and intestine, stimulation of inflammatory mechanisms plays an active role in the development of diseases including insulin resistance, obesity, type 2 diabetes and hepatic steatosis. The increased level of knowledge about the ‘crosstalk’ between any organ and distant organs will facilitate the early diagnosis of the disease as well as the management of the treatment practices in the short- and long-term organ dysfunction.

Immunoexpression of canonical Wnt and NF-κB signaling pathways in the temporomandibular joint of arthritic rats Abstract Objective To investigate the participation of canonical Wnt and NF-κB signaling pathways in an experimental model of chronic arthritis induced by methylated bovine serum albumin (mBSA) in rat temporomandibular joint (TMJ). Materials and methods Wistar rats were sensitized by mBSA+Complete Freund Adjuvant (CFA)/Incomplete Freund Adjuvant (IFA) on the first 14 days (1 ×/week). Subsequently, they received 1, 2 or 3 mBSA or saline solution injections into the TMJ (1 ×/week). Hypernociceptive threshold was assessed during the whole experimental period. 24 h after the mBSA injections, the TMJs were removed for histopathological and immunohistochemical analyses for TNF-α, IL-1β, NF-κB, RANKL, Wnt-10b, β-catenin and DKK1. Results The nociceptive threshold was significantly reduced after mBSA injections. An inflammatory infiltrate and thickening of the synovial membrane were observed only after mBSA booster injections. Immunolabeling of TNF-α, IL-1β and Wnt-10b was increased in the synovial membrane in arthritic groups. The immunoexpression of nuclear β-catenin was significantly higher only in the group that received 2 booster TMJ injections. However, NF-κB, RANKL and DKK1 immunoexpression were increased only in animals with 3 mBSA intra-articular injections. Conclusion Our results suggest that canonical Wnt and NF-κB signaling pathways participate in the hypernociception and inflammatory response in TMJ synovial membrane during the development of rheumatoid arthritis in rats.

The increased T helper cells proliferation and inflammatory responses in patients with type 2 diabetes mellitus is suppressed by sitagliptin and vitamin D3 in vitro Abstract Objective The probably effects of sitagliptin and vitamin D3 (VitD3) on proliferation capacity and cytokines production were investigated in type 2 diabetes mellitus (T2DM) in vitro. Materials and methods Peripheral blood mononuclear cells (PBMCs) were isolated from 35 patients with T2DM and 26 healthy controls (HCs). CFSE-labeled PBMCs stimulated with phytohamagglutinin (PHA, 5 μg/mL) in the presence/absence of sitagliptin (200 mg/mL) with/without VitD3 (10−8 M) for 4 days. The proliferation of CD4+ T helper cells and non-CD4+ cells was analyzed using flow cytometry. The supernatant levels of IFN-γ, IL-17, IL-4, TGB-β and IL-37 were detected using ELISA. Results The proliferation of CD4+ T cells in response to PHA was higher in T2DM patients compared with HCs. The production of IFN-γ and IL-17 in PHA-stimulated cultures was higher, and the levels of IL-4 and IL-37 were lower in T2DM patients compared to HCs. The addition of sitagliptin or VitD3 to the cultures decreased the CD4+ T cells and non-CD4+ cells proliferation in patients and HCs. Sitagliptin with VitD3 was more effective in suppression of proliferation, decreasing of IL-17 and enhancing of IL-37 production. Conclusion Sitagliptin plus VitD3 effectively reduces the proliferative T cells response and modulates pro-inflammatory/anti-inflammatory cytokines production.

MiR-138-5p exacerbates hypoxia/reperfusion-induced heart injury through the inactivation of SIRT1-PGC-1α Abstract Objectives A drastic reduction in myocardial cell apoptosis plays a crucial role in the treatment/management of myocardial infarction, a major cardiovascular health challenge confronting the world, especially the Western world. Accumulating evidence indicates that the cardiotoxicity caused by the apoptotic machinery is partly regulated by miRNAs. The aim of this research is to investigate the role of miR-138-5p on hypoxia/reperfusion-induced heart injury. Methods The expression of miR-138-5p was determined in heart tissue from myocardial infarction patients and rats. Rats were transfection with a miR-138-5p inhibitor to silence miR-138-5p. The cardiac function of rats was detected via echocardiography. SIRT1 and PGC-1α expression in cardiac infarction was detected via quantitative Real-time PCR (qPCR) and Western blot analysis, while the TUNEL assay was used to determine myocardial apoptosis. Results Our observations showed that miR-138-5p expression was upregulated after the induction of myocardial infarction. The miR-138-5p inhibitor significantly improved cardiac function, increased the expression of SIRT1 and PGC-1α, and decreased the rate of myocardial apoptosis, whereas siRNA-SIRT1 reversed these protective effects. Conclusions In conclusion, our study demonstrated that miR-138-5p could promote cardiac ischemia injury via inhibition of the silent information regulator 1 and peroxisome proliferator-initiated receptor gamma and coactivator 1 alpha (SIRT1–PGC-1α) axis.

VE-cadherin regulates migration inhibitory factor synthesis and release Abstract Objective Vascular endothelial (VE)-cadherin-mediated adherens junction is critical to maintain endothelial integrity. Besides its role of homophilic intercellular adhesion, VE-cadherin also has a role of outside-in signaling with functional consequences for vascular physiology. However, the nature of these signals remains not completely understood. Materials and methods Human umbilical vein endothelial cells (HUVECs) were used in cell culture experiments. Confluent HUVECs were treated with VE-cadherin function-blocking antibodies BV9 (50 μg/ml) or IgG control. Antibody array was used to screen for cytokine/chemokine in supernatant. For VE-cadherin knockdown, siRNA transfection was used. ELISA, Western blot, and qRT-PCR were used to confirm the expression of screened cytokine/chemokine. To explore the possible mechanisms, Scr phosphorylation was detected and Scr inhibitor PP2 (1 μM) was used. To investigate in vivo relevance of the findings, BV9 and the indicated neutralizing antibodies were injected into mice and then lung vascular leak and inflammation were examined by Evans blue assay and lung tissue H&E, respectively. Results Using a non-biased, high-throughout human cytokine/chemokine antibody array, we first found that disruption of VE-cadherin-mediated adhesion by function-blocking antibody BV9 triggered the release of migration inhibitory factor (MIF). This VE-cadherin-mediated release of MIF further confirmed by ELISA with both VE-cadherin blocking antibody and siRNA technique was due to enhanced expression of MIF mRNA, which was mediated by Src kinase activation. In addition, in vivo lung vascular leak induced by VE-cadherin function-blocking antibody was partly alleviated by neutralizing MIF. Conclusions VE-cadherin regulates MIF synthesis and release via Src kinase. Our data provide additional evidence to the concept that VE-cadherin transfers intracellular signals to coordinate the state of cell–cell adhesion with gene expression.

Regulation of C-reactive protein conformation in inflammation Abstract C-reactive protein (CRP) is a non-specific diagnostic marker of inflammation and an evolutionarily conserved protein with roles in innate immune signaling. Natural CRP is composed of five identical globular subunits that form a pentamer, but the role of pentameric CRP (pCRP) during inflammatory pathogenesis remains controversial. Emerging evidence suggests that pCRP can be dissociated into monomeric CRP (mCRP) that has major roles in host defenses and inflammation. Here, we discuss our current knowledge of the dissociation mechanisms of pCRP and summarize the stepwise conformational transition model to mCRP to elucidate how CRP dissociation contributes to proinflammatory activity. These discussions will evoke new understanding of this ancient protein.

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.

TIPE2 ameliorates lipopolysaccharide-induced apoptosis and inflammation in acute lung injury Abstract Objective Tumour necrosis factor-α-induced protein 8-like 2 (TIPE2) has strong anti-inflammatory properties. However, it is unknown whether increased TIPE2 is protective against lipopolysaccharide (LPS)-induced ALI. In the current study, we aimed to investigate whether increased TIPE2 can exert protective effects in a mouse model of ALI induced by LPS. Methods We administered TIPE2 adeno-associated virus (AAV-TIPE2) intratracheally into the lungs of mice. Three weeks later, ALI was induced by intratracheal injection of LPS into BALB/c mice. Twenty-four hours later, lung bronchoalveolar lavage fluid (BALF) was acquired to analyse cells and protein, arterial blood was collected for arterial blood gas analysis and the determination of pro-inflammatory factor levels, and lung issues were collected for histologic examination, transmission electron microscopy (TEM), TUNEL staining, wet/dry (W/D) weight ratio analysis, myeloperoxidase (MPO) activity analysis and blot analysis of protein expression. Results We found that TIPE2 overexpression markedly mitigated LPS-induced lung injury, which was evaluated by the deterioration of histopathology, histologic scores, the W/D weight ratio, and total protein expression in the BALF. Moreover, TIPE2 overexpression markedly attenuated lung inflammation, as evidenced by the downregulation of polymorphonuclear neutrophils (PMNs) in the BALF, lung MPO activity, and pro-inflammatory cytokine levels in the serum. Moreover, TIPE2 overexpression not only dramatically prevented LPS-induced pulmonary cell apoptosis in mice but also blocked LPS-activated JNK phosphorylation and NF-κB p65 nuclear translocation. Conclusions Our study shows that the increased expression of AAV-mediated TIPE2 in the lungs of mice inhibits acute inflammation and apoptosis and suppresses the activation of NF-κB and JNK in a murine model of ALI.