Inflammation Research

HMGB1 decreases CCR-2 expression and migration of M2 macrophages under hypoxia Abstract Objective The hypoxic milieu at tumor microenvironment is able to drive the behavior of infiltrating tumor cells. Considering that hypoxia-mediated HMGB1 release is known to promote tumor growth, as well to enhance the pro-tumoral profile of M2 macrophages by a RAGE-dependent mechanism, it is tempting to evaluate the potential contribution of HMGB1 under hypoxia to restrain M2 macrophages mobility. Methods CCR-2 expression was evaluated in M2 polarized macrophages by western blotting and immunocytochemistry. The secreted levels of CCL-2 and the migration capability were evaluated using an ELISA and a chemotaxis assay, respectively. Results HMGB1, under hypoxic conditions, markedly reduce both the production of CCL-2 and the expression of its receptor CCR-2; and reduced the migration capacity of M2 macrophages. Conclusions These results provided new insights into the mechanisms that regulate M2 macrophages mobility at the tumor microenvironment.

Salsalate ameliorates the atherosclerotic response through HO-1- and SIRT1-mediated suppression of ER stress and inflammation Abstract Objective and design Inflammation plays a causative role in atherosclerosis development. Salsalate is an anti-inflammatory drug used to treat atherosclerosis, but the mechanisms by which it affects atherosclerotic progression remain unclear. Methods Human umbilical vascular endothelial cells (HUVECs) and THP-1 human monocytes were treated with salsalate. Heme oxygenase 1 (HO-1) and sirtuin 1 (SIRT1) small interfering RNAs (siRNAs) were used to suppress each gene expression. Protein analyses were performed for measuring the expression of HO-1, SIRT1, nuclear factor kappa B (NFκB), cell adhesion molecules, and endoplasmic reticulum (ER) stress markers. Furthermore, cell adhesion assay, caspase 3 activity assay, and ELISA were also performed. Results In this study, we show that salsalate increases the expression of HO-1 and SIRT1 in HUVEC and suppresses lipopolysaccharide (LPS)-induced atherosclerotic responses via HO-1- and SIRT1-mediated pathways. Salsalate treatment of HUVEC and THP-1 cells reduced LPS-induced phosphorylation of NFκB and secretion of the proinflammatory cytokines TNFα and MCP-1. Salsalate treatment of HUVEC reduced the expression of the adhesion molecules ICAM, VCAM, and E-selectin and the LPS-induced adhesion of THP-1 cells to HUVEC. Salsalate treatment also attenuated LPS-induced ER stress and cell apoptosis. These anti-atherosclerotic effects were reversed by treating cells with siRNA for HO-1 and SIRT1. Conclusions Salsalate ameliorates LPS-induced atherosclerotic reactions via HO-1 and SIRT1-dependent reduction of inflammation and ER stress. Activation of these pathways by salsalate may provide therapeutic strategies for treating atherosclerosis.

Different intensity of autophagy regulate interleukin-33 to control the uncontrolled inflammation of acute lung injury Abstract Objectives Cytokines participate in the progression of acute respiratory distress syndrome (ARDS), and uncontrolled inflammation is a central issue of acute lung injury (ALI). Interleukin (IL)-33 is a nuclear protein that has been reported to have a proinflammatory role in ARDS. Studies have shown that excessive autophagy may lead to the increased mortality of patients with ARDS, while several investigations indicated that IL-33 and autophagy interact with one another. The present study sought to clarify the relation between autophagy and IL-33’s proinflammatory role in ARDS. Methods We built a lipopolysaccharide (LPS)-induced lung injury mouse model. To study the relationship between IL-33 and autophagy, mice were pretreated with rapamycin (RAPA; a promoter of autophagy) and 3-methyladenine (3-MA; an inhibitor of autophagy) prior to LPS administration. The expression of IL-33 in serum and bronchoalveolar lavage fluid (BALF) was measured. Immunohistochemistry of IL-33 in lung tissue was examined. Th1,Th2 cytokines/chemokine levels in serum and BALF were tested. Further, the severity of lung injury was evaluated. And the nuclear factor-kappa B (NF-κB)'s nuclear translocation in lung tissue was detected. Results In comparison with the control group, the levels of IL-33 in serum and BALF were increased after LPS injection. Th1 cytokines/chemokine levels were significantly increased in serum and BALF, while Th2 cytokine levels changed only a little. The levels of IL-33 in serum and BALF of the RAPA group was significantly increased after LPS was injected as compared with the LPS group; additionally, the levels of IL-33 in serum and BALF of the 3-MA group was significantly reduced after LPS was injected as compared with the LPS group, and that lung injury was ameliorated after 3-MA pretreatment. Th1 cytokines and chemokines in both serum and BALF were also decreased in the 3-MA group. Furthermore, we found that the nuclear translocation of NF-κB increased after LPS administration, and NF-κB’s nuclear translocation was significantly increased in comparison with the LPS group after RAPA pretreatment. In contrast, NF-κB’s nuclear translocation decreased after 3-MA pretreatment as compared with the LPS group. Conclusions These findings showed that autophagy might regulate IL-33 by activating or inhibiting NF-κB to control the uncontrolled inflammation of acute lung injury.

microRNA-520c-3p suppresses NLRP3 inflammasome activation and inflammatory cascade in preeclampsia by downregulating NLRP3 Abstract Background The pathogenesis of preeclampsia (PE) is suggested to be a consequence of inflammation. Previously conducted investigations on nod-like receptor pyrin domain-containing 3 (NLRP3) have shed light to its crucial role in PE. Furthermore, microRNA-520c-3p (miR-520c-3p) is observed to be implicated in inflammation. Therefore, the current study aimed to explore the role of miR-520c-3p in inflammatory cascade of PE by targeting NLRP3. Methods Microarray analyses were performed to screen differentially expressed genes associated with PE, and the potential relationship between miR-520c-3p and NLRP3 was analyzed. PE and normal placenta tissues were collected to determine the levels of inflammatory cytokines (IL-18, IL-33, IL-1β, IL-10, and TNF-α), miR-520c-3p and NLRP3. Hypoxic HTR8/SVneo cells were transfected with oe-NLRP3, si-NLRP3 or miR-520c-3p mimic to elucidate the functional role of NLRP3 or miR-520c-3p in the inflammatory cascade in PE, followed by the evaluation of levels of inflammatory cytokines and NLRP3 inflammasomes (NLRP3, ASC and caspase-1). Additionally, the HTR8/SVneo cell migration and invasion were evaluated. Results An upregulation of NLRP3, IL-18, IL-1β and TNF-α, and downregulation of miR-520c-3p, IL-33 and IL-10 were observed in PE placenta tissues. NLRP3 was found to be a target gene of miR-520c-3p. HTR8/SVneo cells after hypoxia transfected with si-NLRP3 or miR-520c-3p mimic exhibited decreased levels of inflammatory cytokines and NLRP3 inflammasomes, in addition to increased IL-10 and IL-33 levels. Moreover, enhanced migration and invasion abilities were observed in cells transfected with si-NLRP3. Conclusion Collectively, miR-520c-3p could potentially inhibit NLRP3 inflammasome activation and inflammatory cascade in PE by downregulating NLRP3, highlighting the potential of miR-520c-3p as a therapeutic target for PE treatment.

BLK and BANK1 polymorphisms and interactions are associated in Mexican patients with systemic lupus erythematosus Abstract Objectives The BLK and BANK1 genes have been consistently associated with systemic lupus erythematosus (SLE), primarily in European or Asian-derived populations. However, this finding has not been replicated in Latin-American patients. Methods Our study included 881 women from Mexico: 487 healthy controls and 394 SLE patients. The BLK rs13277113A/G-rs2736340T/C as well as BANK1 rs10516487G/A (R61H)-rs3733197G/A (A383T) single nucleotide polymorphisms (SNPs) were evaluated using a TaqMan® SNP genotyping assay. Results Our data showed that the BLK rs2736340T/C and rs13277113A/G polymorphisms are associated with susceptibility to SLE (C vs T, OR 1.60, p = 2×10−5; G vs A, OR 1.53, p = 9 × 10−5, respectively). We also identified an association between the functional BANK1 R61H polymorphism and SLE (A vs G, OR 1.56, p = 0.002). In addition, we observed a genetic interaction between BLK (rs2736340T/C, rs13277113A/G) and BANK1 (R61H and A383T) associated with susceptibility to SLE. Conclusion This is the first study documenting an association between BLK and BANK1 and SLE in a Latin-American population. Our data confirm previous reports: BLK and BANK1 are factors associated with SLE. Thus, both genes are universal loci for this autoimmune disease.

High-mobility group box-1 inhibition stabilizes intestinal permeability through tight junctions in experimental acute necrotizing pancreatitis Abstract Background In acute necrotizing pancreatitis (ANP), bacterial translocation (BT) from the gastrointestinal tract is the essential pathogenesis in the development of septic complications. Although high-mobility group box-1 (HMGB1) is associated with BT and organ dysfunction in ANP, the mechanism of HMGB1 in the intestinal barrier dysfunction and BT has not been well addressed. In this study, we intend to address the role of HMGB1 in ANP involving BT and intestinal barrier dysfunction. Methods Experimental ANP was achieved in male Sprague–Dawley rats through a retrograde injection of taurocholate into the common biliopancreatic duct following a laparotomy operation. HMGB1 blockade intervention was conducted with a subcutaneous injection of anti-HMGB1 antibody immediately before the laparotomy procedure. Twenty-four hours after ANP induction, pancreatic and intestinal tissues and blood samples were collected for a histopathological assessment and lipid peroxidation or glutathione (GSH) evaluation. AP-induced barrier dysfunction was determined by an intestinal permeability assessment. Tight junction proteins and autophagy regulators were investigated by western blotting, immunohistological analysis and confocal immunofluorescence imaging. Results ANP developed as indicated by microscopic parenchymal necrosis and fat necrosis, which were associated with intestinal mucosal barrier dysfunction. HMGB1 inhibition played a protective role in intestinal mucosal barrier dysfunction, protected against microbiome changes in ANP, and relieved intestinal oxidative stress. Additionally, HMGB1 inhibition attenuated intestinal permeability; preserved the expression of TJs, such as claudin-2 and occludin; and decreased autophagy. Furthermore, the autophagy regulator LC3 and TJ protein claudin-2 were both upregulated in ANP according to dual immunofluorescence analysis. Conclusion HMGB1 inhibition ameliorated the severity of experimental ANP though beneficial effects on BT, mainly involving in TJ function.

Mucosal-associated invariant T cells: new players in CF lung disease? Abstract The past decade has witnessed a surge in research centered around exploring the role of the enigmatic innate immune-like lymphocyte MAIT cell in human disease. Recent evidence has led to the elucidation of its role as a potent defender at mucosal surfaces including lungs due to its capacity to mount a formidable immediate response to bacterial pathogens. MAIT cells have a unique attribute of recognizing microbial ligands in conjunction with non-classical MHC-related protein MR1. Recent studies have demonstrated their contribution in the pathogenesis of chronic pulmonary disorders including asthma and chronic obstructive pulmonary disease. Several cellular players including innate immune cells are active contributors in the immune imbalance present in cystic fibrosis(CF) lung. This immune dysregulation serves as a central pivot in disease pathogenesis, responsible for causing immense structural damage in the CF lung. The present review focuses on understanding the role of MAIT cells in CF lung disease. Future studies directed at understanding the possible relationship between MAIT cells and regulatory T cells (Tregs) in CF lung disease could unravel a holistic picture where a combination of antimicrobial effects of MAIT cells and anti-inflammatory effects of Tregs could be exploited in synergy to alleviate the rapid deterioration of lung function in CF lung disease due to the underlying complex interplay between persistent infection and inflammation.

Anti-inflammatory activity of extensively hydrolyzed casein is mediated by granzyme B Abstract Objective Nutritional factors such as extensively hydrolyzed casein (eHC) have been proposed to exert anti-inflammatory activity and affect clinical outcomes such as tolerance development in cow’s milk allergy. Granzyme B (GrB) induces apoptosis in target cells and also controls the inflammatory response. Whether eHC could affect the activity of granzyme B and play a role in GrB-mediated inflammatory responses in vitro was unknown. Methods The activity of GrB was measured using the substrate Ac-IEPD-pNA. Inflammatory responses were induced with GrB in HCT-8 and THP-1 cells, and pro-inflammatory cytokines were determined at the transcriptional and protein level. Results GrB could induce the expression of IL-1β in HCT-8 cells, and IL-8 and MCP-1 in THP-1 cells, respectively. Interestingly, GrB acted synergistically on LPS-induced inflammation in HCT-8 cells and eHC reduced pro-inflammatory responses in both GrB and LPS-mediated inflammation. Further analyses revealed that eHC could inhibit the biological activities and cytotoxic activities of GrB and then could reduce GrB-mediated inflammatory response. Conclusion The results from the current study suggest that anti-inflammatory activity of extensively hydrolyzed casein is, to a certain extent, mediated through modulation of granzyme B activity and responses.

Protective effect of galangin against dextran sulfate sodium (DSS)-induced ulcerative colitis in Balb/c mice Abstract Objective and design Inflammatory bowel disease (IBD) is known to cause chronic inflammation in the digestive tract by the immune malfunction. Herein, we demonstrate the protective effect of galangin (GAL), a phytochemical, on LPS-induced inflammation in cultured mouse macrophages (RAW 264.7) and the treatment of DSS-induced ulcerative colitis in Balb/c mice. However, the anti-inflammatory effect of GAL in DSS-exposed experimental colitis has not been investigated. Materials and methods We determined the levels of proinflammatory cytokines by ELISA, biochemical analysis using standard protocols and protein expression level of NF-κB signaling pathway and activation of Nrf2 gene pathway were analyzed by western blot analysis in colitis-induced mice. Results Our in vitro studies showed that LPS-stimulated RAW 264.7 cells treated with GAL reduced the levels of nitrites, IL-6, and TNF-α in a concentration-dependent manner. The results demonstrated that oral administration of GAL at 20 mg/kg (lower dose) and 40 mg/kg (higher dose) significantly reduced the severity of colitis and mitigated the clinical signs of both macroscopic and microscopic of the disease. The levels of proinflammatory cytokines (TNF-α and IL-6) in colonic tissue and serum were reduced significantly and in GAL + DSS-treated group relative to DSS alone treated group.  Increased levels of anti-inflammatory cytokine (IL-10) was detected in colon tissues in GAL + DSS-treated groups relative to DSS alone treated group. We also observed decreased levels of myeloperoxidase (MPO), nitrites and TBARS with increased SOD in colonic tissue of GAL + DSS group. Besides, GAL + DSS-treated animals significantly suppressed protein expressions of p-NF-κB and p-Ikk-βα, COX-2, iNOS, Nrf2 and increased HO-1 levels in colon tissues by inhibiting inflammation and oxidative stress. Conclusion Our study highlights the protective effect of galangin as an anti-inflammatory agent against the severe form of colitis in pre-clinical models suggesting its potency for the treatment of IBD in humans.

Under inflammatory stimuli mesenteric mesothelial cells transdifferentiate into macrophages and produce pro-inflammatory cytokine IL-6 Abstract Objective Inflammatory stimuli inducing epithelial-to-mesenchymal transition (EMT) can transdifferentiate mesenteric mesothelial cells into macrophages. Methods Sprague Dawley rat mesenteric mesothelial cells were used as a model. 1 ml Freund adjuvant was injected into the peritoneal cavity of rat and GM-CSF treatment was used to induce inflammation. IL-10 and IL-6 expression were studied by immunocytochemistry and Western blot analysis both in vivo and in vitro. Results Control mesothelial cell express anti-inflammatory IL-10, but no pro-inflammatory IL-6 expression could be detected in them. By the time of inflammation, IL-6 expression increased (reached the maximum level at the fifth day of inflammation), parallel to this the IL-10 entirely disappeared from these cells. In vitro GM-CSF treatment resulted in similar changes. As the mesothelial cells started to recover (at the eighth day of inflammation) IL-6 expression decreased and IL-10 level started to increase again. Conclusion These data show that under inflammatory stimuli mesothelial cells—like macrophages—can produce pro-inflammatory cytokines.