Attenuative Effects of Platelet-Rich Plasma on 30 kDa Fibronectin Fragment-Induced MMP-13 Expression Associated with TLR2 Signaling in Osteoarthritic Chondrocytes and Synovial Fibroblasts

Proteolytic fragments of fibronectin can have catabolic effects on cartilage, menisci, and synovium. Previous studies have reported that Toll-like receptor (TLR) signaling pathways might be associated with joint inflammation and joint destruction. Platelet-rich plasma (PRP) is increasingly being used to treat a range of joint conditions; however, it has yet to be determined whether PRP influences fibronectin fragment (FN-f) procatabolic activity and TLRs. In this study, human primary culture cells were treated with 30 kDa FN-f with/without PRP co-incubation, and then analyzed using real-time PCR to determine gene expression levels in articular chondrocytes, meniscal fibrochondrocytes, and synovial fibroblasts. Protein levels were evaluated by Western immunoblotting. This study observed an increase in the protein expression of matrix metalloproteinases (MMPs), Toll-like receptor 2 (TLR2), nitric oxide synthase 2 (NOS2), prostaglandin-endoperoxide synthase (PTGS2), and cyclooxygenase 2 (COX2) in articular chondrocytes, meniscal fibrochondrocytes, and synovial fibroblasts following insult with 30 kDa FN-f. Upregulation of these genes was significantly attenuated by PRP treatment. TLR2 and matrix metalloproteinase 13 (MMP-13) were also significantly attenuated by cotreatment with 30 kDa FN-f + PRP + TLR2 inhibitor. PRP treatment was shown to attenuate the 30 kDa FN-f-induced MMP-13 expression associated with the decreased expression of TLR2 in osteoarthritic chondrocytes and synovial fibroblasts. PRP treatment was also shown to attenuate procatabolic activity associated with MMP-13 expression via the TLR2 signaling pathway.

A51 ENTERIC PARASITE INFECTION-INDUCED ALTERATION OF THE GUT MICROBIOTA REGULATES INTESTINAL GOBLET CELL BIOLOGY AND MUCIN PRODUCTION VIA TLR2 SIGNALING

Background Goblet cells (GCs) are the major source of mucin which are the main components of the mucus layer that represents the front line of innate defense in the gastrointestinal (GI) tract. Hyperplasia of GCs and increased mucin production are observed in many enteric nematode infections such as Trichuris muris infection. Increased mucin production contributes to parasite clearance by trapping in mucus and inhibiting motility. The GI tract contains trillions of commensal microbes, and these microbes control mucin production from GCs by activating different signaling cascades. During nematode parasite infection due to the coexistence of parasites and microbiota in close proximity of GCs in gut, it is likely that this nematode-microbiota interaction plays an important role in mucin production. Toll-like receptors (TLRs), components of the innate immune system, sense gut microbiota stimuli. The human GC-like cell line, LS174T, expresses TLR2 mRNA which was enhanced by stimulation with synthetic TLR2 ligands. We hypothesize T. muris-induced altered microbiota modulates GC response and mucin production via TLR2 signaling. Aims To elucidate the role of T. muris-induced altered gut microbiota in the regulation of intestinal GC response and mucin production via host TLR2 signaling. Methods C57BL/6 mice were infected by gavage with ~300 T. muris eggs and infectivity was confirmed by worm burden. Microbiota was analyzed by 16s rRNA sequencing. Colonic GCs response, mucins and TLR2 expression and cytokines production were assessed in germ-free (GF) mice receiving non-infected and T. muris-infected microbiota (collected on day 36 post-infection to exclude worms). Muc2 and Muc5ac expression were assessed in wild-type (WT) and TLR2 deficient (TLR2-/-) mice transplanted with T. muris-infected microbiota following antibiotic treatment. Results We observed a difference in microbial composition between non-infected and T. muris infected mice. Transfer of T. muris-infected microbiota into GF mice significantly increased GC numbers and TLR2 expression as well as up-regulated Muc2 and Muc5ac expression and IL-4, IL-13 production compared to GF mice with non-infected microbiota. Antibiotic-treated TLR2-/- mice after receiving microbiota from T. muris-infected mice showed significantly decreased expression of Muc2 and Muc5ac compared to antibiotic-treated WT mice receiving the same microbiota. Conclusions T. muris-induced altered microbiota influences intestinal GC response and mucin production via TLR2. In addition to enhancing our understanding on the interaction of parasite with resident microbiota in host defense, this study provides new information on TLR2 based innate signaling in the regulation of GC biology and mucin production. Funding Agencies NSERC

Peptidoglycan Switches Off the TLR2-Mediated Sperm Recognition and Triggers Sperm Localization in the Bovine Endometrium

In mammals, the uterine mucosal immune system simultaneously recognizes and reacts to most bacteria as well as allogenic sperm mainly through the Toll-like receptors (TLR)2/4 signaling pathway. Here, we characterized the impact of pathogen-derived TLR2/4 ligands (peptidoglycan (PGN)/lipopolysaccharide (LPS)) on the immune crosstalk of sperm with the bovine endometrial epithelium. The real-time PCR analysis showed that the presence of low levels of PGN, but not LPS, blocked the sperm-induced inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. Immunoblotting analysis revealed that PGN prevented the sperm-induced phosphorylation of JNK in BEECs. Activation or blockade of the TLR2 system in the endometrial epithelium verified that TLR2 signaling acts as a commonly-shared pathway for PGN and sperm recognition. The impairment of endometrial sperm recognition, induced by PGN, subsequently inhibited sperm phagocytosis by polymorphonuclear neutrophils (PMNs). Moreover, using an ex vivo endometrial explant that more closely resembles those in vivo conditions, showed that sperm provoked a mild and reversible endometrial tissue injury and triggered PMN recruitment into uterine glands, while PGN inhibited these events. Of note, PGN markedly increased the sperm attachment to uterine glands, and relatively so in the surface epithelium. However, addition of the anti-CD44 antibody into a PGN-sperm-explant co-culture completely blocked sperm attachment into glands and surface epithelia, indicating that the CD44 adhesion molecule is involved in the PGN-triggered sperm attachment to the endometrial epithelium. Together, these findings demonstrate that, the presence of PGN residues disrupts sperm immune recognition and prevents the physiological inflammation induced by sperm in the endometrial epithelium via the MyD88-dependent pathway of TLR2 signaling, possibly leading to impairment of uterine clearance and subsequent embryo receptivity.

Lactobacillus Murinus Alleviates Intestinal Ischemia/Reperfusion Injury Through Promoting the Release of Interleukin-10 From M2 Macrophages via Toll-like Receptor 2 Signaling

BackgroundIntestinal ischemia/reperfusion (I/R) injury has high morbidity and mortality. Gut microbiota is one of the potential key factors affecting intestinal I/R injury. Different populations exhibit different sensitivities to intestinal I/R injury, but whether this is related to differences in gut microbiota is unclear. Here, to elucidate the interaction between the gut microbiome and intestinal I/R injury, we conducted 16S rRNA gene sequencing and fecal microbiota transplantation experiment in an I/R mouse model. Toll-like receptor 2 (TLR2)-deficient mice, interleukin-10 (IL-10)-deficient mice as well as the transwell co-culture system of small intestine organoids and macrophages were used to explore the potential mechanism of bacteria attenuating intestinal I/R injury.ResultsThe intestinal I/R-sensitive (Sen) and resistant (Res) mice were first defined according to different survival outcomes of mice suffering from intestinal I/R. Fecal microbiota composition and diversity prior to intestinal ischemia were different between in Sen and Res mice. The relative abundance of Lactobacillus murinus (L. murinus) at the species level was higher in Res mice than in Sen mice, and the fold change was the largest. Clinically, the abundance of L. murinus in preoperative feces of patients undergoing cardiopulmonary bypass surgery was closely related to the degree of intestinal I/R injury after surgery. Treatment with L. murinus significantly prevented from intestinal I/R-induced intestinal injury and improved the survival of mice, which depends on the participation of macrophages. Further, in vivo and vitro experiments indicated that L. murinus promoted the release of IL-10 from M2 macrophages via activating TLR2 signaling to alleviate intestinal I/R injury. ConclusionGut microbiome is involved in the postoperative outcome of intestinal I/R. L. murinus promotes the release of IL-10 from M2 macrophages through TLR2 signaling to alleviate intestinal I/R injury, revealing a novel mechanism of intestinal I/R injury and new therapeutic strategy for clinical practice.

Activation of Toll-like receptor 2 via specific agonists promotes OXPHOS and glycolysis in gastric cancer cells rather than other Toll-like receptors

Background Toll-like receptor (TLR)2 activation via microbial and host stimuli plays important roles in the regulation of metabolic reprogramming in immune cells. We recently showed that ligand-induced TLR2 activation enhanced OXPHOS and glycolytic activity of gastric epithelial (cancer) cells, thus promoting tumour progression. Objective To investigate the full spectrum of TLR family members whose ligand-dependent activation can cause metabolic reprogramming in gastric cancer (GC) cells. Methods The expression of TLR family members was measured by RT-qPCR and Western blotting in a panel of human GC cells. Metabolic changes in human GC cells which were induced by agonists for different TLRs (TLR2, 4, 9) were identified by performing the Seahorse bioenergetic assay, as well as by measuring L-lactate and ROS production. The expression of genes involved in oxidative phosphorylation and glycolysis was also profiled in stimulated GC cells by RT-qPCR. Western blot further characterized the expression of SOD2, a key downstream target of TLR2 signaling in GC. Results TLR2 signaling activated by either synthetic molecules or whole pathogen antigen enhanced glycolytic activity and mitochondrial respiration in TLR2-high expressing cells, whereas ligand-induced TLR4 and TLR9 activation inhibited mitochondrial respiration or extracellular acidification rate. Furthermore, genes implicated in the regulation of glucose metabolism and the redox system, such as HIF1A, PFKFB3 and SOD2, were upregulated downstream of TLRs. Conclusion Our study reveals that ligand-induced activation of specific TLRs mediates diverse metabolic phenotype in human GC cells. TLR2 is the only family member that promotes both OXPHOS and glycolysis, which may result in tumor progression.