Trichomonas vaginalis induces IL-1β production in a human prostate epithelial cell line by activating the NLRP3 inflammasome via reactive oxygen species and potassium ion efflux.

Trichomonas vaginalis is a sexually transmitted protozoan parasite that causes vaginitis in women, and urethritis and prostatitis in men. IL-1β is synthesized as immature pro-IL-1β, which is cleaved by activated caspase-1. Caspase-1 is, in turn, activated by a multi-protein complex known as an inflammasome. In this study, we investigated the inflammatory response of a prostate epithelial cell line (RWPE-1) to T. vaginalis and, specifically, the capacity of T. vaginalis to activate the NLRP3 inflammasome. RWPE-1 cells were stimulated by live T. vaginalis, and subsequent expression of pro-IL-1β, IL-1β, NLRP3, ASC and caspase-1 was determined by real-time PCR and Western blotting. IL-1β and caspase-1 production was also measured by ELISA. To evaluate the effects of NLRP3 and caspase-1 on IL-1β production, the activated RWPE-1 cells were transfected with small interfering RNAs to silence the NLRP3 and caspase-1 genes. Activation of the NLRP3 inflammasome was observed by fluorescence microscopy. Intracellular reactive oxygen species (ROS) were evaluated by spectrofluorometry. When RWPE-1 cells were stimulated with live T. vaginalis, the mRNA and protein expression of IL-1β, NLRP3, ASC, and caspase-1 increased. Moreover, silencing of NLRP3 and caspase-1 attenuated T. vaginalis-induced IL-1β secretion. The NADPH oxidase inhibitor DPI and high extracellular potassium ion suppressed the production of IL-1β, caspase-1, and the expression of NLRP3 and ASC proteins. The specific NF-κB inhibitor, Bay 11-7082, inhibited IL-1β production, and also inhibited the production of caspase-1, ASC and NLRP3 proteins. T. vaginalis induces the formation of the NLRP3 inflammasome in human prostate epithelial cells via ROS and potassium ion efflux, and this results in IL-1β production. This is the first evidence for activation of the NLRP3 inflammasome in the inflammatory response by prostate epithelial cells infected with T. vaginalis. Prostate 76:885-896, 2016. © 2016 Wiley Periodicals, Inc.

Gu NY ，Kim JH ，Han IH ，Im SJ ，Seo MY ，Chung YH ，Ryu JS ... -  《-》

Reactive oxygen species activated NLRP3 inflammasomes initiate inflammation in hyperosmolarity stressed human corneal epithelial cells and environment-induced dry eye patients.

In studies on dry eye (DE) disease, an association has been identified between tear film hyperosmolarity and inflammation severity elicited through receptor-induced increases in proinflammatory cytokine and chemokine release. These immune reactions might be mediated by inflammasomes, macromolecular complexes mounted around the NLRP3 protein and can be activated by reactive oxygen species (ROS) over-generation. Hence in this study we determine whether: a) ROS activated NLRP3 inflammasomes mediate hyperosmotic stress-induced inflammation in human corneal epithelial cells (HCECs); b) the ROS-NLRP3-IL-1β axis activation is associated with environment-induced DE. Immortalized HCECs were exposed to 500 mOsm medium in the presence and absence of a ROS inhibitor, N-acetyl-l-cysteine (NAC). HCECs transfected with NLRP3 siRNA or a negative control (NC) siRNA. Intracellular ROS was measured by fluorometric analysis using the probe 2',7'-dichlorofluorescin diacetate (DCFH-DA). Real-time PCR evaluated NLRP3, ASC, pro-caspase-1 and pro-IL-1β mRNA levels. Western blot analysis assessed NLRP3 protein expression whereas caspase-1 activity was determined with a fluorometric assay. Bioactive IL-1β release was assessed by ELISA. ROS production, NLRP3 inflammasome and pro-IL-1β gene expression as well as IL-1β secretion were also evaluated in the conjunctival epithelial cells and tear fluid samples of environment-induced DE patients and normal subjects. NAC suppressed hyperosmolarity-induced rises in ROS levels, NLRP3 inflammasome formation and activation, caspase-1 activity and IL-1β release. On the other hand, NLRP3 siRNA knockdown inhibited hyperosmotic stress-induced NLRP3 activation, which led to ASC, pro-caspase-1 and pro-IL-1β mRNA down-regulation followed by suppression of associated caspase-1 activity and IL-1β secretion. In addition, in ocular surface samples of environment-induced DE patients, ROS generation, NLRP3, ASC, pro-caspase-1 and pro-IL-1β gene expression as well as IL-1β secretion were upregulated. Taken together, NLRP3 mediated innate immune responses triggered by rises in ROS generation induce inflammation in hyperosmotic stressed HCECs. ROS-NLRP3-IL-1β signaling pathway might play a priming role in environment-induced DE development.

Zheng Q ，Ren Y ，Reinach PS ，Xiao B ，Lu H ，Zhu Y ，Qu J ，Chen W ... -  《-》

Inflammatory response of prostate epithelial cells to stimulation by Trichomonas vaginalis.

Trichomonas vaginalis is known as the most common cause of sexually transmitted infection. However, its prevalence may have been underestimated. Trichomonads are detected in prostatic tissue in benign prostatic hyperplasia, prostatitis, and prostate cancer. Our objective was to investigate whether T. vaginalis could induce an inflammatory response in prostate epithelium. The cytokine production by human prostate epithelial cell (RWPE-1) activated with T. vaginalis was determined by ELISA and real-time PCR. Intracellular ROS was evaluated by flow cytometry or spectrofluorometry. The protein levels of MAP kinase, NF-κB were analyzed by Western blot. The migration of neutrophil and monocyte were performed in 24-well microplates with filter insert. Incubation of cells of a human prostate epithelial cell line with a live T. vaginalis T016 isolate increased expression of the inflammatory mediators IL-1β, CCL2, and CXCL8. In addition, ROS, MAPK, and NF-κB activities increased, while inhibitors of ROS, ERK, and NF-κB reduced IL-1β production. Medium conditioned by incubation of RWPE-1 cells with T. vaginalis contained IL-1β and stimulated the migration of human neutrophils and monocytes (THP-1 cell line). We conclude that T. vaginalis may increase IL-1β expression in human prostate epithelium through activation of ROS, ERK, and NF-κB, and this in turn may induce the migration of neutrophils and monocytes and lead to an inflammatory response. This research is the first attempt to confirm inflammatory reaction caused by T. vaginalis in prostate epithelial cell.

Seo MY ，Im SJ ，Gu NY ，Kim JH ，Chung YH ，Ahn MH ，Ryu JS ... -  《-》

TLR2/MyD88/NF-κB pathway, reactive oxygen species, potassium efflux activates NLRP3/ASC inflammasome during respiratory syncytial virus infection.

Segovia J ，Sabbah A ，Mgbemena V ，Tsai SY ，Chang TH ，Berton MT ，Morris IR ，Allen IC ，Ting JP ，Bose S ... -  《PLoS One》

Mechanisms that lead to the regulation of NLRP3 inflammasome expression and activation in human dental pulp fibroblasts.

The NLRP3 inflammasome plays an important role in the cellular defense against invading pathogens and is reported to be expressed in human dental pulp fibroblasts (HDPFs). However, the role of the NLRP3 inflammasome in HDPFs during pulpal infection and inflammation remains unclear. To elucidate the function of the NLRP3 inflammasome and the mechanisms that lead to its expression and activation in HDPFs. The test model used lipopolysaccharide (LPS) and adenosine triphosphate (ATP) to simulate an inflammatory environment. Lentiviral vectors encoding short hairpin RNAs were used to knock down NLRP3 and caspase-1 in HDPFs. Specific inhibitors were used to determine whether the toll-like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), or nuclear factor-kappa B (NF-κB) pathways were involved in the regulation of NLRP3 expression. Reactive oxygen species (ROS) production was measured by fluorescent microscopy and flow cytometry using the total ROS/superoxide detection kit. Gene and protein expression were quantified by real-time polymerase chain reaction and Western blot, while cytokine release was measured by an enzyme-linked immunosorbent assay. LPS up-regulated NLRP3 and IL-1β expression while ATP induced the activation of caspase-1 and the release of IL-1β in LPS-primed HDPFs. The knockdown of NLRP3 or caspase-1 expression significantly inhibited IL-1β secretion. Pretreatment with a TLR4 inhibitor, a MyD88 inhibitory peptide, or an I Kappa B alpha (IκBα) phosphorylation inhibitor significantly inhibited LPS-induced NLRP3 and IL-1β expression. ATP potently promoted ROS generation in HDPFs; N-acetyl cysteine inhibited ROS production, caspase-1 activation and IL-1β secretion induced by ATP. Our results demonstrated that the NLRP3 inflammasome in HDPFs is crucial for IL-1β secretion in response to LPS plus ATP. LPS engaged the TLR4/MyD88/NF-κB pathway to enhance NLRP3 and pro-IL-1β expression in HDPFs. ATP promoted the generation of ROS and activated the NLRP3 inflammasome in a ROS-dependent manner.

Zhang A ，Wang P ，Ma X ，Yin X ，Li J ，Wang H ，Jiang W ，Jia Q ，Ni L ... -  《-》