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 ... -  《-》

Reactive oxygen species activated NLRP3 inflammasomes prime environment-induced murine dry eye.

Tear film hyperosmolarity along with exposure to oxidant stress are factors that can induce chronic ocular surface inflammation and pain. However, there is limited information on how increases in reactive oxygen species (ROS) generated by oxidant exposure can induce inflammation. There is emerging evidence in other tissues that innate immune responses to a variety of environmental stresses stem from ROS-induced cytosolic NLRP3 inflammasome activation. Once this occurs, pro-caspase-1 is converted into its catalytic active form, which in turn cleaves pro-IL-1β thereby generating its bioactive form. We determined the role of ROS generation in mediating increases in IL-1β secretion through caspase-1 activation caused by NLRP3 inflammasome activation in an environment-induced murine dry eye (DE) model. An intelligently controlled environmental system (ICES) induced evaporative DE in female 4-6 week old C57BL/6J mice. Increases in ROS production preceded rises in corneal and conjunctival gene expression of NLRP3 inflammasome components and IL-1β that were identified using real-time PCR. Confocal microscopy evaluated concomitant increases in NLPR3, caspase-1 and IL-1β immunostaining. Increases in caspase-1 activity were used as an indicator of inflammasome activation. Rises in ROS generation occurred after 1 week of ICES exposure, which preceded increases in gene expression of three NLRP3 inflammasome components (i.e. NLRP3, ASC and caspase-1) leading to rises in bioactive IL-1β release. Increases in caspase-1 activity occurred after 2 weeks of ICES exposure. Eyedrops containing 0.3% N-acetyl-l-cysteine (NAC) were applied to quench ROS generation by mice kept in the ICES for 2 weeks. This scavenger reduced corneal fluorescein staining and decreased ROS production. NAC also down-regulated both increases in NLRP3, ASC, caspase-1 and IL-1β mRNA levels, along with their immunostaining. It robustly attenuated rises in inflammasome mediated increases in caspase-1 catalytic activity. We show in a dessicating DE disease murine model that rises in ROS generation trigger NLRP3 inflammasome complexation and activation leading to increases in bioactive IL-1β secretion. These results prompt us to suggest that the ROS-NLRP3-IL-1β signaling pathway might play a priming role in environment-induced DE progression. Finally, our findings provide a basis for developing novel strategies that may improve the management of patients requiring treatment for environment-induced dry eye disease.

Zheng Q ，Ren Y ，Reinach PS ，She Y ，Xiao B ，Hua S ，Qu J ，Chen W ... -  《-》

MiR-223 inhibits hyperosmolarity-induced inflammation through downregulating NLRP3 activation in human corneal epithelial cells and dry eye patients.

We previously showed that increases in reactive oxygen species (ROS) generation upregulate NLRP3 inflammasome and inflammation through increases in both caspase-1 activity and rises in IL-1β expression levels in animal models of dry eye (DE). As changes in microRNA (miRNAs) expression levels can modulate inflammasome function, we determine here if there is a relationship in DE between changes in miR-223 expression levels and NLRP3 activation induced in an intelligent controlled environmental system (ICES) in mice. In parallel, ROS, miR-223 and NLRP3 expression levels were assessed in conjunctival impression cytology and tear fluid samples obtained from DE patients and normal subjects. MiR-223 expression levels were modulated by transfection of either a mimic or its negative control (NC) in a human corneal epithelial cell line (HCECs) exposed to a 500 mOsm hyperosmotic medium for 4 h. The dual-luciferase reporter assay confirmed that miR-223 controls NLRP3 gene expression readout through directly interacting with the 3' UTR of its mRNA. Hyperosmolarity-induced NLRP3 activation was confirmed based on recruitment and colocalization of NLRP3 with ASC as well as increases in IL-1β expression. The miR-223 expression level decreased by 55% in the conjunctiva and cornea of the murine DE model from the level in the control group (P ≤ 0.047), while NLRP3 protein expression rose by 30% (P ≤ 0.017). In DE patients, miR-223 expression decreased in conjunctival impression cytology samples (P = 0.002), whereas IL-1β tear content rose significantly (P < 0.001).The relevance of this decline was confirmed by showing that exposure to a 500 mOsm stress decreased the miR-223 expression level whereas ROS generation as well as the NLRP3, and IL-1β expression levels rose in HCECs (P ≤ 0.037). In contrast, miR-223 mimic transfection reduced the NLRP3 protein expression level by 30% (P = 0.037), whereas both ROS generation and IL-1β secretion rose compared to their corresponding levels in the control group (P ≤ 0.043). Thus, miR-223 negatively regulates NLRP3 inflammasome activity via suppressing NLRP3 translation in DE. This inverse regulation between miR-223 and NLRP3 expression levels suggests that selective upregulation of miR-223 expression may be a novel option to suppress chronic inflammation in DE.

Ren Y ，Feng J ，Lin Y ，Reinach PS ，Liu Y ，Xia X ，Ma X ，Chen W ，Zheng Q ... -  《-》

Hyperosmotic Stress-Induced TRPM2 Channel Activation Stimulates NLRP3 Inflammasome Activity in Primary Human Corneal Epithelial Cells.

Zheng Q ，Tan Q ，Ren Y ，Reinach PS ，Li L ，Ge C ，Qu J ，Chen W ... -  《-》

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 ... -  《-》