The role of the P2X7 receptor in injury-induced calcium dynamics and cell migration in the corneal epithelium

Wound healing in the corneal epithelium is an essential process to maintain corneal clarity and organism health. The earliest events of cellular injury response include the release of nucleotides and the activation of P2 purinergic receptors. While the purinergic receptor P2X7 has been shown to promote cell migration, its role in corneal epithelial wound healing is still poorly understood. The goal of this work is to better understand the role of P2X7 in the injury response. We analyzed P2X7 expression after epithelial injury in rat corneal organ cultures and found that the receptor localizes to the leading edge of the corneal epithelium. However, overall mRNA and protein expression of P2X7 decreased after injury. Inhibition of P2X7 activation significantly delayed wound closure and prevented the leading edge-localization after injury. We found that P2X7 inhibition altered the wound-induced calcium wave in epithelial cells and altered the number and distribution of focal adhesions in the migrating cells. Live cell imaging of epithelial cells showed that P2X7 inhibition led to altered actin rearrangement, with thick actin bundles in the treated cells. In order to determine the importance of P2X7 in epithelial differentiation and stratified cell migration, we developed a stratified culture model. The cells in the stratified model expressed proliferative and differentiation markers similar to organ cultured corneas, as well as similar P2X7 expression and localization after injury. Together, these results show the importance of P2X7 in the overall purinergic response to injury, and provide tools to study P2X7 in stratified corneal cell migration. To determine if P2X7 may contribute to pathologic delayed wound healing in diseases such as type 2 diabetes, we analyzed P2X7 expression in diabetic human corneas and diabetic model rodent corneas. We showed that P2X7 expression is significantly elevated in unwounded diabetic corneas, and that wound healing is delayed in the rodent model. These data show that elevated P2X7 expression may contribute to the delayed healing in disease and may be a possible therapeutic target

NLRP3 Selectively Drives IL-1β Secretion by Pseudomonas aeruginosa Infected Neutrophils and Regulates Corneal Disease Severity

Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-dependent, but GSDME independent IL-1β secretion and pyroptosis. Here we examine inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1β secretion by neutrophils requires the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT (ΔexoST) require NLRC4 for IL-1β secretion. While IL-1β release from ΔexoST infected neutrophils is also NLRC4-dependent, infection with PAO1 is instead NLRP3-dependent and driven by the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches using MCC950 reveal that NLRP3 is also essential for bacterial killing and disease severity in a murine model of P. aeruginosa corneal infection (keratitis). Overall, these findings reveal a function for ExoS ADPRT in regulating inflammasome subtype usage in neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis

Effectiveness of physiotherapy exercise following hip arthroplasty for osteoarthritis: a systematic review of clinical trials

Background: Physiotherapy has long been a routine component of patient rehabilitation following hip joint replacement. The purpose of this systematic review was to evaluate the effectiveness of physiotherapy exercise after discharge from hospital on function, walking, range of motion, quality of life and muscle strength, for osteoarthritic patients following elective primary total hip arthroplasty. Methods: Design: Systematic review, using the Cochrane Collaboration Handbook for Systematic Reviews of Interventions and the Quorom Statement. Database searches: AMED, CINAHL, EMBASE, KingsFund, MEDLINE, Cochrane library (Cochrane reviews, Cochrane Central Register of Controlled Trials, DARE), PEDro, The Department of Health National Research Register. Handsearches: Physiotherapy, Physical Therapy, Journal of Bone and Joint Surgery (Britain) Conference Proceedings. No language restrictions were applied. Selection: Trials comparing physiotherapy exercise versus usual/standard care, or comparing two types of relevant exercise physiotherapy, following discharge from hospital after elective primary total hip replacement for osteoarthritis were reviewed. Outcomes: Functional activities of daily living, walking, quality of life, muscle strength and range of hip joint motion. Trial quality was extensively evaluated. Narrative synthesis plus meta-analytic summaries were performed to summarise the data. Results: 8 trials were identified. Trial quality was mixed. Generally poor trial quality, quantity and diversity prevented explanatory meta-analyses. The results were synthesised and meta-analytic summaries were used where possible to provide a formal summary of results. Results indicate that physiotherapy exercise after discharge following total hip replacement has the potential to benefit patients. Conclusion: Insufficient evidence exists to establish the effectiveness of physiotherapy exercise following primary hip replacement for osteoarthritis. Further well designed trials are required to determine the value of post discharge exercise following this increasingly common surgical procedure

Corneal Epithelium Expresses a Variant of P2X7 Receptor in Health and Disease

Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X7 receptors leading to cation fluxes and cytotoxicity. The corneal epithelium is an excellent model to study the expression of both the full-length P2X7 form (defined as the canonical receptor) and its truncated forms. When Ca2+ mobilization is induced by BzATP, a P2X7 agonist, it is attenuated in the presence of extracellular Mg2+ or Zn2+, negligible in the absence of extracellular Ca2+, and inhibited by the competitive P2X7 receptor inhibitor, A438079. BzATP enhanced phosphorylation of ERK. Together these responses indicate the presence of a canonical or full-length P2X7 receptor. In addition BzATP enhanced epithelial cell migration, and transfection with siRNA to the P2X7 receptor reduced cell migration. Furthermore, sustained activation did not induce dye uptake indicating the presence of truncated or variant forms that lack the ability to form large pores. Reverse transcription-polymerase chain reaction and Northern blot analysis revealed a P2X7 splice variant. Western blots identified a full-length and truncated form, and the expression pattern changed as cultures progressed from monolayer to stratified. Cross-linking gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X7 mRNA from diabetic corneal epithelium compared to non-diabetic controls and an increased trend in expression of P2X7variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X7, which ultimately allows P2X7 to function as a multifaceted receptor that can mediate cell proliferation and migration or cell death