PurposeRetinal detachment (RD) separates the retina from the underlying retinal pigment epithelium, resulting in a gradual degeneration of photoreceptor (PR) cells. It is known that RD also results in an inflammatory response, but its contribution to PR degeneration is unknown. In this study we examine the inflammatory responses to RD in patient vitreous and validate a mouse experimental RD as a model of this phenomenon.MethodsMultiplex bead arrays were used to examine cytokine levels in vitreous samples from 24 patients with macula-off rhegmatogenous retinal detachment (RRD) undergoing reattachment surgery and from 10 control patients undergoing vitrectomy for vitreous opacities or epiretinal membrane. Activation of the innate immune response was then examined in a mouse model of RD.ResultsTwenty-eight factors were significantly increased in vitreous from RD patients versus controls. Notable were the cytokines MCP-1 (CCL2), IP-10 (CXCL10), fractalkine (CX3CL1), GRO (CXCL1), MDC (CCL22), IL-6, and IL-8, which all exhibited relatively high concentrations and several-fold increases in the vitreous of RD patients. Concentrations of various analytes correlated with a range of clinical variables such as duration of detachment and visual acuity. Retinal detachment in the mouse resulted in cytokine mRNA expression changes consistent with human RD vitreous results, as well as microglial proliferation and migration toward the outer retina.ConclusionsThe findings suggest that an inflammatory response involving microglia is a component of the reaction to retinal detachment that may impact visual acuity after surgical repair and that mouse experimental RD can serve as a model to study this effect.

PurposeISG15, a di-ubiquitin-like protein, is critical for controlling certain viral and bacterial infections. We sought to determine if ISG15 plays a role in corneal innate immunity against Candida albicans (C. albicans) using a C57BL/6 (B6) mouse model of human fungal keratitis.MethodsScarified corneas of adult B6 mice were pretreated with TLR5 ligand flagellin and then inoculated with C. albicans. The expression of ISG15 and other genes involved in ISG15 conjugation (ISGylation) was determined by real-time PCR. ISG15 expression and distribution in infected corneas were assessed by immunohistochemistry. ISGylation was examined by Western blotting. siRNA knockdown and recombinant ISG15 were used to elucidate the effects of ISG15 on controlling fungal keratitis by clinical scoring, fungal number plate counting, ELISA cytokine determination, and polymorphonuclear leukocytes (PMN) infiltration measurement.ResultsHeat-killed C. albicans induced expression of ISG15, and hBD2 was markedly enhanced by flagellin-pretreatment in cultured human primary corneal epithelial cells (CECs). In vivo, C. albicans infection induced the expression of ISG15, ISGylation-associated genes (UBE1L, UBCH8, and HERC5), and ISGylation in mouse CECs, all of which were enhanced by flagellin-pretreatment. siRNA knockdown of ISG15 increased keratitis severity, dampened flagellin-induced protection, and greatly suppressed the expressions of ISGylation enzymes, IFN-γ, but not CXCL2 in B6 mouse CECs. Recombinant ISG15, on the other hand, enhanced corneal innate immunity against C. albicans and suppressed infection-induced IL-1β, but not IL-Ra expression. ISG15 alone induced the expression of IL-1Ra, CXCL10, and CRAMP in mouse CECs. ISG15 was upregulated and secreted in cultured human CECs in response to challenge in a type 1 IFN-dependent manner.ConclusionsOur data, for the first time, demonstrate that ISG15 acts as an immunomodulator in the cornea and plays a critical role in controlling fungal keratitis.