Uveitis, an intraocular inflammation which primarily involves the retina and the uvea, is a major cause of blindness due to retinal photoreceptor degeneration from mitochondrial oxidative stress (1, 2). The mitochondrial oxidative stress and damage primarily takes place in the retinal photoreceptors (3, 4). Experimental Autoimmune Uveitis (EAU) is well- established animal model of uveitis. This model has been used to decipher and study the efficacy of novel anti- inflammatory therapeutics and their mode of action to investigate the molecular mechanisms of the disease progression (5). We have earlier reported a novel finding of selective αA crystallin upregulation in the photoreceptors during the early phase of EAU (6). This upregulation occurred at the sites of the mitochondrial oxidative stress in the retina, mainly in the photoreceptor cells. Moreover, systemic administration of αA during the EAU development resulted in protection of the retina and amelioration of the inflammatory process by down regulating the TLRs, Th1- and Th17 cytokines required for induction of oxidative stress and perpetuation of the intraocular inflammation (7). However, the molecular mechanism of such αA crystallin mediated suppression of proinflammatory cytokines and TLRs was unclear. Recent studies in inherited retinal degeneration mouse model also reported a similar finding indicating that loss of α crystallins resulted in increased intraretinal inflammation, activation of apoptosis, necroptosis and photoreceptor death suggesting its important role in regulating photoreceptor survival (8). Crystallins, the major structural proteins of the eye lens, consist of three distinct families: α, β and γ (9). The α-crystallins constitute two similar crystallins with about 60 percent homology: αA and αB, and these are the principal members of the small heat shock proteins (sHsps) family of molecular chaperones which are known to be expressed in multiple tissues, including retina, brain, heart, kidney, spinal cord, and lungs (10–15). Although αA- and αB-crystallin have related amino acid sequences with similar structural properties, they vary significantly in their tissue specificity and phosphorylation sites and have different functions (16, 17). Since α -crystallins are induced by a temperature upshift in many organisms, they are often referred to as small heat shock proteins (sHsps) or, more accurately, alpha-Hsps. sHSPs are found to modulate microRNA expression and through such modulatory effect they could suppress autoimmune process (18). MicroRNAs (miRNAs) are a class of noncoding RNAs 18-24 nucleotides long and suppress the expression of protein-coding genes at the post transcriptional level by directing translational repression, or by destabilizing mRNA or by a combination of the two (19–22). They are transcribed in the nucleus through transcription factor NFκB and are exported into the cytoplasm where they control gene expression at the post transcriptional level by binding to target messenger RNAs (23). As binding sites of these microRNAs are not fully complementary; each miRNA may target several different mRNA transcripts. As a result, modulation of individual miRNAs can change the expression of hundreds of genes. These RNAs are known to play key roles in a wide variety of biological processes, including cell differentiation and growth, development, metabolism, cell signaling, apoptosis and disease processes linked to cancer and inflammation (20, 24–26). MicroRNAs are also promising candidates for new, targeted therapeutic approaches and as biomarkers of diseases like cancers, heart disease and chronic inflammatory diseases (23). miRNA-based therapies involve modulation of pathogenetic pathways by antagonists and mimics. MicroRNA biogenesis occurs through activation of NFκB, and studies have shown that the small heat shock protein Hsp27, a close member of the αA-crystallin family, regulates NFκB activity through degradation of IκB leading to upregulation of miRNA biogenesis (18). We postulated that the beneficial effect of αA administration in amelioration of uveitis could be from the αA modulation of NFκB transcription factor. Although uveitis is caused by various infectious agents and develops in association with autoimmune systemic disorders, the later uveitis entities are commonly encountered in clinical practice and they are usually treated with non-specific immune modulators including corticosteroids, cytotoxic agents, and biologicals such as anti-TNFα (1, 27, 28). However, such therapeutic interventions are associated with significant systemic complications such as systemic immune suppression, infections, malignancies and ocular complications, glaucoma, and cataract (29). Interestingly, the above therapeutic agents were delivered locally in the uveitis eyes by intravitreal injections and such treatments were effective, however such local delivery was also associated with ocular complications, cataract development and glaucoma (30). In contrast microRNAs by virtue of their function in suppressing the proinflammatory cytokines genes (21) could offer advantages in treatment of uveitis by local delivery because of low toxicity, absence of degradation and efficient delivery to the retina and uvea due to their low molecular size. In this study of EAU, we carried out modulation of microRNA expression profile followed by bioinformatics analysis combined with validated microRNA target data to detect specifically upregulated miRNAs which are known to suppress inflammatory cytokines. Moreover, we determined the abrogation of EAU by systemic and local administration of the miRNAs.

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Association of Research in Vision and Ophthalmology. The protocol was approved by the Committee of the Ethics of Animal Experiments of the University of Southern California (Protocol Number: 11218). All surgery was performed under ketamine and xylazine anesthesia, and all efforts were made to minimize suffering of animals.

Statistical analyses were performed by a student t-test using Prism (GraphPad Software, La Jolla, CA). A p value of < 0.05 was considered statistically significant.

Development of experimental autoimmune uveitis requires activation of innate immune response leading to T cell mediated induction of organ specific autoimmune process directed at the retina. Recent studies on target prediction analyses have indicated that a significant number of innate immune response genes could be under the direct control of microRNAs (19, 49). Therefore we investigated the microRNA expression profile in the retina of the EAU mice treated with αA crystalline. Such treatment revealed upregulation of a few selective microRNAs in the retina. Moreover, these microRNAs were downregulated significantly in EAU retina without αA Crystallin treatment suggesting that αA Crystallin could upregulate select few microRNAs. (Table 2) However further studies with additional experiments are required to support the suggested role of αA crystallin in modulation of microRNAs.

Novel bioinformatics combined with validated microRNA targets of upregulated microRNAs from αA administration revealed that among these, microRNA 146a and 155 could play a significant role in inhibition/suppression in immune reactive genes resulting in the amelioration of the inflammatory process involved in uveitis (50–52). Computational bioinformatics analysis using validated microRNA targets showed that microRNA146a targets the mRNAs of key innate immune response genes of TLRs signaling molecules IRAK1 and TRAF6. These signaling molecules are crucial in initiation of robust innate immune response. This microRNA is known to negatively regulate important factors in the MyD88/NFkB signaling pathway, namely TRAF6 and IRAK1, thereby dampening the pro-inflammatory response mediated through MyD88/NFkB signaling (45, 51, 53–55). microRNA146a was also shown to reduce intraocular inflammation in Experimental Autoimmune Anterior Uveitis (EAAU) in rats through the inhibition of NFκB (56). Previous studies on TLRs signaling blocking experiments showed inhibition of EAU development implicating important role played by the innate immune response in induction of EAU (57). Blocking of various TLRs using TLR knockout mice did not prevent EAU however studies in MYD88 KO mice have shown that these mice were completely resistant to EAU as they fail to generate the proinflammatory and Th1 responses (58). However, inhibition of the downstream key signaling molecules of TLRs such as TRAF6 and IRAK1 could result in robust suppression of the innate immune response by blocking both the MYD88 as well as TRIF signaling pathways. In our earlier report, we showed that αA crystallin was upregulated in early EAU, specifically in the photoreceptors of the retina (6, 7) Intravenous administration of αA crystallin downregulated genes involved in the immune response such as TLR signaling as well as Th1, Th2 and Th17 cytokines. Similarly in the present study, systemic administration of microRNA146a, revealed suppression of TLRs and their signaling molecules IRAK1 and TRAF6. Interestingly previously validated in-vitro studies showed that microRNA146a targets IRAK1 and TRAF6 (59–61). To determine biological functions of microRNA 146a, several techniques have been employed. One common approach used in vitro or in vivo was with either miRNA mimics (for overexpression) or complementary inhibitors (to block miRNA function). Therefore, in the current study we evaluated the effects of systemic administration of microRNA146a and 155 by using microRNA mimics of them on the inhibition of the EAU. Moreover, we used the qPCR array to detect the mRNA levels of TLRs and their signaling molecules and the Th1/Th2/Th17 cytokines by Multi-Analyte ELISArray as we reported earlier (7). The innate as well as adaptive immune response genes were suppressed with miRNA 146a mimic administration in the retina as well as in the spleen (Figure 5 and Table 3). We also determined the levels of the T reg activation marker FOXP3 in the spleen since T regs cells are known to suppress EAU (51). The microRNA 146a treatment resulted in upregulation of T regs. The combination of suppression of innate, adaptive immune response genes and upregulation of T reg from the microRNA 146a administration could have abrogated EAU. microRNA146a was also reported to suppress exacerbation of inflammation in allergic conjunctivitis by upregulation of FOXP3 and downregulation of pSTAT3 thus demonstrating a protective mechanism (62). Transgenic mice with altered miRNA expression have also been used successfully to study miRNA function, and have been designed to target miRNA expression at certain stages during development, as well as in a tissue- or cell type- specific manner (63, 64). Therefore in our study we showed the protective function of microRNA 146a in EAU, using microRNA146a KO mice. Interestingly our results revealed that the microRNA 146a KO mice had severe inflammation compared to the WT mice ( Figures 2, 3) which substantiated our findings that microRNA146a can suppress uveitis and absence of this microRNA can lead to severe intraocular inflammation and retinal damage ( Figures 4, 5). As local antigen presentation in the retina is critical for uveitis development, we designed a study to investigate the protective role of microRNA146a through local delivery and to see if such delivery would inhibit the TLRs signals in the retina resulting in inhibition of EAU. Our data indicated that similar to the protective effects of systemic administration of microRNA146a, the intravitreal local delivery of this microRNA also prevented development of uveitis. This finding indicated that the protection from local delivery could be from suppression of TLR signals required for local antigen presentation in the retina. To confirm the entry of the microRNA into the retina of these mice, localization of the microRNA after intravitreal delivery was carried out by confocal microscopy by using microRNA mimics labeled with a fluorescent dye DY547 as described by others (65). This enabled us to trace the injected microRNA and localize it in the retina ( Figure 6). Finally, to investigate the molecular mechanism of amelioration of uveitis by intravitreal local delivery of microRNA146a, we designed an experiment to study the immune response genes by qPCR array. We analyzed the TLRs and its signaling molecules as well as the Th1/Th2/Th17 cytokines in the spleen and the retina after intravitreal local delivery of microRNA146a into the eyes of EAU mice and compared them to the scrambled RNA delivered EAU eyes. In the spleen the TLRs signaling molecules IRAK1 and TRAF6 were upregulated in both microRNA146a, and scrambled RNA treated mice whereas in the retina these molecules were downregulated with microRNA 146a delivery and not with scrambled RNA ( Figure 8). The results indicate that the most likely mechanism of abrogation of uveitis with local delivery of microRNA146a could be due to suppression of local innate immune response that could translate into suppression of Th1/Th2/Th17 immune response in EAU. These data suggests that intravitreal delivery of microRNA146a may abrogate EAU, however additional experimental evidence is required to determine the mechanism required for such abrogation of EAU inflammatory process. Overexpression of microRNA146a also showed a similar result by demonstrating decreased expression of proinflammatory mediators TRAF6 and IRAK1 and downstream target NFkB and other inflammatory cytokines such as IL-1α, IL-1 β and IL-6 in diabetic cornea (66). A similar study in EAAU in rats following miR-146a injections downregulation of interleukin- (IL-) 1β, IL-6, IL-12 and IFN γ and upregulation of IL-10 and IL-17 were noted (56). MicroRNAs are known to be transcribed in the nucleus by the transcription factor NFkB (45) and are exported to the cytoplasm where they control the expression of various target mRNAs. Through this mechanism they serve as a new class of novel target specific immune suppressors and can be used as a novel therapeutic agent in autoimmune diseases (54). In our study, administration of αA crystallin in EAU upregulated the microRNA146a, therefore indicating a possible mechanism of interaction of αA crystallin with NFκB. Since microRNA biogenesis occurs through activation of NFκB, in our EAU model αA crystallin may activate this transcription factor. NFκB activation can occur through the TLR signaling pathway by the activation of its signaling molecules IRAK1 and TRAF6. In addition to TLR signaling, TNF α is also known to activate NFκB. However, till date the role of αA crystallin in activation of NFκB is unknown. A recent report with Hsp27 a close member of crystallin family had demonstrated that Hsp27 regulates NFkB activity through the degradation of iκB (18). Similar to this observation, αA crystallin might also activate NFκB through iκB degradation. To investigate this function of αA crystallin, we further tested the activation of NFκB by αA crystallin administration in αA KO mice with EAU. αA KO mice proved to be a good model for this study since there is no endogenous αA crystallin and the molecular action we detect is solely due to the exogenous αA crystallin administration through intravenous injection. The results showed that, in vivo administration of αA crystallin in αA KO mice resulted in NFκBp65 activation. There was increased phosphorylation of iκB and activated NFκBp65 in these mice administered with αA crystallin compared to the control animals ( Figure 1), and such activation of NFκB by αA crystallin is known to lead to the upregulation of microRNA146a as reported by others (54). However further studies are required to confirm its role in our experimental conditions. In depth studies in the IRBP-induced EAU model will be done to investigate whether NF-kB pathway plays a role in this model and if it does, how the administration of α a crystallin affect its pathway.

Current treatments for uveitis are associated with various side effects because of their broader immune suppression and the targeting of immune cells or cytokines. Therefore, microRNAs as modulators of cytokine genes offer an innovative approach to the treatment of uveitis. Such studies on microRNAs also have a broader implication in the treatment of various ocular, neurologic and other organ specific inflammatory diseases. Identifying the targets of miRNAs in inflammation, which have multicellular components is a great challenge, and one of the most important goals to advance our understanding of how miRNAs contribute to health and disease. Continued research into the regulatory powers of these small non-coding RNAs is required to fully determine their potential for the treatment of disease which will enable them to be successfully targeted and delivered to patients and bring new therapies to the clinic.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Guide for the Care and Use of Laboratory Animals of the Association of Research in Vision and Ophthalmology. The protocol was approved by the Committee of the Ethics of Animal Experiments of the University of Southern California (Protocol Number: 11218).

SS: Designed and Performed all experiments. Contributed in analyzing the data, data compilations, preparing images for figures and writing the manuscript. NR: Guidance throughout the project in designing the experiments and reviewing the results, and analyzing data and review the manuscript. All authors contributed to the article and approved the submitted version.