RP is a group of hereditary disorders affecting the retina, and microglia activation has been observed in both experimental and clinical instances of RP. [37, 38]. In the current study, we found that BMSC-Exos significantly improved the activation status of microglia. Importantly, after treatment with IFN-γ, this effect became stronger and was also more effective in protecting the degenerative retina of RP rats. Our results showed that IFNγ pretreatment increased the expression of CD81 and CD9 and the particle size and quantity of exosomes. In-depth analysis showed that the high expression of the tsRNA Other-1_17-tRNA-Phe-GAA-1-M3 in the exosomes of the IFNγ-treated group was regard as targeted to regulate the expression of Src, which may serve to modulate the immune status of microglia. By adding Other-1_17-tRNA-Phe-GAA-1-M3 mimics to the microglia culture system and targeting Src expression, we further demonstrated that the regulation of Src by Other-1_17-tRNA-Phe-GAA-1-M3 improved microglial activation.
MSCs have promising efficacy for treating retinal degeneration, partially through the regulation of microglia or macrophages[39, 40]. IFNγ, a cytokine secreted by activated natural killer (NK) cells and T cells, serves as a pivotal player in adaptive immune responses[41]. MSCs that have been stimulated with IFN-γ demonstrate potent immunomodulatory effects by inducing the elevation of immune-activating molecules[42]. Nevertheless, there remains a significant knowledge gap regarding alterations in microglial subtypes after treatment with exosomes derived from IFNγ-pretreated MSCs and the functional improvement in RCS rats. Our findings indicated that IFNγ induces MSCs to produce more exosomes with larger diameters, and the expression of the exosomal signature protein CD9 was significantly upregulated, which was consistent with the report of Zhang’s group[43]. Our results demonstrated that injection of IFNγ-Exos into the vitreous body of RCS rats produced better protection of photoreceptors and retinal functions than the native MSC-derived exosomes. This is consistent with the findings of a previous report, in which exosomes derived from human MSCs stimulated by IFNγ produced better therapeutic effects on EAE mice[18]. These findings suggest that IFNγ preconditioning may represent a strategy to optimize the exosomes of MSCs, providing new insight into improving exosome performance for treating related diseases.
To explore the mechanisms underlying the neuroprotective effect of IFNγ-Exos on RCS rats, we compared the proteomic cargo between IFNγ-Exos and Native-Exos. As functional small non-coding RNAs, tRFs and tiRNAs are abundantly distributed in exosomes and maintain a stable structure and highly conserved characteristics[30, 44]. Multiple studies have shown that tRFs and tiRNAs are involved in the development of various neurodegenerative diseases[45], immune-related diseases, metabolic diseases, cancers, and viral infections[30]. However, little is known about exosomal tRFs, especially their roles in neuroprotective effects. In this study, we demonstrated that three tRFs and tiRNAs were highly expressed in IFNγ-Exos; this was related to neuroinflammation. Among them, the expression level of Other-1_17-tRNA-Phe-GAA-1-M3 was the highest. Given the large number of tRFs that were significantly different between IFNγ-Exos and Native-Exos, we performed target enrichment analysis to investigate the targets of the complete EV tRF cargo. The top targets of the IFNγ-Exo-tRF cargo included Src and several genes involved in cell proliferation, migration, and development, such as the adaptor protein transducin beta-like 1 (TBL1X), the Clip1, and the ligand Delta-like 4 (Dll4). The proto-oncogene tyrosine-protein kinase SRC (Src) is an important target gene of Other-1_17-tRNA-Phe-GAA-1-M3, which modulates the MAPK pathway, and MAPK regulates macrophage activation, cytokine production, and chemotaxis[46]. Notably, Src-related signaling participates in the factor-regulated interaction between inflammatory cells. Src is sufficient for developing microgliosis[47].
Microglia, which are immune cells situated in the retina, play a crucial role in RP development. New investigations have uncovered the potential for varied phenotypic expressions in microglia, rendering them highly diverse and adaptable. The manifestation of these phenotypes is contingent upon the disease's severity and stage, as well as the distinct inflammatory environment[48]. Our previous research demonstrated that resident microglia in the OPL effectively inhibit ectopic neuritogenesis and delay vision deterioration by engulfing synapses in the early stages of RP[49]. Furthermore, the activation of disease-associated microglia (DAM) in the outer retina of RCS rats reduces secondary photoreceptor degeneration caused by the accumulation of dead cells and infiltrated neutrophils[35]. These findings suggest that microglia can respond to various photoreceptor insults and transform into different phenotypes, both pro- and anti-inflammatory, each exerting distinct functions[50]. Therefore, redirecting microglial activation toward beneficial and neuroprotective phenotypes has the potential to halt the progression of RP.
To verify the regulatory effect of IFNγ-Exos on microglia, we injected them into the vitreous body of RCS rats and co-cultured them with LPS-induced BV2 microglia in vitro. The results demonstrated that IFNγ-Exos promoted activated microglia polarization to homeostasis and inhibited the expression of proinflammatory genes in vivo. Importantly, treatment with IFNγ-Exos significantly upregulated LPS-induced Src expression. We also used lentivirus and mimics to knock down Src expression and found that the anti-inflammatory effect of IFNγ-Exos was blocked. Hence, our results suggest that Other-1_17-tRNA-Phe-GAA-1-M3/Src may represent a novel target in the investigation of IFNγ-Exo treatment, implying a potential molecular mechanism by which IFNγ-Exos polarizes microglia homeostasis in RP.
Next, to establish whether IFNγ-Exos have immune-regulatory functions, we injected them into the vitreous body of RCS rats. For the in vitro experiments, we chose BV2 microglia and used LPS to induce microglial neuroinflammation in vitro. Microglia can exert double-edged effects depending on their intrinsic subtypes, including disease-associated and homeostasis phenotypes. A previous study demonstrated that miR-124 could reduce inflammation by modulating microglial polarization in intracerebral hemorrhage[51]. Dou et al. proved that MSC-Exos inhibited the M1-type polarization of macrophages, possibly by transferring tsRNA-21109, in systemic lupus erythematosus[32]. In this study, the IFNγ-Exo group was shown to promote microglia to restore homeostasis and reduce pro-inflammatory cytokines both in vivo and in vitro. Based on these results, we conclude that IFNγ-Exos represent a promising bioagent to improve functional behavioral recovery by promoting the microglia to restore homeostasis in RCS rats.
To better understand the phenomenon underlying the ability of IFNγ-Exos to promote microglia to restore homeostasis in vivo and in vitro, we chose the target tsRNA Other-1_17-tRNA-Phe-GAA-1-M3 and its target gene Src for further study. Src kinases are present in glial cells and are upregulated following neurological insults in both human and animal models. BV2 activated and showed increased TNF-a levels, which were attenuated by a Src kinase inhibitor, suggesting that Src plays a role in neuroinflammation and microglia polarization[47]. A previous study on the retina showed that Src inhibition in an ischemia-reperfusion injury model significantly reduced microglial activation, changed the morphology of microglia to a resting phenotype, and prevented neuronal apoptosis[52]. We also performed loss-of-function experiments to further confirm Src as the target gene of the identified tsRNA. The results demonstrated that knockdown of Src in BV2 cells could abolish the beneficial effects observed from the anti-inflammatory effect in IFNγ-Exos. Taken together, we conclude that exosomal Other-1_17-tRNA-Phe-GAA-1-M3 derived from IFNγ-pretreated MSCs can suppress microglial-induced neuroinflammation by promoting microglia from DAM to homeostasis and inhibiting Src expression in RCS rats.