Retinitis pigmentosa (RP) is a category of inherited retinal dystrophies marked by vision loss and, ultimately, blindness. More than 3000 mutations in over 80 distinct genes or loci have been identified as causes of non-syndromic RP (1, 2). These mutations can be transmitted in an autosomal-dominant, autosomal-recessive, or X-linked manner. There are also syndromic forms of RP, such as Usher syndrome and Bardet-Biedl syndrome (3). The prevalence of RP is reported to be 1/3,000 to 1/5,000 (https://www.orpha.net/consor/cgi-bin/index.php?lng=EN). Early in the progression of RP, patients experience night blindness and difficulty with dark adaptation. They gradually lose peripheral vision and develop tunnel vision as the disease advances, indicating the loss of rod function. Cone involvement contributes to visual acuity decline over time, and finally, typically in middle age, central vision loss occurs (2). Due to its clinical and genetic heterogeneity, RP has limited therapeutic options. It has long been recognized that inflammation and immune responses are associated with RP, and this theme has recently gained increasing attention (4–6). Greater understanding of the molecular processes driving RP inflammation is expected to provide new therapeutic approaches independent of the genetic background.

Inflammation activation is a prominent feature of RP. In RP, inflammation is characterized by activation of the innate immune system, including dysfunction of the immune barrier, activation and infiltration of immune cells, and upregulation of topical and peripheral inflammatory factors. Bone-spicule pigmentation, attenuated retinal vessels and waxy pallor of the optic disc are typical clinical manifestations of RP. In addition, inflammatory cells are commonly observed in the vitreous due to the collapse of the blood−retina barrier (BRB). Higher cell density correlates with younger age and impaired visual function (6). In addition, it has been reported that increased aqueous flare in RP patients correlates closely with visual function and the extent of global retinal degeneration (7–11). Aqueous flare is generally seen in individuals with inflammatory ocular disorders, indicating deficits of the blood–aqueous barrier and inflammatory protein/cell leakage (12, 13). BRB disruption begins early in the disease. Prior to the infiltration of inflammatory cells and photoreceptor (PR) starvation, the tight junctions of the retinal pigment epithelium (RPE) and the retinal vasculature become leaky, thereby promoting the formation of an inflammatory milieu and the degeneration of PRs (14–17).

Microglia are essential components of the retinal innate immune system and play a pivotal role in retinal inflammatory responses. Gupta et al. reported that microglial activation is engaged in human RP. With thinning of the PR layer, microglia were observed to infiltrate degenerative foci; these microglia were enlarged amoeboid cells containing rhodopsin-positive cytoplasmic inclusions (18). Microglia promote retinal inflammation via infiltration, phagocytosis, and secretion of proinflammatory mediators, whereas genetic ablation or inhibition of microglial phagocytosis ameliorates PR degeneration in RP model mice (19).

Several studies (6, 20–22) have reported elevated levels of inflammatory factors in serum, vitreous, and aqueous humor, indicating a proactive inflammatory response in RP patients. Immunologic disorders observed in patients with RP are summarized in a previous work (23).

Animal models are essential for the study of RP (24, 25). Activation of the immune system has been detected in various rodent RP animal models (26–28). Due to this similarity, animal models are indispensable for clarifying the pathogenesis of RP and developing treatments. Clinically, synthetic corticosteroids with potent anti-inflammatory and immunosuppressive properties are used in treatments for RP-related cystoid macular edema (29). In RP models, it also works. In RCS and rhodopsin mutant model S334ter-4 rats, fluocinolone acetonide treatment markedly protects PR from degeneration and suppresses microglial activity (30, 31). In combination with polyamidoamine dendrimers, fluocinolone acetonide selectively targets the microglia localized in the outer retina where degeneration is ongoing (32). Dexamethasone administration to rd10 mice reduces retinal inflammation, restores cone structure and function, and preserves RPE integrity by preserving ZO-1 density (15, 33).

Microglial activation is a sign of neuroinflammation. Retinal resident microglia arise from yolk sac erythromyeloid progenitors, comprising 85% of total retina macrophages (34–36). They colonize the developing retina during embryogenesis, shape the retina by secreting neurotrophic factors, engulf and eliminate unwanted neurons and synapses, and engage in vascular development of the eye (37–39). In postnatal retinas, microglia are maintained throughout life independent of circulating monocytes and by self-renewal (34, 40, 41).

Microglia express a variety of receptors (e.g., CX3CR1, TLRs, IL-1R, and TNFR) that allow them to detect environmental changes and initiate the inflammatory signal cascade (42). Microglia activation induces a robust inflammatory response, including the release of proinflammatory factors, phagocytosis, and inflammatory cell recruitment. Excessive microglial phagocytosis contributes to local inflammation and neurodegeneration (43, 44).

Classically, activated microglia were categorized into two groups: M1 (classically activated) and M2 (alternatively activated), with the belief that M1 microglia secrete proinflammatory factors such as TNFα, IL-1β, IL-6, and inducible nitric oxide synthase (iNOS) that fuel inflammation, whereas M2 microglia produce anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-13, IL-18) that are beneficial for damage repair (45). Recent research, however, suggests that the microglial phenotype varies in response to environmental changes (46).

In healthy retinas, microglia tile the inner and outer plexiform layers without overlapping (37), where they are ramified cells responsible for immune surveillance and maintenance of synaptic structure and transmission (47–50). In response to insults, microglia rapidly change into an amoeboid appearance and migrate into lesion areas, removing dead/dying neurons and neuronal debris while concurrently releasing proinflammatory factors as well as protective cytokines and trophic factors to repair damage and restore homeostasis (51), after which microglia recover to the “resting” state; this process usually results in minimal retinal remodeling.

In RP retinas, initial mutation-driven PR degeneration increases extracellular signal molecules (e.g., ATP, HSPs, HMGB1, DNA, and many others) termed damage-associated molecular patterns (DAMPs) (52–56). The “eat-me” signal, phosphatidylserine, appears on stressed rods (19). Microglia proliferate and infiltrate the PR layer and subretinal space, where they function as reactive phagocytes, phagocytose dead and stressed PRs, secrete proinflammatory cytokines (e.g., TNFα and IL-1β) and chemokines (e.g., CCL2 and RANTES), and recruit infiltrating immune cells (19, 24, 57, 58). Due to this mutant genetic background, however, microglial activation persists, and the continuous production of inflammatory and cytotoxic factors exacerbates PR loss until the late stage, at which point PRs mostly die and the retinal structure is severely damaged (59).

Müller glia are another group of retinal cells engaged in degeneration. Müller glia are retinal macroglia that provide homeostasis, metabolism, and functional support for neurons (60). Depending on the severity, the Müller glial response to injury refers to reactive gliosis accompanied by Müller proliferation or not. Reactive gliosis can be beneficial because it releases protective factors such as neurotrophic factors, whereas prolonged gliosis is detrimental and generally results in neurodegeneration (61). Müller glia are potential modulators of retinal inflammation. Upon BRB disruption, Müller glia compensate for RPE deficiency by sealing the leaky choroid and inducing claudin-5 expression (14). Müller glia share characteristics with immune cells. Müller glia express multiple cytokine receptors and are a major source of cytokines and inflammatory factors (62). Proteomic evidence supports the capacity of Müller glia for antigen presentation and inflammatory signaling transduction in response to immune stimulation (63, 64). Müller glia contribute to the phagocytic clearance of dead PRs (65). Moreover, the interaction between Müller glia and microglia modulates retinal inflammation and degeneration (66–68).

As the predominant glial population of the retina, Müller glia are abundant and widely distributed. Müller glia traverse the thickness of the neuroretina structurally, allowing them to keep touch with all types of retinal cells. Due to its neurotrophic function and regeneration potential, the Müller cell has been studied in a variety of degenerative retinal disorders (69). In actuality, Müller glia are also intimately linked to retinal inflammation. For more information about how Müller glia interact with the innate immune system and monitor retinal inflammation, we refer the reader to this article (70).

Complicated mechanisms are involved in the regulation of retinal inflammation in RP. Microglia and Müller glia are major cellular populations that express and modulate these signaling pathways (Figure 1). Here, we review treatment strategies from the perspective of inflammation management (Table 1), focus on molecular mechanisms related to immunomodulation, and discuss new findings regarding epigenetic modification and the gut microbiome as novel therapies for RP.

Tumor necrosis factor α (TNFα) is a strong proinflammatory cytokine that plays vital roles in immune modulation, cell proliferation, differentiation, and apoptosis. TNFα is produced predominantly by T and innate immune cells and is initially synthesized as transmembrane protein (tmTNFα), a precursor that requires proteolytic cleavage by TNFα-converting enzyme (also ADAM17) to release a soluble form (sTNFα) (124). Both tmTNFα and sTNFα are implicated in the inflammatory response.

TNFα initiates a signal cascade by binding to its receptors, TNFR1 and TNFR2. TNFR1 is activated by both tmTNFα and sTNFα, whereas TNFR2 is proposed to be fully activated primarily by tmTNFα (125). Ligand binding to TNFR1 recruits the adaptor molecule TNFR1-associated death domain protein, which then leads to the assembly of several signaling complexes known as complexes I, IIa, IIb, and IIc. Complex I formation stimulates nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs). Complex IIa and IIb assembly activates caspase-8 and facilitates apoptosis, and complex IIc formation activates the mixed lineage kinase domain-like protein and induces necroptosis and inflammation. TNFR2 stimulation activates NF-κB, MAPKs, and protein kinase B (125).

TNFα is postulated to participate in the pathogenesis of RP (74). TNFα and TNFR expression levels are elevated in the retina of RP models and in the aqueous humor of RP patients (24, 74, 126–128); microglia (129) and Müller glia (130, 131) are the primary cellular sources of TNFα. TNFα signaling has been found to mediate PR death via RIP1/3-related necrosis and caspase3/7-dependent apoptosis, in addition to triggering proinflammatory signaling in the RP retina (73, 126).

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) responsible for identifying pathogen-associated molecular patterns (PAMPs) and DAMPs and mediating immune responses; the generation of PAMPs or DAMPs prompts pathogen invasion or tissue injury. TLR expression is conserved among species, and to date, 10 TLRs (TLR1–10) in humans and 12 TLRs (TLR1–9 and TLR11–13) in mice have been described. TLRs are predominantly but not exclusively expressed on immune cells (146–148).

TLRs serve as the first line of defense for the innate immune system. Upon recognition of DAMPs or PAMPs, TLRs dimerize and initiate recruitment of Toll/IL-1 receptor (TIR) domain-containing adaptor molecules, including myeloid differentiation primary response 88 (MyD88), TIR-domain-containing adaptor-inducing interferon-β (TRIF), MyD88 adaptor-like protein (Mal), and TRIF-related adaptor molecule (TRAM), thereby initiating intracellular signaling cascades: the MyD88- or TRIF-dependent pathways (146). TLR activation facilitates the transduction of NF-κB and MAPK (149–151), as well as the release of proinflammatory cytokines (TNFα, IL-6, IL-1β, and IFNβ), chemokines, and cluster of differentiation 80 (CD80), CD86, CD40, and major histocompatibility complex class II (146).

Activation of TLR signaling has been shown to worsen inflammation and accelerate the course of RP (77, 152, 153). Microglia highly express TLRs (154), and TLR activation in the retina facilitates microglial activation and infiltration (77, 155). Moreover, microglia in the rd1 retina undergo RIP1/RIP3-dependent necroptosis mediated by TLR4 activation, which amplifies retinal inflammation and destruction with large amounts of proinflammatory cytokines (TNFα and CCL2) (152).

Inflammasomes are cytosolic multiprotein complexes that facilitate the release of mature IL-1β, IL-18, and cleaved caspase-1. The intracellular PRRs, NOD-like receptors (NLRs), are important components of the inflammasome complex. Some NLRs oligomerize upon activation to form multiprotein complexes that function as caspase-1-activating scaffolds (171). NLRP3 is the most well-studied NLR; NLRP3 inflammasome assembly requires two signals: a priming signal that activates NF-κB, followed by transcription of NLRP3, pro-IL-1β, and pro-IL-18. A second activation signal facilitates the recruitment and oligomerization of NLRP3, adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), and pro-caspase-1. Once the inflammasome is assembled, it stimulates pro-caspase-1 self-cleavage and activation, and cleaved caspase-1 catalyzes pro-IL-1β and pro-IL-18 maturation and induces the release of their mature forms. The recognition of DAMPs or PAMPs that act through PRRs such as TLRs or cytokines that act through particular receptors (TNFR, IL-1R) exemplifies the priming signal. The activation signal encompasses a wide range of stimuli, including ion flux (K+, Cl-, Ca2+), lysosomal instability, mitochondrial dysfunction, reactive oxygen species generation, and trans-Golgi disassembly, with K+ efflux being the upstream event in almost all NLRP3 activations (172, 173).

Inflammasome activation initiates the host’s defense response to endogenous or external damaging stimuli and aids in homeostasis maintenance. Nevertheless, chronic inflammasome activation and the subsequent overproduction of caspase-1, IL-1β, and IL-18 can be detrimental.

Canine models of RP upregulate NLRP3 inflammasome-related genes (26). NLRP3 was detected in cone PRs and one-third of reactive microglia in P23H rhodopsin mutant retinas, which also upregulates the expression of mature IL-1β and IL-18, as well as cleaved caspase-1, indicating inflammasome activation during retinal degeneration. In rd10 mice, administration of the antioxidant N-acetylcysteine prevented PR loss and suppressed inflammatory factors and microglial activation (24). Studies conducted on P23H mice demonstrated that N-acetylcysteine lowered NLRP3 expression by 50% and decreased microglial infiltration, hence improving cone survival and retinal function (87).

The JAK/STAT signaling pathway is a ubiquitously expressed intracellular signal transduction system implicated in a wide range of biological functions. Various ligands, including cytokines, growth hormones, growth factors, and their receptors, can activate the JAK/STAT pathway (203). Briefly, ligand binding to specific receptors induces receptor multimerization and JAK activation, activated JAKs phosphorylate the receptors, activate and phosphorylate their primary substrate STAT, and phosphorylated STAT dimerizes and translocates into the nucleus, where it binds to particular regions to either activate or inhibit the transcription of target genes. Suppressor of cytokine signaling (SOCS) is a negative modulator of JAK/STAT signaling, and its expression is promoted by stimulation of JAK/STAT signaling (203, 204). Numerous studies on the JAK/STAT pathway have revealed its significance in neoplastic and inflammatory disorders (203, 205).

Histone acetylation and methylation are the two most well-studied types of histone modification, with acetylation typically resulting in increased gene expression and methylation being related to either increased or decreased gene transcription. Histone acetylation is regulated by histone acetyltransferases and histone deacetylases (HDACs), while histone methylation is regulated by lysine methyltransferases and arginine methyltransferases and histone demethylation by histone demethylases. Enzymes that add or remove epigenetic marks on histones are known as “writers” and “erasers.” In addition, there are “readers” containing bromodomains, chromodomains, or Tudor domains that are able to decipher histone codes (229).

RP retinas exhibit excessive HDAC activity (104, 105, 230), and HDAC inhibition delays retinal degeneration in RP animal models (rd1 and rd10 mice and zebrafish) (104–107). In rd10 mice, the HDAC inhibitor romidepsin prevented rod degeneration and enhanced retinal function. Two molecular mechanisms contribute to this neuroprotective effect. First, by acting on histone targets in PRs, increasing chromatin accessibility and upregulating neuroprotective genes, and second, by acting on nonhistone targets in microglia and resident and invading immune cells, it suppresses inflammatory gene transcription and inflammation (108).

Microglial activity is related to histone methylation levels. LPS-activated microglia increase HDAC expression, which is accompanied by an increase in inflammatory gene expression (231). HDAC inhibition or knockdown promotes a protective microglial phenotype and reduces neuroinflammation (232–234).

Valproic acid is an HDAC inhibitor that reduces PR degeneration in rd1 and P23H RP models (109, 110). Valproic acid increases the expression of STAT1 by inhibiting HDAC3 expression; subsequently, acetylated STAT1 forms a complex with nuclear NF-κB p65, preventing NF-κB p65 DNA-binding activity (235).

Moreover, suppression of the “read” (bind) behavior to histone acetylation marks of bromodomain and extraterminal domain proteins by JQ1 ameliorated PR degeneration and maintained electroretinographic function in rd10 mice. This protection seems to be partially mediated by the inhibition of retinal microglial proliferation, migration, and cytokine production (111).

Several studies, including our previous report, have reported altered histone methylation in RP retinas (112, 236, 237). Lysine demethylase 1 inhibition attenuated PR degeneration in rd10 mice, in part by inhibiting microglial-related inflammation (108). DZNep (3-deazaneplanocin A) specifically inhibits Ezh2 (H3K27 trimethyltransferase) and mediates neuroprotective effects in rd1 mice by inhibiting H3K27me3 deposition (112). Ezh2 reportedly mediates TLR-induced inflammatory gene expression (238) and activation of multiple types of inflammasomes in microglia (239), hence promoting microglial-related pathologies.

MicroRNAs (miRNAs) are small noncoding RNAs that modify gene expression post-transcriptionally by targeting messenger RNAs, long noncoding RNAs, and pseudogenes and circular RNAs. MiRNAs can be packed into exosomes or microvesicles to perform long-distance cell-to-cell communication. MiRNAs play a critical role in gene expression modulation and are therefore interesting candidates for the development of biomarkers and therapeutic targets (240). Throughout development, miRNAs are required for retinal neuron differentiation (241, 242). Dysregulated miRNAs were found in the retinas of mouse and canine models of RP (243, 244), indicating the involvement of miRNAs in the etiology of RP.

MiRNAs regulate microglial phenotypes, as evidenced by various studies on retinal and neurodegenerative disorders (245, 246). Inhibition of miR-6937-5p preserved the outer nuclear layer thickness and promoted the ERG wave response in rd10 mice (113), and AAV-miR-204 attenuated retinal degeneration in two different mouse models. By downregulating microglial activation and PR mortality, miR-204 alters the expression profiles of transgenic retinas toward those of healthy retinas (114). In addition, miR-223 is required for the regulation of microglial inflammation and the maintenance of normal retinal function (247).

DNA methylation refers to the addition of a methyl group to the 5′-carbon of a cytosine (C) ring, resulting in the formation of 5-methylcytosine (5mC), which mainly occurs in the promoter regions. Typically, methylation modifications result in gene repression, and global genomic hypermethylation relates to heterochromatin formation and inhibits transcription (248). Aberrant regulation of DNA methylation results in PR degeneration and neuronal loss in the retina. In the absence of DNA methyltransferase 1, the initiation of PR differentiation is severely hindered (249). In RP retinas, binding sites of several important transcription factors for retinal physiology were hypermethylated (250). The role of DNA methylation in the development of retinitis pigmentosa has been reviewed in detail elsewhere (251) and will not be repeated here.

We argue that trained immunity regulates the microglial phenotype in RP by plasticizing microglial reactivity via epigenetic modification.

Trained immunity, also known as innate immune memory, refers to the phenomenon in which innate immunity modifies its function after an initial insult and reacts more vigorously to subsequent stimuli. Epigenic reprogramming determines the immune phenotype of immune cells and leads to long-lasting functional alterations (252, 253) (Figure 2). Using macrophages as an illustration, in the resting state, the promoter regions of inflammatory genes are enriched with repressive epigenetic marks, called epigenetic barriers, to prevent activation in the absence of stimuli. Upon stimulus, repressive epigenetic marks are removed, and activating epigenetic marks are introduced to the promoters and enhancers of specific genes in an attempt to encourage inflammatory molecule synthesis and phagocytosis to eliminate the insult. After stimulus elimination, activating epigenetic marks are partly retained (254). The innate immune system may become overly trained in chronic inflammatory diseases as a result of such mechanisms, resulting in pathological tissue damage.

In the context of neurodegenerative disorders of the CNS, the relationship between trained immunity and microglial phenotype has been discussed (255, 256). Low-dose LPS intraperitoneally administered to mice induced long-lasting innate immune memory in brain microglia and exacerbated Alzheimer’s disease pathology. Activated microglia are enriched with the epigenetic marks H3K4me1 and H3K27ac, which define active enhancers (256). In RP model P23H rats, intraperitoneal injection of low-dose LPS increased microglial activation and the number of infiltrating microglia, as well as elevated the expression levels of several inflammation-related genes (257). In addition to the activation of retinal microglia, elevated levels of serum cytokines show the activation of peripheral immune cells in RP (22, 23). Recent work by Su et al. revealed that monocytes from patients with autosomal recessive RP exhibit a trained-like phenotype. Upon stimulation, these monocytes produce more TNF-α, IL-6, and IL-1β and upregulate inflammatory pathways such as NF-κB (258). Current evidence supports a role for trained immunity in RP pathogenesis by epigenetic reprogramming of microglia and peripheral macrophages to modulate the immune phenotype and trigger an active immune response, although many details remain to be confirmed.