The innate immune receptor Nlrp12 suppresses autoimmunity to the retina

Uveitis, or intraocular inflammatory disease, accounts for a significant proportion of blindness in the US [1, 2]. Autoimmune responses that overcome the protective mechanisms of ocular immune privilege constitute one of a number of possible etiologies of uveitis. Ocular immune privilege is normally maintained by physical sequestration of ocular antigens such as interphotoreceptor retinoid-binding protein (IRBP) from the systemic immune system by the blood-retinal barrier, active immunosuppressive microenvironment of the eye [3], and peripheral tolerance mechanisms. Together, these mechanisms suppress immune/inflammatory processes that would otherwise destroy the terminally differentiated photoreceptors of the neuroretina that are critical for vision [4]. Uveitis can occur as an isolated entity or in association with systemic autoimmune diseases [5, 6], including multiple sclerosis (MS) [7‐9]. Many clinical and histopathological aspects of uveitis are mimicked in an animal model, experimental autoimmune uveitis (EAU), which is induced by immunization with retinal proteins such as IRBP [4, 10]. To date, discoveries of adaptive immune mechanisms in uveitis using this model have led to therapies that target T cells, such as CD4⁺ effector T cells polarized to the Th17 phenotype, a driving pathogenic mechanism of EAU [11].

Innate immune sensors such as Nod-like receptors (NLRs) detect an array of potential pathogens and activate microbicidal and inflammatory responses required for their elimination [12]. NLRs reside in the cytosol where their activation leads to their oligomerization, formation of large molecular scaffolds, signal transduction, and subsequent restoration of cellular homeostasis [13]. Interestingly, a number of NLRs are linked to human inflammatory disorders including ones accompanied by ocular manifestations such as uveitis, thereby underscoring their importance in immune homeostasis and disease. The NLR, NLRP12 has been linked to inflammatory diseases with ocular involvement [14‐16], including MS [17]. While its agonist is yet to be identified, Nlrp12 has been shown to negatively regulate inflammation through suppression of inflammatory pathways, e.g., canonical and non-canonical NF-κB signaling [18‐27], although there are also contrasting reports of Nlrp12 function in promotion of inflammatory pathways [24, 28‐30].

Nlrp12 has recently been implicated in regulation of neuroinflammation in the context of a murine model of MS, experimental autoimmune encephalomyelitis (EAE). Nlrp12^−/− mice were shown to be highly susceptible to EAE, which was attributed to an exacerbated Th1 response [31] and microglia activation [32]. Conversely, Nlrp12 has also been found to promote EAE. Nlrp12^−/− mice developed a milder form of EAE that was mediated by T cell-intrinsic control of cytokine production [33]. This would be consistent with another report showing that Nlrp12 promotes development of a spontaneous form of EAE in 2D2 T cell receptor (TCR) transgenic (Tg) mice and its role in suppressing TCR signaling pathways [31]. These contradictory findings not only indicate an incomplete understanding of how Nlrp12 modulates EAE, but also highlight potential counter-regulatory functions of NLRs within T cells in shaping autoinflammatory disease. Here, we sought to investigate the function of Nlrp12 using EAU, a T cell-mediated model of CNS autoimmunity similar to EAE.

Our findings demonstrated a role for Nlrp12 in downregulation of inflammation and protection against EAU. The mechanism is not regulated inherently by T cells, but may be attributed to a multi-cellular mechanism involving BM-derived myeloid cellular responses as well as local Nlrp12 function in the neuroretina. These data provide novel insight into the functioning of Nlrp12 in suppression of T cell-mediated uveitis through integration of innate immune and ocular responses.

While NLRs play critical roles in health and disease, their functions have been poorly defined in uveitis. We used the EAU model, which has been instrumental in defining contributions of inflammatory mediators and T cell responses to pathogenesis of uveitis, to study Nlrp12 in integration of innate and T cell-mediated responses in ocular inflammation. We show, somewhat surprisingly, that Nlrp12 exerts an anti-inflammatory role in EAU to suppress uveitis. The exacerbated uveitis observed in Nlrp12^−/− mice was not attributed to inherent T cell dysfunction, but was due rather, to multi-cellular mechanisms involving BM-derived myeloid cellular responses as well as non-hematopoietic cell contributions. Nlrp12 was observed within ocular tissues, most notably within neuroretina, where it mediated induction of cytokines and chemokines.

A T cell-intrinsic mechanism for Nlrp12 is as yet to be resolved, as there have been conflicting findings in its role in EAE [31‐33], a murine model for multiple sclerosis in which the CNS is the target organ. It is possible that differences in methodology, such as the amount of MOG_35–55 peptide used for immunization (either 100 μg [33] or 200 μg [31, 32]), could affect patterns of cytokine production that might influence disease development. It is also conceivable that mice with genetically-engineered Nlrp12 alterations located in exon 2 [25] (used previously [33]) or exon 3 [24] (used in studies here and by others [32]) resulted in different animal phenotypes. Although Nlrp12^−/− mice have been reported to develop an attenuated form of classical EAE [33], we and others [32] found Nlrp12^−/− mice to be more susceptible to autoimmunity of the CNS. In any case, our data also did not support a T cell-intrinsic mechanism in protection against EAU.

Nlrp12 is predominately expressed in BM-derived cells of the myeloid lineage where it has been reported to negatively regulate inflammatory pathways [19, 47]. Hematopoietic-derived Nlrp12 has been reported to be important for host defense against Klebsiella infection, where bacterial clearance and neutrophil influx into the lung are dependent on Nlrp12 [48]. Our findings indicate that Nlrp12 is important in controlling responses of BM-derived myeloid cells such as neutrophils and macrophages, as a protective mechanism during EAU. These observations would be consistent with those of others using experimental models of colitis and colitis-associated colon cancer (CAC) [20, 25, 33, 49], where an inhibitory role for Nlrp12 is also involved in suppression of BM-derived macrophage recruitment and cytokine/chemokine expression.

In the eye, enhanced neutrophil and macrophage accumulation could, in turn, further potentiate IRBP-reactive T cell responses and more severe uveitis in Nlrp12^−/− mice. Thus, Nlrp12 may serve as an important immunomodulatory checkpoint in restricting autoimmune disease. This hypothesis would be substantiated by mutations (e.g. p.Arg284X) in NLRP12 that result in impaired ability to suppress NF-κB activity [16] and inflammatory syndromes [50], and other missense mutations (p.Asp294Glu or Arg352Cys) that result in hyperactivation of the caspase-1 inflammasome [14]. Still, the precise molecular pathway by which Nlrp12 controls myeloid responses that protect against EAU remains to be determined in future studies. Historically, Nlrp12-mediated myeloid cellular responses may have been under-appreciated as a consequence of a missense mutation in Nlrp12 in the C57BL/6J mouse strain, which recently came to light [51]. The Nlrp12 missense mutation was found to result in diminished neutrophil recruitment by macrophages. Our observations of heightened myeloid responses in Nlrp12^−/− mice and a BM-cellular mechanism, further lend support to the possibility of a Nlrp12-myeloid signaling axis as a dominant mechanism in uveitis.

The anti-inflammatory function of Nlrp12 has been previously documented in response to Mycobacterium tuberculosis [19, 23]. Accordingly, we detected molecular changes after peripheral injection of CFA-adjuvant alone (without IRBP), with retinal expression of Cxcl1 and Tlr4 increased in Nlrp12^−/− mice. This would support an additional function for Nlrp12 in retinal cells in protection against peripheral factors that may trigger breaches in the blood-ocular barrier and loss of immune privilege. Indeed, we not only showed Nlrp12 expression within the retina but also demonstrated a non-hematopoietic contribution of Nlrp12-mediated protection against EAU. Such combinatorial BM and non-BM derived roles for Nlrp12 have also been described in suppression of colon inflammation and cancer [20]. Collectively, our data support distinct tissue-specific functions for Nlrp12 involving suppression of retinal inflammation and BM-derived macrophage responses, which together protect against perpetuation of T cell-mediated uveitis.

The studies presented here are not without potential caveats. First, given the overlapping phenotypes between monocyte-derived macrophages and microglia, it has long been technically challenging to distinguish between their functions, particularly in scenarios of inflammation. However, it was recently discovered that monocyte-derived macrophages are of hematopoietic origin, whereas microglia are yolk-sac-derived and have a unique phenotypic signature that is conserved under steady-state and tissue injury conditions [52‐54]. In the brain, microglia express Nlrp12, which functions to suppress production of reactive oxygen species and cytokines [32]. Networks of microglia also reside within the uveal tract and retina [41, 55], where they not only help maintain ocular homeostasis but also initiate inflammation in EAU [56]. Given the anatomical and developmental connection between the neuroretina and brain, it would be resonable to expect that retinal microglia also express Nlrp12. Based on our BM chimera studies and the markers used to phenotype the cell infiltrate in uveitic eyes of Nlrp12^−/− mice (CD45^highCD11c^loMHCII⁺F4/80⁺), we have strong indication for a BM-monocyte-derived macrophage response rather than a microglia response. Examination of the microglia population by flow cytometry based on them being defined as CD45^loCD11b^loCD11c^lo, did not show any difference between WT and Nlrp12^−/− (both being < 5% of the cells). However, we cannot exclude how their activation and/or function may be controlled by Nlrp12, as has been reported in purified microglia cultures from the brain [32]. This is notable since the retinal environment itself can also have potent influence over characteristics of infiltrated myeloid cells that render them to resemble endogenous microglia [57]. Thus, future investigation of how Nlrp12 controls microglia function would be of interest. Second, Nlrp12 expression was also found to be particularly high in the RPE/choroid complex. The RPE is known to play an important role in suppression of macrophage activation within the eye and protection against EAU [58]. Thus, our studies did not consider any putative mechanism of Nlrp12 that might be imparted by the RPE. Third, given several differences of cytokine/chemokine transcript regulation (e.g. of Cxcl1 and Cxcl10) using RT-qPCR, it would be of interest to confirm these findings at the protein level, as well as future mechanistic studies to explore Nlrp12-mediated protection against uveitis. Last, Nlrp12 has recently been recognized to be involved in regulation of gastrointestinal commensal organisms that promote intestinal homeostasis [49, 59]. Nlrp12-deficient mice have increased susceptibility to colitis [20, 25] which can be partially mitigated with antibiotic treatment [49]. There is increasing evidence that disruption of commensal microflora (dysbiosis) is also connected to predisposition to ocular inflammation [60, 61]. While we found the mechanism for Nlrp12 protection against autoimmune uveitis to be predominantly of hematopoietic over non-hematopoietic origin (such as would be for the gastrointestinal tract), the extent to which Nlrp12 control of microbiota and/or intestinal cells contributes to protection against EAU remains to be fully determined.

In summary, these findings uncover a novel role for Nlrp12 as a key genetic determinant of uveitis susceptibility, and the cellular mechanism by which Nlrp12 protects against uveitis could be targeted as an alternative approach for treatment of ocular autoimmunity.