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The ocular microenvironment has adapted several negative regulators of inflammation to maintain immune privilege and health of the visual axis. Several constitutively produced negative regulators within the eye TGF-β2, α-melanocyte stimulating hormone (α-MSH), Fas ligand (FasL), and PD-L1 standout because of their capacity to influence multiple pathways of inflammation, and that they are part of promoting immune tolerance. These regulators demonstrate the capacity of immune privilege to prevent the activation of inflammation, and to suppress activation of effector immune cells even under conditions of ocular inflammation induced by endotoxin and autoimmune disease. In addition, these negative regulators promote and expand immune cells that mediate regulatory and tolerogenic immunity. This in turn makes the immune cells themselves negative regulators of inflammation. This provides for a greater understanding of immune privilege in that it includes both molecular and cellular negative regulators of inflammation. This would mean that potentially new approaches to the treatment of autoimmune disease can be developed through the use of molecules and cells as negative regulators of inflammation., (©2018 Society for Leukocyte Biology.)

The recovery of EAU, a mouse model of endogenous human autoimmune uveitis, is marked with the emergence of autoantigen-specific regulatory immunity in the spleen that protects the mice from recurrence of EAU. This regulatory immunity is mediated by a melanocortin-driven suppressor APC that presents autoantigen and uses adenosine to activate an antigen-specific CD4(+) Tregs through the A2Ar. These cells are highly effective in suppressing uveitis, and they appear to be inducible Tregs. In this study, we determined whether they are inducible or natural Tregs and identified the dependent mechanism for the function of these post-EAU Tregs. The post-EAU spleen CD25(+)CD4(+) T cells were sorted for NRP-1 expression and transferred to recipient mice immunized for EAU. The sorted NRP-1(-), but not the NRP-1(+), Tregs suppressed EAU. These NRP-1(-) Tregs coexpress PD-1 and PD-L1. Treatment of naive APCs with α-MSH promoted a regulatory APC that induced CD25(+) CD4(+) Tregs in a CD73-dependent manner. These Tregs were PD-L1(+) PD-1(+) NRP-1(-) FOXP3(+) HELIOS(-) and suppressed EAU when transferred to recipient mice. In contrast, PD-1(-) T cells did not suppress EAU, indicating that PD-1 is necessary for the suppressive activity of iTregs. Moreover, these Tregs did not suppress effector T cells when the PD/-1/PD-L1 pathway was blocked. These results demonstrate that post-EAU Tregs are inducible Tregs, which use a PD-1/PD-L1 mechanism to suppress disease., (© Society for Leukocyte Biology.)

The evolutionarily conserved TGF-beta proteins are distributed ubiquitously throughout the body and have a role in almost every biological process. In immunity, TGF-beta has an important role in modulating immunity. Much is understood about the process of TGF-beta production as a latent molecule and of the consequences and the intercellular signaling of active TGF-beta binding to its receptors; however, there is little discussed between the production and activation of TGF-beta. This review focuses on what is understood about the biochemical and physiological processes of TGF-beta activation and identifies the gaps in understanding immune cell activation of TGF-beta. A mechanistic understanding of the process activating TGF-beta can lead to regulating multiple biological systems by enhancing or inhibiting TGF-beta activation.

The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) is a powerful suppressor of inflammation mediated by macrophages, which express at least two receptors, melanocortin 1 and 3 receptors (MC1r and MC3r) that bind alpha-MSH. Albeit, the anti-inflammatory activity of alpha-MSH has been well documented in macrophages, the mechanisms of alpha-MSH activity in macrophages are not clearly understood. This study is to investigate which of the MCr expressed on macrophages is associated with the immunosuppressive activities of alpha-MSH on LPS-stimulated macrophages. To address this question, we transfected RAW264.7 macrophage cells with MC1r small interfering (si)RNA, which specifically targets mouse MC1r mRNA. The diminution of MC1r mRNA expression was 82% at 24 h and 67% at 48 h after transfection. There was a significant loss in alpha-MSH suppression of NO generation and TNF-alpha production by MC1r siRNA-transfected macrophages stimulated with LPS. There was an equally diminished alpha-MSH suppression of LPS-stimulated intracellular activation of NF-kappaB and p38 phosphorylation. In addition, the diminishment of MC1r expression by siRNA transfection had no influence on MC3r expression and function in the macrophages. These findings demonstrate that alpha-MSH suppression of LPS-induced inflammatory activity in macrophages requires expression of MC1r. The results imply that although all of the MCr are G-coupled proteins, they may not necessarily function through the same intracellular pathways in macrophages.

T regulatory (Treg) cells have been studied for more than 30 years. Recently, changing technology and attitudes have led to new interest in T cell regulation of the immune responses. The eye is an immune-privileged site with unique mechanisms for the prevention of damaging immune inflammation. The eye fashions its Treg cells in novel ways to prevent immune inflammation locally and systemically. The purpose of this mini-review is to condense and summarize reports of Treg cells dependent on the eye in the context of the Treg literature in general.

The immune-privileged eye has the potential to induce regulatory immunity along with local mechanisms of immunosuppression. Rodent models of human autoimmune uveoretinitis [experimental autoimmune uveoretinitis (EAU)] recover without spontaneous recurrence of uveitis, which differs from uveitis in some humans. This raises the possibility that the mechanism of immune privilege in the rodent eye can reimpose itself during autoimmune uveoretinitis and re-establish tolerance to autoantigen. To investigate this possibility, we examined the spleens of EAU-recovered mice for regulatory immunity. We detected regulatory immunity when we adoptively transferred post-EAU spleen cells into other mice immunized for EAU. We could not detect this regulatory immunity in enucleated mice nor in naive mice. Moreover, unlike the mechanisms of anterior chamber-associated immune deviation, the suppression was only mediated by post-EAU CD4+ T cells, which required activation with autoantigen presented by post-EAU spleen antigen-presenting cells (APC). Our results demonstrate that when the immune-privileged ocular microenvironment recovers from an autoimmune response, it has influenced systemic immunity to retinal autoantigen by affecting APC and mediating induction of potential regulatory CD4+ T cells laying in wait in the post-EAU spleen for restimulation.

To evaluate the potential role of NK1.1 (CD161c) cells in autoimmune uveoretinitis, we treated experimental autoimmune uveoretinitis (EAU)-susceptible mice with anti-CD161c antibodies (PK136) to deplete natural killer (NK) cells. Injection of anti-CD161c antibodies deleted NK cells from the peripheral blood of EAU-susceptible mice. The T cell proliferative response against the ocular autoantigen K2 was not suppressed in mice treated with anti-CD161c antibody when compared with T cells from control mice. Although mice treated with anti-CD161c developed EAU, the clinical severity on days 17 and 19 after induction of EAU was significantly mild in anti-CD161c-treated mice compared with control mice. In addition, the histopathological severity of EAU was significantly milder in mice treated with anti-CD161c antibodies than controls 21 days after induction of EAU. Our results indicate that the severity of EAU is augmented by NK1.1(+) NK cells.

Recently, we have reported that the cytokines alpha-melanocyte-stimulating hormone (alpha-MSH) and transforming growth factor-beta2 (TGF-beta2) work in synergy to induce the activation of regulatory T (Treg) cells. When we used alpha-MSH and TGF-beta2 to generate ocular autoantigen-specific Treg cells and adoptively transferred them into mice susceptible to experimental autoimmune uveoretinitis (EAU), there was suppression in the incidence and severity of EAU. Specificity to a retinal autoantigen was required for the Treg cells to suppress EAU. When stimulated, these Treg cells produced TGF-beta1, and their production of interferon-gamma, interleukin (IL)-10, and IL-4 was suppressed. Also, the Treg cells are suppressed in their proliferative response. Our results demonstrate that alpha-MSH with TGF-beta2 induce Treg cells that can subdue a tissue-specific autoimmune response. This also promotes the possibility of using these immunomodulating cytokines to purposely induce antigen-specific Treg cells to prevent and suppress autoimmune disease.