OGR1/GPR68 Modulates the Severity of Experimental Autoimmune Encephalomyelitis and Regulates Nitric Oxide Production by Macrophages.

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a proton-sensing molecule that can detect decreases in extracellular pH that occur during inflammation. Although OGR1 has been shown to have pro-inflammatory functions in various diseases, its role in autoimmunity has not been examined. We therefore sought to determine whether OGR1 has a role in the development of T cell autoimmunity by contrasting the development of experimental autoimmune encephalomyelitis between wild type and OGR1-knockout mice. OGR1-knockout mice showed a drastically attenuated clinical course of disease that was associated with a profound reduction in the expansion of myelin oligodendrocyte glycoprotein 35-55-reactive T helper 1 (Th1) and Th17 cells in the periphery and a reduced accumulation of Th1 and Th17 effectors in the central nervous system. We determined that these impaired T cell responses in OGR1-knockout mice associated with a reduced frequency and number of dendritic cells in draining lymph nodes during EAE and a higher production of nitric oxide by macrophages. Our studies suggest that OGR1 plays a key role in regulating T cell responses during autoimmunity.

Cell-extrinsic CTLA4-mediated regulation of dendritic cell maturation depends on STAT3.

Regulatory T (Treg) cells suppress immune responses by downregulating the expression of costimulatory molecules CD80 and CD86 on dendritic cells (DCs) through cytotoxic T lymphocyte antigen 4 (CTLA4). However, it is unclear whether inducible Treg (iTreg) cells can hamper immune responses via the same mechanism. Moreover, whether a reverse signal sent by CTLA4 alone is sufficient to prevent maturation of DCs has never been evaluated. Here, we demonstrate that stimulation of DCs with CTLA4, either expressed by inducible Treg cells or by cross-linking with CTLA4Fc fusion protein, can significantly inhibit LPS-induced CD80 and CD86 mRNA and protein expression in both mouse and human DCs. Importantly, CTLA4Fc-treated DCs had reduced ability to stimulate CD4(+) and CD8(+) T-cell proliferation and cytokine production in both syngeneic and allogeneic settings. We also investigated the molecular mechanism involved in the induction of tolerogenic DCs by CTLA4. We determined that the interaction of CTLA4 with its high affinity ligand CD80 on DCs induces STAT3 phosphorylation followed by reduction of NF-κB activity, leading to suppression of CD80 and CD86 gene transcription and protein production. Our work opens new windows for the generation of tolerogenic DCs that could ultimately be used for treating autoimmune diseases and transplant rejection.

Pretransplant infusion of donor B cells enhances donor-specific skin allograft survival.

Pretransplant donor lymphocyte infusion (DLI) has been shown to enhance donor-specific allograft survival in rodents, primates and humans. However, the cell subset that is critical for the DLI effect and the mechanisms involved remain elusive. In this study, we monitored donor cell subsets after DLI in a murine MHC class I L(d)-mismatched skin transplantation model. We found that donor B cells, but not DCs, are the major surviving donor APCs in recipients following DLI. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Furthermore, mice that had received donor B cells showed higher expression of Ly6A and CD62L on antigen-specific TCRαß(+)CD3(+)CD4(-)CD8(-)NK1.1(-) double negative (DN) regulatory T cells (Tregs). B cells presented alloantigen to DN Tregs and primed their proliferation in an antigen-specific fashion. Importantly, DN Tregs, activated by donor B cells, showed increased cytotoxicity toward anti-donor CD8(+) T cells. These data demonstrate that donor B cells can enhance skin allograft survival, at least partially, by increasing recipient DN Treg-mediated killing of anti-donor CD8(+) T cells. These findings provide novel insights into the mechanisms underlying DLI-induced transplant tolerance and suggest that DN Tregs have great potential as an antigen-specific immune therapy to enhance allograft survival.

Trogocytosis of CD80 and CD86 by induced regulatory T cells.

Trogocytosis is a process which involves the transfer of membrane fragments and cell surface proteins between cells. Various types of T cells have been shown to be able to acquire membrane-bound proteins from antigen-presenting cells and their functions can be modulated following trogocytosis. However, it is not known whether induced regulatory T cells (iTregs) can undergo trogocytosis, and if so, what the functional consequences of this process might entail. In this study, we show that iTregs can be generated from CD80(-/-)CD86(-/-) double knockout (DKO) mice. Using flow cytometry and confocal fluorescence microscopy, we demonstrate that iTregs generated from DKO mice are able to acquire both CD80 and CD86 from mature dendritic cells (mDCs) and that the acquisition of CD86 occurs to a higher extent than that of CD80. Furthermore, we found that after co-incubation with iTregs, dendritic cells (DCs) downregulate their surface expression of CD80 and CD86. The trogocytosis of both CD80 and CD86 occurs in a cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), CD28 and programmed death ligand-1 (PDL1)-independent manner. Importantly, we showed that iTregs that acquired CD86 from mDCs expressed higher activation markers and their ability to suppress naive CD4(+) T-cell proliferation was enhanced, compared to iTregs that did not acquire CD86. These data demonstrate, for the first time, that iTregs can acquire CD80 and CD86 from mDCs, and the acquisition of CD86 may enhance their suppressive function. These findings provide novel understanding of the interaction between iTregs and DCs, suggesting that trogocytosis may play a significant role in iTreg-mediated immune suppression.

Reciprocal effects of alpha-synuclein overexpression and proteasome inhibition in neuronal cells and tissue.

Defects in the 20S/26S proteasome and conformational changes in alpha-synuclein (alpha-syn) are implicated in the development of sporadic and familial cases of PD. The objective of this study was to evaluate whether alpha-syn affects proteolysis by the proteasome and, reciprocally, whether proteasome inhibition affects alpha-syn solubility and localization. Although alpha-syn directly inhibited purified 20S proteasomes reversibly in vitro, its overexpression in neuroblastoma (SH-SY5Y and SK-N-BE), embryonic kidney (HEK293) cells, or mouse brain did not affect proteasome activity. Proteasome inhibition with MG132 and epoxomicin in SH-SY5Y cells failed to induce alpha-syn aggregation, although it increased membrane bound forms of endogenous and overexpressed wild-type, but not mutant, alpha-syn. Concomitantly this treatment generated cytoplasmic alpha-syn inclusions devoid of polyubiquitin in a small percentage of cells. The combination of proteasome inhibition with serum deprivation, which induced oxidative stress and autophagy, caused the appearance of high molecular weight alpha-syn species, such as those found in Lewy bodies. Our data suggest that high concentrations of alpha-syn do not affect proteasome function in vivo, whereas proteasome inhibition can modify synuclein solubility, most prominently under conditions of cell stress which occur during aging. These results have implications for the convergence of age-related oxidative stress and impaired protein degradation in neurodegeneration.

Differences in susceptibility of MBP charge isomers to digestion by stromelysin-1 (MMP-3) and release of an immunodominant epitope.

Charge microheterogeneity of myelin basic protein is known to affect its conformation and function. Here, the citrullinated myelin basic protein charge isomer, component-8, was shown to be more susceptible to stromelysin-1 cleavage than myelin basic protein component-1. Since levels of component-8 are increased in multiple sclerosis brain, the increased susceptibility of component-8 to proteolytic digestion may play a role in the pathogenesis of multiple sclerosis. Interestingly, component-1 isolated from multiple sclerosis patients was digested at a faster rate by stromelysin-1 than component-1 isolated from normal individuals. The reason for this difference is not clear, but likely reflects conformational differences between the two proteins as a result of post-translational modifications. Stromelysin-1 was able to cleave myelin basic protein in the presence of lipids and within the context of myelin and released several peptides including peptides containing the immunodominant epitope.

Autocatalytic cleavage of myelin basic protein: an alternative to molecular mimicry.

Although multiple sclerosis (MS) is thought to be an autoimmune disease, the mechanisms by which immunodominant epitopes are generated and lymphocytes are activated are not known. Here, myelin basic protein-component 1 (MBP-C1) from MS tissue was shown to undergo autocatalytic cleavage at slightly alkaline pH. Importantly, one of the major peptides released contained the immunodominant epitope 84-89. Interestingly, MBP isolated from MS patients showed a faster time course of cleavage and a more robust release of epitope 84-89 than MBP isolated from normal individuals. The cleavage reaction was not inhibited by protease inhibitors, except for phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor. Since PMSF inhibition suggested a role for a serine residue in the cleavage, we labeled myelin basic protein with diisopropyl fluorophosphate (DFP), known to bind active site serine residues. Mass spectrometry was used to identify the labeled peptide, which consisted of residues 140-152. Since this peptide contained a single serine residue, we concluded it to be the active serine. The importance of this cleavage mechanism is that it provides for a ready source of the immunodominant peptide for sensitization of T-cells. It is not necessary to invoke other mechanisms such as molecular mimicry.

The up-regulation of stromelysin-1 (MMP-3) in a spontaneously demyelinating transgenic mouse precedes onset of disease.

The matrix metalloproteinases (MMPs) are a family of endoproteinases that degrade various components of the extracellular matrix and have been implicated in the pathogenesis of multiple sclerosis. To determine whether up-regulation of MMP-3, or stromelysin-1, was a causative factor during the development of demyelination, we have examined the expression of MMP-3 mRNA and protein in brain tissue of a spontaneously demyelinating mouse model overexpressing DM20 (ND4 line) prior to and during the progression of disease. Stromelysin-1, but not other MMP mRNA was elevated approximately 10-fold in transgenic mice between 5 days and 1 month of age, more than 2 months before the onset of disease, and was coordinately expressed with the DM20 transgene. Stromelysin-1 protein levels were also up-regulated as was tissue inhibitor of metalloproteinase-1 (TIMP-1), an in vivo regulator of stromelysin-1 mRNA. When we crossed our ND4 mice with a line of transgenic mice overexpressing TIMP-1 in brain, clinical signs in these mice were attenuated, and the level of stromelysin-1 protein was reduced. Thus, in this transgenic model of demyelinating disease up-regulation of DM20, MMP-3, and TIMP-1 represent important changes in the chemical pathogenesis in brain, which precede the onset of disease.