An intracameral injection of antigen induces in situ chemokines and cytokines required for the generation of circulating immunoregulatory monocytes.

Anterior Chamber-Associated Immune Deviation (ACAID) induced by an intracameral injection of antigen generates antigen-specific regulatory splenic T cells that suppress specifically cell-mediated immunity specific for the injected antigen. Circulating F4/80(+) cells recovered from mice receiving an intracameral injection of antigen are thought to be ocular in origin and induce the development of thymic and splenic regulatory T cells. We have shown previously that after the intracameral injection of antigen there is a CCR2/CCL2-dependent infiltration of circulating F4/80(+) cells into the anterior chamber associated with the generation of circulating, ACAID-inducing F4/80(+) monocytes. Here we tested the hypothesis that the intracameral injection of antigen induces events in the anterior chamber that are associated with the induction of circulating immunoregulatory monocytes that induce the suppression of cell-mediated immunity. The intracameral injection of antigen resulted in aqueous humor (i) a time- dependent increase of CCL2 and CCL7, (ii) a transient increase in TNF-α, and (iii) an infiltration of CD11b(hi), Gr1(hi) and F4/80(+) as well as F4/80(-) and Gr1(hi) peripheral blood cells into the anterior chamber. Further characterization of these F4/80(+) cells revealed that they are Ly 6C(hi), LY6G(lo) or negative, 7/4 (LY6B)(hi), CD115(+), CD45(+), CD49B(+), and CD62 L(+). Antibody-mediated neutralization of TGF-ß in situ in the anterior chamber prevented the induction of circulating, ACAID-inducing monocytes and ACAID. These cells did not increase in the irides of ACAID-refractory CCR2-/- and CCL2-/- mice that received an intracameral injection of antigen. Our results extend our suggestion that ACAID is initiated as the result of a mild proinflammatory response to intracameral injection that results in the infiltration of a CCR2(+) subset of monocytes into the anterior chamber where there is a TGF-ß-dependent induction of an immunosuppressive phenotype in the infiltrated monocytes that recirculate to induce antigen-specific regulatory T cells.

Antigen-specific splenic CD4+ and CD8+ regulatory T cells generated via the eye, suppress experimental autoimmune encephalomyelitis either at the priming or at the effector phase.

The injection of antigen into the ocular anterior chamber (AC) induces the generation of splenic CD4(+) and CD8(+) regulatory T (Treg) cells, specific for the antigen injected into the AC. These Treg cells inhibit the induction (CD4(+)) and also the expression (CD8(+)) of a delayed-type hypersensitivity response. The ability of AC-induced self-antigen-specific Treg cells in modulating autoimmunity is not well defined. Here we show that an injection of encephalitogenic myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide into the anterior chamber of the eye (AC-MOG), before the induction of or during established experimental autoimmune encephalomyelitis (EAE) induced by MOG(35-55), suppresses the induction or progression of EAE, respectively. CD4(+) or CD8(+) splenic Treg cells induced by an injection of AC-MOG prevent EAE either at the inductive (priming) or at the progressive (effector) phase, respectively. This suppression of EAE by an AC-MOG injection or by intravenous transfer of splenic regulatory cells induced by an AC-MOG injection is specific for the antigen injected into the AC. Additionally, our data suggest that splenic CD8(+) Treg cells that suppress active EAE may use a transforming growth factor (TGF)-ß-dependent suppression mechanism while the suppression of the induction of EAE by the AC-induced CD4(+) Treg cells is independent of TGF-ß. Thus, we show for the first time that regulation of EAE at the priming or the chronic phase requires different phenotypes of Treg cells. Hence, it is important to consider the phenotype of Treg cells while designing effective cell-based therapies against autoimmune disorders.

Paradoxical effect of pertussis toxin on the delayed hypersensitivity response to autoantigens in mice.

BACKGROUND: Pertussis toxin (PTX), an exotoxin of Bordetella pertussis, enhances the development of experimental autoimmune diseases such as experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE) in rodent models. The mechanisms of the promotion of experimental autoimmune diseases by PTX may be based upon PTX-induced disruption of the blood eye/brain barriers facilitating the infiltration of inflammatory cells, the modulation of inflammatory cell migration and the enhancement of the activation of inflammatory cells. We hypothesized that the facilitation of experimental autoimmunity by PTX suggests that its influence on the in vivo immune response to auto-antigen may differ from its influence on non-self antigens. METHODOLOGY/PRINCIPAL FINDINGS: We have evaluated the effect of PTX on the simultaneous generation of delayed type hypersensitivity (DTH) responses and autoimmune responses to uveitogenic interphotoreceptor retinoid binding protein peptide (IRBP161-180), encephalitogenic myelin oligodendrocyte glycoprotein peptide (MOG35-55) or ovalbumin (OVA). PTX injection of mice immunized to IRBP peptide161-180 led to (i) the development of EAU as shown by histopathology of the retina, (ii) pro-inflammatory cytokine production by splenocytes in response to IRBP peptide161-180, and (iii) symptomatic EAE in mice immunized with encephalitogenic MOG peptide35-55. However, mice that received PTX had a reduced DTH response to IRBP161-180 peptide or MOG peptide35-55 when challenged distal to the site affected by autoreactive T cells. Moreover, footpad challenge with MOG35-55 peptide reduced EAE in mice immunized with MOG peptide. In contrast, the use of PTX when immunizing with OVA protein or an OVA immunogenic peptide did not affect the DTH response to OVA. CONCLUSIONS/SIGNIFICANCE: The results suggest that that the reduced DTH response in mice receiving PTX may be specific for autoantigens and autoantigen-reactive T cells are diverted away from ectopic sites that received the autoantigen and towards the tissue site of the autoantigen.

Cbl-b(-/-) T cells demonstrate in vivo resistance to regulatory T cells but a context-dependent resistance to TGF-beta.

Cbl-b is an E3 ubiquitin ligase that negatively regulates T cell activation. Cbl-b(-/-) mice develop spontaneous autoimmunity, and Cbl-b dysregulation has been described in both murine and human autoimmune diseases. Although the mechanisms underlying the development of autoimmunity in Cbl-b(-/-) mice are not yet clear, we have reported that Cbl-b(-/-) CD4(+)CD25(-) effector T cells (Teffs) are resistant to CD4(+)CD25(+) regulatory T cell (Treg)-mediated suppression in vitro and have suggested that this may be an important mechanism in the development of autoimmunity. To confirm the relevance of this resistance to autoimmune disease, we now show that Cbl-b(-/-) Teffs are resistant to suppression by Tregs in vivo and that this involves a resistance of truly naive Cbl-b(-/-) Teffs. Additionally, we show that Cbl-b(-/-) Tregs are fully functional in vivo, further suggesting that the regulatory abnormalities in Cbl-b(-/-) mice are related to defects in Teff, not Treg, function. To characterize the relevance of TGF-beta sensitivity in Treg resistance, we examined in vivo Th17 generation and report that Cbl-b(-/-) mice are able to mount a normal Th17 response in vivo. As Cbl-b(-/-) Teffs have been shown to be insensitive to the suppressive effects of TGF-beta in other in vivo models, the present results suggest that Cbl-b(-/-) Teffs demonstrate a context-dependent sensitivity to TGF-beta in vivo. Overall, our results suggest that resistance to Tregs may be a bona fide mechanism underlying autoimmunity and that Cbl-b(-/-) mice offer unique approaches for studying the interrelationships between Treg function, TGF-beta-mediated responses, and the development of autoimmunity.

The Induction of Circulating, ACAID-Inducing Monocytes Requires CCR2/CCL2-Dependent Migration of Circulating F4/80(+) Cells into the Anterior Chamber.

To determine the origin of peripheral blood mononulclear cells (PBMC) that activate regulatory T cells in anterior chamber-associated immune deviation (ACAID), fluorescein-labeled PBMC were intravenously injected into mice before the mice received an intracameral injection of antigen. Six-24 hr after intracameral injection, fluorescein-labeled PBMC increased in the iris. Twenty-four-48 hr labeled cells decreased in the iris and increased in the thymus and spleen. The entry of the labeled PBMC into the anterior chamber and subsequent production of PBMC that transfer ACAID required the expression of CCR2 by the PBMC and the production of the chemokine CCL2 by the recipient of the PBMC. The results suggest that the intracameral injection of antigen induces i) the infiltration of F4/80(+) PBMC into the AC, ii) where these PBMC are converted to a regulatory phenotype, and iii) recirculate to activate T cells that suppress cell-mediated immunity.

The sympathetic nervous system modulates CD4(+)FoxP3(+) regulatory T cells via a TGF-beta-dependent mechanism.

CD4(+)FoxP3(+) Tregs are essential mediators of the peripheral immune response to self-antigens. Accordingly, the homeostatic regulation of Treg activity and number would impact on the immune response to both self- and non-self antigens. Because the sympathetic nervous system (SNS) interacts chemically and physically with the central and peripheral immune system and exerts a direct influence on antigen-presenting cells and effector lymphocytes, we have investigated the effect of chemical ablation of the SNS on the number and function of peripheral Treg. Removal of murine peripheral sympathetic innervation by 6-hydroxydopamine induced an increase in splenic and lymph node CD4(+)FoxP3(+) Tregs by a TGF-beta-dependent mechanism. Further, this increase in Tregs coincides with an inhibition of the induction of experimental autoimmune encephalomyelitis. Our results demonstrate that the SNS is an important contributor to the maintenance of peripheral Treg and TGF-beta acts as a bridge between the immune system and the nervous system. Neurological events mediated by the SNS, such as a stress response, may affect the number of T cells that regulate an immune response. Additionally, targeting Tregs via the SNS may be a novel approach to the prevention or treatment of autoimmune diseases.

Neuroprotection conferred by astrocytes is insufficient to protect animals from succumbing to Japanese encephalitis.

Astrocytes play a key role in regulating aspects of inflammation and in the homeostatic maintenance of the central nervous system (CNS). However, the role of astrocytes in viral encephalitis mediated inflammation is not well documented. As Japanese encephalitis virus (JEV) infection is localized to neurons and considering the importance of astrocytes in supporting neuronal survival and function, we have exploited an experimental model of Japanese encephalitis (JE) to better understand the role of astrocytes in JE. Suckling mice pups were inoculated with the virus and 2 and 4 days later we analyzed a panel of molecules characteristic of reactive astrogliosis. We show that JEV infection increases the expression of astrocyte-specific glial fibrillary acidic protein (GFAP), the glutamate aspartate transporter (GLAST), glutamate transporter-1 (GLT-1) and ceruloplasmin (CP). The transcript levels of growth factors produced predominantly by activated astrocytes such as nerve growth factor (NGF) and ciliary neurotrophin factor (CNTF) were elevated following JEV infection. The transcript level of brain-derived neurotrophic factor (BDNF) was also elevated following JEV infection. Both NGF and CNTF were capable of preventing ROS mediated neuronal death following in vitro JEV infection to a certain extent. Taken altogether, these data indicate that increased astrogliosis following JEV infection is accompanied by the enhanced ability of astrocytes to detoxify glutamate, inactivate free radical and produce neurotrophic factors that are involved in neuronal protection. However, this elevated physiological state of astrocyte is insufficient in conferring neuroprotection, as infected animals eventually succumb to infection. The response of astrocytes to JE can be amplified to modulate the adaptive response of brain to induce neuroprotection.

Induction of IP-10 (CXCL10) in astrocytes following Japanese encephalitis.

Chemokines and their receptors are important elements for the selective attraction and activation of various subsets of leukocytes. Interferon-gamma inducible protein (IP-10 or CXCL-10) is a potent chemoattractant and has been suggested to enhance the severity of virus infection and neuronal injury. In order to assess functional importance of this chemokine in viral encephalitis, we have exploited an experimental model of Japanese encephalitis. We report for the first time that in Japanese encephalitis, astrocytes are the predominant source of IP-10. A progressive increase in IP-10 induction following viral infection is concomitant with the increase in IFN-gamma a known inducer of IP-10. However, this increase in IFN-gamma level is not sufficient to confer protection as animals eventually succumb to the infection.