Type II Collagen-Specific B Cells Induce Immune Tolerance in Th1-Skewed, Th2-Skewed, and Arthritis-Prone Strains of Mice

Antigen-specific regulatory T cells play key immune suppressive roles in autoimmune disease models and regulate the peripheral tolerance achieved via anterior chamber-associated immune deviation (ACAID). Articular cartilage has type II collagen (CII), which is a potent autoantigen protein in arthritis. There has not been much research on the clinical importance of CII-associated diseases. Moreover, the capability of CII to induce immune tolerance has not been previously assessed. We reported that delivery of CII either directly into the eye or via intravenous injection of CII-specific ACAID antigen presenting cells (APCs) can induce ACAID. Here, we hypothesized that peripheral tolerance can be induced following adoptive transfer of in vitro generated CII-specific ACAID B cells to naive mice. Delayed hypersensitivity (DTH) assays were used to assess the suppressive ability of adoptively transferred B cells. Immune responses of ACAID B cell-injected mice were significantly suppressed following challenges with CII as compared to positive controls. This effect was replicated in three different strains of mice (C57BL/6, BALB/c, and DBA/1). Thus, CII-specific ACAID B cells were able to induce immune tolerance in Th1-skewed, Th2-skewed, and arthritis-prone mice. ACAID B cell-mediated tolerance induced by CII could have therapeutic implications for the treatment of CII-mediated autoimmune diseases

In vitro-induced cell-mediated immune deviation to encephalitogenic antigens

The injection of antigens into the Anterior Chamber (AC) of the eye induces Anterior Chamber Associated Immune Deviation (ACAID), which is a potent form of immune deviation that is largely attributed to the effect of TGF beta 2 in the aqueous humor on ocular antigen-presenting cells (APCs). ACAID antigen presentation via APCs and B cells leads to the generation of antigen-specific T regulatory cells. The encephalitogenic antigens Myelin oligodendrocyte glycoprotein (MOG) and Myelin basic protein (MBP) have an obvious clinical relevance. We hypothesized that the intravenous injection of in vitro-generated ACAID APCs or in vitro-generated ACAID B cells specific to the encephalitogenic antigens MOG(35-55)/MBP induces specific peripheral tolerance in recipient BALB/c mice. We examined the suppression of MOG(35-55)-specific/MBP-specific inflammatory responses using delayed-type hypersensitivity (DTH) assays and Local Adoptive Transfer (LAT) assays. Results indicated that MOG(35-55)-specific/MBP-specific tolerance was generated after the intravenous injections of MOG(35-55)-specific/MBP-specific ACAID APCs, MOG(35-55)-specific/MBP-specific ACAID B cells, and MOG(35-55)-specific/MBP-specific ACAID T regulatory cells. The specific immune deviation was in vitro-induced, cell-mediated, and specific to the encephalitogenic antigens MOG(35-55)/MBP. This in vitro-mediated approach for the generation of MOG(35-55)/MBP-specific tolerance opens up avenues for the application of ACAID as a tool for the therapy of Multiple Sclerosis, Schizophrenia, and other diseases

<i>In vivo</i>-generated spleen cells (as a result of CII injected in the AC) inhibit CII-induced DTH responses in BALB/c mice (LAT assay).

<p>A LAT assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048635#s2" target="_blank">materials and methods</a> section. Seven days after AC injection, mice were s.c. immunized with CII/CFA. On day 14, putative regulatory spleen cells were isolated and mixed with immune spleen cells (isolated from s.c. immunized mice) and 500 µg CII before injection into the left ear pinna of naïve mice. Positive control mice received immune and naïve spleen cells along with CII. Two negative controls were included; one received only naïve spleen cells along with CII and the other received only CII. The regulatory activity of spleen cells was confirmed by inhibition of ear swelling responses induced by immune spleen cells after 24 hr (panel ‘a’) and 48 hr (panel ‘b’).</p

<i>In vivo</i>-generated splenic CD8⁺ T cells (as a result of CII injected in the AC) inhibit CII-induced DTH responses in BALB/c mice (LAT assay).

<p>A LAT assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048635#s2" target="_blank">materials and methods</a> section. Seven days after AC injection, mice were s.c. immunized with CII/CFA. On day 14, putative regulatory splenic CD8⁺ T cells were isolated (CD8⁺ microBeads) and mixed with immune spleen cells (isolated from s.c. immunized mice) and 500 µg CII before injection into the left ear pinna of naïve mice. Positive control mice received immune and naïve spleen cells along with CII. Two negative controls were included; one received only naïve spleen cells along with CII and the other received only CII. The regulatory activity of splenic CD8⁺ T cells was confirmed by inhibition of ear swelling responses induced by immune spleen cells after 24 hr (panel ‘a’) and 48 hr (panel ‘b’).</p

AC injection of CII induced ACAID in BALB/c mice via the suppression of DTH

<p><b>(DTH assay).</b> A DTH assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048635#s2" target="_blank">materials and methods</a> section. To induce ACAID, mice received an AC injection of CII followed by immunization (s.c.) on day 7 with CII/CFA. On day 14, mice were challenged with CII (500 µg in 20 µl) intradermally in the left ear pinna, and 20 µl PBS alone was injected into the right ear pinna as an internal control. Induction of ACAID was confirmed by inhibition of ear-swelling responses after 24 hr (panel ‘a’) and 48 hr (panel ‘b’). The positive control mice were immunized s.c. with CII/CFA on day 7 and with CII on day 14 whereas the negative control mice only received the day 14 intradermal injection of CII.</p

Eye-mediated immune tolerance to Type II collagen in arthritis-prone strains of mice

Type II collagen (CII) is a cartilage structural protein that plays important roles in joint function, arthritis and ageing. In studying the ability of CII to induce eye-mediated specific immune tolerance, we have recently proven that CII is capable of inducing anterior chamber-associated immune deviation (ACAID) in Balb/c mice. Here, we study the ability of CII to induce eye-mediated immune tolerance in strains of mice that are prone to the induction of rheumatoid arthritis. Thus, we hypothesized that CII induces ACAID in DBA/1 mice and in C57BL/6 mice through the AC route (direct injection) or the intravenous route (adoptive transfer of in vitro-generated CII-specific ACAID macrophages or of CII-specific in vitro-generated T regulatory cells). Specific immune tolerance induction was assessed using both delayed-type hypersensitivity (DTH) and local adoptive transfer (LAT) assays. Results indicated the ability of CII to generate CII-specific ACAID-mediated immune tolerance in vivo and in vitro in both DBA/1 mice and C57BL/6 mice. These findings could be beneficial in studies of immune tolerance induction using CII

The in vivo and in vitro induction of anterior chamber associated immune deviation to myelin antigens in C57BL/6 mice

Introduction of antigens into the anterior chamber (AC) of the eye generates a specific systemic form of tolerance that is termed AC-associated immune deviation (ACAID). Experimental autoimmune encephalomyelitis (EAE) is an animal model of the human CNS demyelinating diseases, including multiple sclerosis (MS) and acute disseminated encephalomyelitis. We investigated whether the encephalitogenic antigens myelin oligodendrocyte glycoprotein (MOG35–55) or myelin basic protein (MBP) induce ACAID in the EAE-prone C57BL/6 mice. We hypothesized that injection of MOG35–55/MBP induces antigen-specific tolerance whether via the AC route, the adoptive transfer of in vitro-generated MOG35–55-specific/MBP-specific ACAID antigen presenting cells (APCs), or the adoptive transfer of MOG35–55-specific/MBP-specific ACAID T regulatory cells (Tregs). ACAID is characterized by the specific impairment of delayed-type hypersensitivity (DTH) responses. Thus, DTH assays were used to test for ACAID following the AC injection of MOG35–55/MBP, or the intravenous injection of MOG35–55-specific/MBP-specific ACAID APCs. The functional local adoptive transfer (LAT) assays were used to examine the putative regulatory functions of in vitro generated MOG35–55-specific/MBP-specific Tregs. This report is the first to demonstrate the in vivo and in vitro induction of MOG35–55-specific/MBP-specific ACAID-mediated tolerance in C57BL/6 mice. These findings highlight the need for novel immunotherapeutic strategies for MS and optic neuritis

Therapeutic Effect of Blocking CXCR2 on Neutrophil Recruitment and Dextran Sodium Sulfate-Induced Colitis

Dextran sodium sulfate (DSS)-induced colitis in mice is characterized by polymorphonuclear neutrophil (PMN) infiltration into the colonic mucosa and lumen. The mechanism by which this occurs is unclear. To begin to understand the mechanism, we determined the role of the PMN chemokine receptor, CXCR2, in DSS-induced colitis by using CXCR2(-/-) mice or by neutralizing CXCR2. DSS was administered through drinking water to CXCR2(-/-) and BALB/c mice for 5 days followed by regular water for 1 day. In the neutralization study, mice were injected with control serum or goat anti-CXCR2 antiserum. BALB/c mice receiving DSS and control serum-injected mice receiving DSS lost weight and showed considerable clinical illness. Histological observation revealed submucosal edema, PMN infiltration into the submucosa and mucosa, extensive crypt damage with abscesses, and ulceration. In contrast, both the CXCR2(-/-) and anti-CXCR2 antiserum-treated mice gained weight and had significantly lower symptom scores. Histology of these mice showed submucosal edema but relatively intact crypt architecture and very few ulcers. Significantly fewer PMNs were found in the mucosa in anti-CXCR2 antiserum compared with control serum-injected inflamed mice, but no significant difference in eosinophil infiltration was observed between the groups. Our experiments identify a role for CXCR2 in DSS-induced colitis and suggest that antagonizing CXCR2 provides some therapeutic efficacy, possibly by impeding PMN recruitment into the mucosa. Antagonizing CXCR2 may form the basis for therapeutic drugs directed at controlling colitis