Pleiotropic consequences of metabolic stress for the major histocompatibility complex class II molecule antigen processing and presentation machinery.

Hyperglycemia and hyperlipidemia are often observed in individuals with type II diabetes (T2D) and related mouse models. One dysmetabolic biochemical consequence is the non-enzymatic reaction between sugars, lipids, and proteins, favoring protein glycation, glycoxidation, and lipoxidation. Here, we identified oxidative alterations in key components of the major histocompatibility complex (MHC) class II molecule antigen processing and presentation machinery in vivo under conditions of hyperglycemia-induced metabolic stress. These modifications were linked to epitope-specific changes in endosomal processing efficiency, MHC class II-peptide binding, and DM editing activity. Moreover, we observed some quantitative and qualitative changes in the MHC class II immunopeptidome of Ob/Ob mice on a high-fat diet compared with controls, including changes in the presentation of an apolipoprotein B100 peptide associated previously with T2D and metabolic syndrome-related clinical complications. These findings highlight a link between glycation reactions and altered MHC class II antigen presentation that may contribute to T2D complications.

Isoflavone-mediated radioprotection involves regulation of early endothelial cell death and inflammatory signaling in Radiation-Induced lung injury.

Purpose: Vascular damage and inflammation are limiting toxic effects of lung cancer radiotherapy, which lead to pneumonitis and pulmonary fibrosis. We have demonstrated that soy isoflavones (SIF) mitigate these toxic effects at late time points after radiation. However, the process by which SIF impacts the onset of radiation-induced inflammation remains to be elucidated. We have now investigated early events of radiation-induced inflammation and identified cellular and molecular signaling patterns by endothelial cells that could be modified by SIF to control vascular damage and the initiation of lung inflammation.Materials and methods: Histopathological, cellular and molecular studies were performed on mouse lungs from C57Bl/6 mice treated with 10 Gy of thoracic radiation (XRT) in conjunction with daily oral SIF treatment given prior and after radiation. Parallel studies were performed in-vitro using EA.hy926 endothelial cell line with SIF and radiation. Immunohistochemistry, western blots analysis, and flow cytometry were performed on lung tissue or EA.hy926 cells to analyze endothelial cells, their patterns of cell death or survival, and signaling molecules involved in inflammatory events.Results: Histopathological differences in inflammatory infiltrates and vascular injury in lungs, including vascular endothelial cells, were observed with SIF treatment at early time points post-XRT. XRT-induced expression of proinflammatory adhesion molecule ICAM-1 cells was reduced by SIF in-vitro and in-vivo in endothelial cells. Molecular changes in endothelial cells with SIF treatment in conjunction with XRT included increased DNA damage, reduced cell viability and cyclin B1, and inhibition of nuclear translocation of NF-κB. Analysis of cell death showed that SIF treatment promoted apoptotic endothelial cell death and decreased XRT-induced type III cell death. In-vitro molecular studies indicated that SIF + XRT increased apoptotic caspase-9 activation and production of IFNß while reducing the release of inflammatory HMGB-1 and IL-1α, the cleavage of pyroptotic gasdermin D, and the release of active IL-1ß, which are all events associated with type III cell death.Conclusions: SIF + XRT caused changes in patterns of endothelial cell death and survival, proinflammatory molecule release, and adhesion molecule expression at early time points post-XRT associated with early reduction of immune cell recruitment. These findings suggest that SIF could mediate its radioprotective effects in irradiated lungs by limiting excessive immune cell homing via vascular endothelium into damaged lung tissue and curtailing the overall inflammatory response to radiation.

T-Cells Specific for a Self-Peptide of ApoB-100 Exacerbate Aortic Atheroma in Murine Atherosclerosis.

On the basis of mouse I-Ab-binding motifs, two sequences of the murine apolipoprotein B-100 (mApoB-100), mApoB-1003501-3515 (designated P3) and mApoB-100978-992 (designated P6), were found to be immunogenic. In this report, we show that P6 is also atherogenic. Immunization of Apoe-/- mice fed a high-fat diet (HFD) with P6 resulted in enhanced development of aortic atheroma as compared to control mice immunized with an irrelevant peptide MOG35-55 or with complete Freund's adjuvant alone. Adoptive transfer of lymph node cells from P6-immunized donor mice to recipients fed an HFD caused exacerbated aortic atheromas, correlating P6-primed cells with disease development. Finally, P6-specific T cell clones were generated and adoptive transfer of T cell clones into recipients fed an HFD led to significant increase in aortic plaque coverage when compared to control animals receiving a MOG35-55-specific T cell line. Recipient mice not fed an HFD, however, did not exhibit such enhancement, indicating that an inflammatory environment facilitated the atherogenic activity of P6-specific T cells. That P6 is identical to or cross-reacts with a naturally processed peptide of ApoB-100 is evidenced by the ability of P6 to stimulate the proliferation of T cells in the lymph node of mice primed by full-length human ApoB-100. By identifying an atherogenic T cell epitope of ApoB-100 and establishing specific T cell clones, our studies open up new and hitherto unavailable avenues to study the nature of atherogenic T cells and their functions in the atherosclerotic disease process.

Emerging role of IL-16 in cytokine-mediated regulation of multiple sclerosis.

Cytokines are pleiotropic soluble mediators of cellular functions. Cytokines are critical in immune pathogenesis of human diseases, including autoimmune CD4(+) T cell mediated chronic inflammatory, demyelinating and neurodegenerative diseases of the central nervous system (CNS), multiple sclerosis (MS). In MS and its experimental model, experimental autoimmune encephalomyelitis (EAE), chronic persistence and/or reoccurrence of inflammation in the CNS causes chronic progressive or relapsing disease, accompanied with demyelination and damage to axons and oligodendrocytes, which ultimately leads to paralysis and disability. As opposed to other cytokines, whose effects are not limited to the CD4(+) T cell subset, IL-16 exerts its biological properties by exclusive binding and signaling through CD4 receptor. IL-16 selectively regulates migration of all CD4 expressing T cells regardless of their activation state, which is of critical importance for immune modulation and potential therapy of MS. Other major biological properties of IL-16 essential for the function of CD4(+) T cells include regulation of: T cell activation, CD25 expression, MHC class II expression, dendritic cell (DC)-T cell cooperation, B cell-T cell and T cell-T cell cooperation, inflammatory cytokine production and modulation of chemokine regulated T cell chemo-attraction. In this article we outline immune pathogenesis of the disease necessary to understand significance of cytokines and IL-16 in MS regulation. We revisit cytokine regulation with emphasis on involvement of IL-16 mechanisms, implicated in MS progression and important for development of new therapies. We emphasize the significance of similar IL-16 mechanisms for other chronic inflammatory CNS diseases.

Overcoming unresponsiveness in experimental autoimmune encephalomyelitis (EAE) resistant mouse strains by adoptive transfer and antigenic challenge.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2(b)) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated. The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 µg/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 µg/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Lessons learned from studies of natural resistance in murine experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a commonly-used animal model of the human demyelinating disease, multiple sclerosis (MS). Similar to MS, EAE is under genetic control in that certain mouse strains are susceptible to disease induction with myelin antigens, while other strains are resistant. In the past, major efforts studying EAE tended to focus on the mechanism of disease susceptibility pertaining to antigen specificities, disease progression and related cytokines. The basis of EAE resistance, on the other hand, had received relatively little attention. It is our contention that EAE resistance is a tightly regulated process and many lessons can be learned from studying its mechanisms. Initially, this laboratory showed that resistance to EAE induced by MBP in B6 mice and many other strains with different H-2 haplotypes could be reversed in an adoptive transfer system by challenging the recipients with MBP-CFA. The disease developed in these mice was very similar to that induced in EAE susceptible mouse strains without the antigenic challenge. This approach of reversing EAE resistance was confirmed by several other laboratories. It was also demonstrated definitively that EAE was mediated by the donor T cells and not by host T cells. Indeed, a "resistant" host environment did not affect the outcome of disease development. The antigenic challenge appeared to induce an anamnestic response in the donor T cells, as the antigen dose used could be as low as only 5µg per mouse. Significantly, the period between adoptive cell transfer and antigenic challenge could be as long as over one year, again indicating that the donor cells persisted in the host for a long period of time. Recently, it has been suggested that EAE resistance can be due to the activities of regulatory T cells (Tregs). Depletion of Tregs with anti-CD25 antibodies prior to immunization with PLP139-151 rendered 30% of resistant B10.S mice to develop EAE. These results were confirmed in SJL.B mice responding to MBP but not in B6 mice responding to the same antigen, suggesting that regulation might vary among EAE resistant mouse strains. In addition, it is noted that while B6 and SJL.B mice are resistant to EAE induction with MBP, these mice are susceptible to disease induction when immunized with MOG, suggesting that EAE susceptibility verses resistance is antigen dependent. This unique mouse model, coupled with advance technologies such as peptide/IA tetramers and microarrays, should provide a powerful tool for further elucidation of the basic mechanisms of EAE resistance.

Differential levels of resistance to disease induction and development of relapsing experimental autoimmune encephalomyelitis in two H-2b-restricted mouse strains.

Besides the major histocompatibility complex (MHC) genes, background genes are believed to influence the encephalitogenicity of SJL(H-2(s)) and B10.S (H-2(s)) mice responding to myelin basic protein (MBP). A new mouse strain was constructed to study the effects of the SJL genetic background in mice responding to H-2(b)-restricted neuroantigens. Although the SJL.B (H-2(b)) mouse remained resistant to MBP in active EAE induction, the disease severity was uniformly higher in MOG-induced active EAE and in MBP-induced adoptive EAE when compared to those of B6 (H-2(b)) mice. Treatment of mice with anti-CD25 antibodies prior to immunization caused 60% of SJL.B mice to become susceptible to MBP-induced EAE while only 14% of B6 mice were converted. In addition, MOG-induced EAE in SJL.B mice followed a remitting-relapsing disease course while B6 mice only exhibited monophasic or chronic episodes. The new SJL.B mouse strain provides a valuable tool for studying EAE resistance and remitting-relapsing disease in H-2(b) mice.

T cells that trigger acute experimental autoimmune encephalomyelitis also mediate subsequent disease relapses and predominantly produce IL-17.

Earlier studies showed that donor T cells that initiated a murine adoptive EAE persisted in the CNS of the recipients throughout the subsequent relapsing cycles. To clarify the functions of the persistent donor T cells in EAE relapsing disease, anti-Thy-1 antibodies were used to deplete these cells. Results showed that such treatment abrogated subsequent relapsing cycles in these animals. In addition, it was evident that a shift in cytokine profile occurred during acute and relapsing disease phases. These results unambiguously support the appropriateness of targeting T cells with specificity for the priming antigen in design of therapeutic approaches for MS.

High cell surface expression of CD4 allows distinction of CD4(+)CD25(+) antigen-specific effector T cells from CD4(+)CD25(+) regulatory T cells in murine experimental autoimmune encephalomyelitis.

Analysis of T regulatory cells (Treg) and T effector cells (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. We demonstrate that encephalitogenic T cells, following antigen recognition, up-regulate cell surface expression of CD4. The CD4(high) sub-population contains all of the antigen response as shown by proliferation and cytokine secretion, and only these cells are capable of transferring EAE to naive animals. On the other hand, a FACS separable CD25(+) sub-population of cells displayed consistent levels of CD4 prior to and after antigen stimulation. These cells displayed characteristics of Treg, such as expressing high levels of the Foxp3 gene and the ability to suppress mitogenic T cell responses.

Adoptive transfer of myelin basic protein-induced experimental autoimmune encephalomyelitis between SJL and B10.S mice: correlation of priming milieus with susceptibility and resistance phenotypes.

To study the mechanisms of EAE resistance, we directly transfer MBP-primed EAE-susceptible SJL lymph node cells into EAE-resistant B10.S recipients and vice versa. These transfers were unsuccessful because of strong alloreactivity between the two strains. Neonatal tolerance to SJL antigens was induced in B10.S mice and in these hosts MBP-primed SJL lymph node cells readily induce development of adoptive EAE. Conversely, transfer of MBP-primed B10.S lymph node cells into EAE-susceptible (SJL x B10.S)F1 recipients failed to induce EAE. These results are consistent with the notion that the priming milieus in the donor mice affect the expression of susceptible and resistant phenotypes.

Distinct immune regulation of the response to H-2b restricted epitope of MOG causes relapsing-remitting EAE in H-2b/s mice.

To find immune mechanisms underlying relapse regulation, we developed a model of relapsing-remitting experimental autoimmune encephalomyelitis (EAE) in (B6xSJL) F1 (H-2(b/s)) mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG(35-55)) and compared with low/non-relapsing B6 (H-2(b)) mice. In relapsing H-2(b/s) mice, inflammatory lesions scattered throughout the white matter with extensive demyelination, consisted of CD4(+) T and B220(+) B cells with fewer Mac3(+) macrophages. Memory T cell proliferation to MOG(35-55) was significantly enhanced. Switch of macrophage chemoattractant protein-1 (MCP-1) production from GFAP(+) astrocytes to CD3(+) T cells was observed. Distinct patterns of inflammation and demyelination, MOG(35-55) memory T cell response and regulation of MCP-1 are associated with relapsing H-2(b/s) phenotype.