Xiangle Sun

Depletion of CD8+ T Cells Exacerbates CD4+ Th Cell-Associated Inflammatory Lesions During Murine Mycoplasma Respiratory Disease

Mycoplasma infection is a leading cause of pneumonia worldwide and can lead to other respiratory complications. A component of mycoplasma respiratory diseases is immunopathologic, suggesting that lymphocyte activation is a key event in the progression of these chronic inflammatory diseases. The present study delineates the changes in T cell populations and their activation after mycoplasma infection and determines their association with the pathogenesis of murine Mycoplasma respiratory disease, due to Mycoplasma pulmonis infection. Increases in T cell population numbers in lungs and lower respiratory lymph nodes were associated with the development of mycoplasma respiratory disease. Although both pulmonary Th and CD8+ T cells increased after mycoplasma infection, there was a preferential expansion of Th cells. Mycoplasma-specific Th2 responses were dominant in lower respiratory lymph nodes, while Th1 responses predominated in spleen. However, both mycoplasma-specific Th1 and Th2 cytokine (IL-4 and IFN-γ) responses were present in the lungs, with Th1 cell activation as a major component of the pulmonary Th cell response. Although a smaller component of the T cell response, mycoplasma-specific CD8+ T cells were also a significant component of pulmonary lymphoid responses. In vivo depletion of CD8+ T cells resulted in dramatically more severe pulmonary disease, while depletion of CD4+ T cells reduced its severity, but there was no change in mycoplasma numbers in lungs after cell depletion. Thus, mycoplasma-specific Th1 and CD8+ T cell activation in the lung plays a critical regulatory role in development of immunopathologic reactions in Mycoplasma respiratory disease.

Co-expression of granulocyte-macrophage colony-stimulating factor with antigen enhances humoral and tumor immunity after DNA vaccination

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to enhance humoral and tumor immunity resulting from DNA immunization. The genes encoding GM-CSF and antigen were cloned onto the same plasmid backbone, but separate promoters drove expression of each gene. beta-Galactosidase was used as the model antigen to generate antibody responses while the human tumor antigen, MAGE-1, was used to monitor tumor resistance. Immunization with a DNA vaccine co-expressing GM-CSF and beta-gal resulted in higher antigen-specific IgG responses than immunization with antigen encoding plasmid alone or co-inoculated with GM-CSF expressing plasmid. Similarly, DNA vaccines expressing both MAGE-1 antigen and GM-CSF were more effective in protecting against B16-MAGE-1 melanoma. However, both GM-CSF co-expressing DNA vaccines and co-inoculation with plasmids encoding the cytokine or antigen enhanced the generation antigen-specific IFN-gamma and IL-6 responses. These results demonstrate that co-expressing both GM-CSF and antigen on a DNA vaccine enhances humoral and tumor immune responses.

Cytokine and Chemokine Transcription Profile during Mycoplasma pulmonis Infection in Susceptible and Resistant Strains of Mice: Macrophage Inflammatory Protein 1β (CCL4) and Monocyte Chemoattractant Protein 2 (CCL8) and Accumulation of CCR5+ Th Cells

ABSTRACT The progression of murine mycoplasma pneumonia is dependent on T cells and other immune cells. The role of cytokines in immunity are complex, and identifying the network of cytokines produced after infection of mice is essential in dissecting the key cytokine cascades involved mycoplasma disease pathogenesis. In the present study, mRNA expression of 143 different cytokines, chemokines, or receptors were evaluated in lung tissues from both susceptible (BALB/c and C3H/HeN) and resistant (C57BL/6) mice after Mycoplasma pulmonis infection. To accomplish this, membrane-based cDNA microarrays were used to monitor changes mRNA expression in lungs. There was a clear association with disease susceptibility and development of cytokine mRNA expression. In addition to proinflammatory cytokines, mRNA expression of an anti-inflammatory cytokine, interleukin-10, increased with disease severity, suggesting an attempt to moderate the severity of the inflammatory response. Furthermore, it is clear that an array of chemokines produced in susceptible mice could contribute to the recruitment and maintenance of inflammatory cells at the site of disease. In support of this, there was an increase in macrophage inflammatory protein 1β (MIP-1β; CCL4) and monocyte chemoattractant protein 2 (MCP-2; CCL8) mRNA levels from mycoplasma-infected mice and a corresponding accumulation of CD4+ Th cells expressing the MIP-1β/MCP-2 receptor, CCR5, in the lungs of mice. Furthermore, MIP-1β- and MCP-2-producing cells and CD4+ T cells were found to be in close association in pulmonary lesions. Thus, there was a significant cytokine response associated with disease pathogenesis, and these studies provide important leads and insights into ongoing cytokine- and chemokine-mediated processes in this persistent inflammatory disease.

Interferon gamma and interleukin 4 have contrasting effects on immunopathology and the development of protective adaptive immunity against mycoplasma respiratory disease.

For vaccine development, it is critical to understand the regulatory mechanisms determining resistance and immunopathology against mycoplasma respiratory diseases. The present study evaluated the contribution of the polarizing cytokines interferon gamma (IFN-gamma) and interleukin 4 (IL-4) in the regulation of mycoplasma-specific immunity. The absence of a single cytokine (either IFN-gamma or IL-4) uniquely altered the expression of multiple chemokines and cytokines in the lungs of uninfected mice and influenced responses to mycoplasma infection. Most importantly, prior nasal-pulmonary immunization of IFN-gamma(-/-) mice led to exacerbated mycoplasma disease, whereas immunized IL-4(-/-) mice were dramatically more resistant than wild-type mice. Helper T cell type 2 responses in IFN-gamma(-/-) mice corresponded to immunopathologic reactions that developed after mycoplasma infection or immunization. Thus, adaptive immunity clearly can independently promote either protection or immunopathology against mycoplasma infection, and optimal vaccination appears to be dependent on promoting protective IFN-gamma-dependent networks (perhaps helper T cell type 1 responses) while minimizing the effect of IL-4-mediated responses, which dampen the generation of protective immunity.

Thiopurine prodrugs mediate immunosuppressive effects by interfering with Rac1 protein function

6-Thiopurine (6-TP) prodrugs include 6-thioguanine and azathioprine. Both are widely used to treat autoimmune disorders and certain cancers. This study showed that a 6-thioguanosine triphosphate (6-TGTP), converted in T-cells from 6-TP, targets Rac1 to form a disulfide adduct between 6-TGTP and the redox-sensitive GXXXXGK(S/T)C motif of Rac1. This study also showed that, despite the conservation of the catalytic activity of RhoGAP (Rho-specific GAP) on the 6-TGTP-Rac1 adduct to produce the biologically inactive 6-thioguanosine diphosphate (6-TGDP)-Rac1 adduct, RhoGEF (Rho-specific GEF) cannot exchange the 6-TGDP adducted on Rac1 with free guanine nucleotide. The biologically inactive 6-TGDP-Rac1 adduct accumulates in cells because of the ongoing combined actions of RhoGEF and RhoGAP. Because other Rho GTPases, such as RhoA and Cdc42, also possess the GXXXXGK(S/T)C motif, the proposed mechanism for the inactivation of Rac1 also applies to RhoA and Cdc42. However, previous studies have shown that CD3/CD28-stimulated T-cells contain more activated Rac1 than other Rho GTPases such as RhoA and Cdc42. Accordingly, Rac1 is the main target of 6-TP in activated T-cells. This explains the T-cell-specific Rac1-targeting therapeutic action of 6-TP that suppresses the immune response. This proposed mechanism for the action of 6-TP on Rac1 performs a critical role in demonstrating the capability to design a Rac1-targeting chemotherapeutic agent(s) for autoimmune disorders. Nevertheless, the results also suggest that the targeting action of other Rho GTPases in other organ cells, such as RhoA in vascular cells, may be linked to cytotoxicities because RhoA plays a key role in vasculature functions.

Involvement of AP-1 and C/EBPβ in Upregulation of Endothelin B (ETB) Receptor Expression in a Rodent Model of Glaucoma

Previous studies showed that the endothelin B receptor (ETB) expression was upregulated and played a key role in neurodegeneration in rodent models of glaucoma. However, the mechanisms underlying upregulation of ETB receptor expression remain largely unknown. Using promoter-reporter assays, the 1258 bp upstream the human ETB promoter region was found to be essential for constitutive expression of ETB receptor gene in human non-pigmented ciliary epithelial cells (HNPE). The −300 to −1 bp and −1258 to −600 bp upstream promoter regions of the ETB receptor appeared to be the key binding regions for transcription factors. In addition, the crucial AP-1 binding site located at −615 to −624 bp upstream promoter was confirmed by luciferase assays and CHIP assays which were performed following overexpression of c-Jun in HNPE cells. Overexpression of either c-Jun or C/EBPβ enhanced the ETB receptor promoter activity, which was reflected in increased mRNA and protein levels of ETB receptor. Furthermore, knock-down of either c-Jun or C/EBPβ in HNPE cells was significantly correlated to decreased mRNA levels of both ETB and ETA receptor. These observations suggest that c-Jun and C/EBPβ are important for regulated expression of the ETB receptor in HNPE cells. In separate experiments, intraocular pressure (IOP) was elevated in one eye of Brown Norway rats while the corresponding contralateral eye served as control. Two weeks of IOP elevation produced increased expression of c-Jun and C/EBPβ in the retinal ganglion cell (RGC) layer from IOP-elevated eyes. The mRNA levels of c-Jun, ETA and ETB receptor were upregulated by 2.2-, 3.1- and 4.4-fold in RGC layers obtained by laser capture microdissection from retinas of eyes with elevated IOP, compared to those from contralateral eyes. Taken together, these data suggest that transcription factor AP-1 plays a key role in elevation of ETB receptor in a rodent model of ocular hypertension.

Dendritic Cells Are the Major Antigen Presenting Cells in Inflammatory Lesions of Murine Mycoplasma Respiratory Disease

Mycoplasmas cause chronic respiratory diseases in animals and humans, and to date, development of vaccines have been problematic. Using a murine model of mycoplasma pneumonia, lymphocyte responses, specifically T cells, were shown to confer protection as well as promote immunopathology in mycoplasma disease. Because T cells play such a critical role, it is important to define the role of antigen presenting cells (APC) as these cells may influence either exacerbation of mycoplasma disease pathogenesis or enhancement of protective immunity. The roles of APC, such as dendritic cells and/or macrophages, and their ability to modulate adaptive immunity in mycoplasma disease are currently unknown. Therefore, the purpose of this study was to identify individual pulmonary APC populations that may contribute to the activation of T cell responses during mycoplasma disease pathogenesis. The present study indeed demonstrates increasing numbers of CD11c− F4/80+ cells, which contain macrophages, and more mature/activated CD11c+ F4/80− cells, containing DC, in the lungs after infection. CD11c− F4/80+ macrophage-enriched cells and CD11c+ F4/80− dendritic cell-enriched populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC containing CD11c+ F4/80− cell populations from the lungs of infected mice were most capable of stimulating mycoplasma-specific CD4+ Th cell responses in vitro. In vivo, these CD11c+F4/80− cells were co-localized with CD4+ Th cells in inflammatory infiltrates in the lungs of mycoplasma-infected mice. Thus, CD11c+F4/80− dendritic cells appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these dendritic cells likely contribute to responses impacting disease pathogenesis.

Interferon γ and Interleukin 4 Have Contrasting Effects on Immunopathology and the Development of Protective Adaptive Immunity against Mycoplasma Respiratory Disease

For vaccine development, it is critical to understand the regulatory mechanisms determining resistance and immunopathology against mycoplasma respiratory diseases. The present study evaluated the contribution of the polarizing cytokines interferon gamma (IFN-gamma) and interleukin 4 (IL-4) in the regulation of mycoplasma-specific immunity. The absence of a single cytokine (either IFN-gamma or IL-4) uniquely altered the expression of multiple chemokines and cytokines in the lungs of uninfected mice and influenced responses to mycoplasma infection. Most importantly, prior nasal-pulmonary immunization of IFN-gamma(-/-) mice led to exacerbated mycoplasma disease, whereas immunized IL-4(-/-) mice were dramatically more resistant than wild-type mice. Helper T cell type 2 responses in IFN-gamma(-/-) mice corresponded to immunopathologic reactions that developed after mycoplasma infection or immunization. Thus, adaptive immunity clearly can independently promote either protection or immunopathology against mycoplasma infection, and optimal vaccination appears to be dependent on promoting protective IFN-gamma-dependent networks (perhaps helper T cell type 1 responses) while minimizing the effect of IL-4-mediated responses, which dampen the generation of protective immunity.

IFN-γ and IL-4 have contrasting effects on immunopathology and development of protective adaptive immunity against mycoplasma respiratory disease

For vaccine development, it is critical to understand the regulatory mechanisms determining resistance and immunopathology against mycoplasma respiratory diseases. The present study evaluated the contribution of the polarizing cytokines interferon gamma (IFN-gamma) and interleukin 4 (IL-4) in the regulation of mycoplasma-specific immunity. The absence of a single cytokine (either IFN-gamma or IL-4) uniquely altered the expression of multiple chemokines and cytokines in the lungs of uninfected mice and influenced responses to mycoplasma infection. Most importantly, prior nasal-pulmonary immunization of IFN-gamma(-/-) mice led to exacerbated mycoplasma disease, whereas immunized IL-4(-/-) mice were dramatically more resistant than wild-type mice. Helper T cell type 2 responses in IFN-gamma(-/-) mice corresponded to immunopathologic reactions that developed after mycoplasma infection or immunization. Thus, adaptive immunity clearly can independently promote either protection or immunopathology against mycoplasma infection, and optimal vaccination appears to be dependent on promoting protective IFN-gamma-dependent networks (perhaps helper T cell type 1 responses) while minimizing the effect of IL-4-mediated responses, which dampen the generation of protective immunity.