In the past year, we have completed a number of studies on the pathogenesis of lymphatic filariasis and the immunology of tuberculosis. We are also continuing our studies on TB-filarial co-infection and immune responses in TB and TB-diabetes. Lymphatic filariasis (LF) pathogenesis: A. Role of Th9 cells: Th9 cells are a subset of CD4+ T cells, shown to be important in allergy, autoimmunity and anti-tumor responses. However, their role in human infectious diseases has not been explored in detail. We identified a population of IL-9 and IL-10 co-expressing cells (lacking IL-4 expression) in normal individuals that respond to antigenic and mitogenic stimulation but are distinct from IL-9+ Th2 cells. We also demonstrate that these Th9 cells exhibit antigen specific expansion in a chronic helminth infection (lymphatic filariasis). Comparison of Th9 responses reveals that individuals with pathology associated with filarial infection exhibit significantly expanded frequencies of filarial antigen induced Th9 cells but not of IL9+Th2 cells in comparison to filarial-infected individuals without associated disease. Moreover, the per cell production of IL-9 is significantly higher in Th9 cells compared to IL9+Th2 cells, indicating that the Th9 cells are the predominant CD4+ T cell subset producing IL-9 in the context of human infection. This expansion was reflected in elevated antigen stimulated IL-9 cytokine levels in whole blood culture supernatants. Finally, the frequencies of Th9 cells correlated positively with the severity of lymphedema (and presumed inflammation) in filarial diseased individuals. This expansion of Th9 cells was dependent on IL-4, TGFb and IL-1 in vitro. We have therefore a identified an important human CD4+ T cell subpopulation co expressing IL-9 and IL-10 but not IL-4 that is whose expansion is associated with disease in chronic lymphatic filariasis and could potentially play an important role in the pathogenesis of other inflammatory disorders. B. Role of IL-10 family members Lymphatic filariasis (LF) is known to be associated with an increased production of IL-10. The role of the other IL-10 family members in the pathogenesis of infection and/or disease is not known. We examined the expression patterns of IL-10 family members - IL-19, IL-24 and IL-26 in LF. We demonstrate that both CD4+ and CD8+ T cells express IL-19, IL-24 and IL-26 and that the frequency of CD4+ T cells expressing IL-19 and IL-24 (as well as IL-10) is significantly increased at baseline and following filarial antigen stimulation in patients with LF in comparison to individuals with filarial lymphedema. This CD4+ T cell expression pattern was associated with increased production of IL-19 and IL-24 by filarial antigen stimulated PBMC. Moreover, the frequency of CD4+ and CD8+ T cells expressing IL-26 was significantly increased at baseline and following filarial antigen stimulation in filarial lymphedema individuals. Interestingly, IL-10 blockade resulted in diminished frequencies of IL-19+ and IL-24+ T cells, whereas the addition of recombinant IL-10 resulted in significantly increased frequency of IL-19+ and IL-24+ T cells as well as significantly up regulated IL-19 and IL-24 gene expression, suggesting that IL-10 regulates IL-19 and IL-24 expression in T cells. In addition, IL-1b and IL-23 blockade also induced a diminution in the frequency of IL-19+ and IL-24+ T cells, indicating a novel role for these cytokines in the induction of IL-19 and IL-24 expressing T cells. Finally, elimination of infection resulted in significantly decreased frequencies of antigen specific CD4+ T cells expressing IL-10, IL-19 and IL-24. Our findings, therefore, suggest that IL-19 and IL-24 are associated with the regulation of immune responses in active filarial infection and potentially with protection against developmentt of pathology, while IL-26 is predominantly associated with pathology in LF. C. Role of Th2 subsets Two different Th2 subsets have been defined recently on the basis of IL-5 expression an IL-5+, Th2 subset and an IL-5-, Th2 subset in the setting of allergy. However, the role of these newly described CD4+ T cells subpopulations has not been explored in other contexts. To study the role of the Th2 subpopulation in a chronic, tissue invasive parasitic infection (lymphatic filariasis), we examined the frequency of IL-5+IL-4+IL-13+ CD4+ T cells and IL-5-IL-4 IL13+ CD4+ T cells in asymptomatic, infected individuals (INF) and compared them to frequencies (Fo) in filarial-uninfected (UN) individuals and to those with filarial lymphedema (CP). INF individuals exhibited a significant increase in the spontaneously expressed and antigen-induced Fo of both Th2 subpopulations compared to the UN and CP. Interestingly, there was a positive correlation between the Fo of IL-5+Th2 cells and the absolute eosinophil and neutrophil counts;in addition there was a positive correlation between the frequency of the CD4+IL-5- Th2 subpopulation and the levels of parasite antigen specific IgE and IgG4 in INF individuals. Moreover, blockade of IL-10 and/or TGFb demonstrated that each of these 2 regulatory cytokines exert opposite effects on the different Th2 subsets. Finally, in those INF individuals cured of infection by anti-filarial therapy, there was a significantly decreased Fo of both Th2 subsets. Our findings suggest that both IL-5+ and IL-5-Th2 cells play an important role in the regulation of immune responses in filarial infection and that these two Th2 subpopulations may be regulated by different cytokine-receptor mediated processes. Tuberculosis and Diabetes Studies: Type 2 diabetes mellitus (T2DM) is a major risk factor for the development of active tuberculosis (TB), although the biological basis underlying this susceptibility remains poorly characterized. To identify the influence of coincident DM on cytokine levels in pulmonary TB, we examined circulating levels of a panel of cytokines and chemokines in the plasma of individuals with TB-DM and compared them with those without DM (TB-NDM). TB-DM is characterized by elevated circulating levels of Type 1 (IFNg, TNFa and IL-2), Type 2 (IL-5) and Type 17 (IL-17A) cytokines but decreased circulating levels of IL-22. This was associated with increased systemic levels of other pro-inflammatory cytokines (IL-1b, IL-6 and IL-18) and an anti inflammatory cytokine (IL-10) but not Type 1 interferons. Moreover, TB antigen stimulated whole blood also showed increased levels of pro-inflammatory cytokines. Finally, Type1 and Type 17 cytokines in plasma exhibit a significant positive correlation with HbA1C levels, indicating that impaired control of diabetes is associated with this pro inflammatory milieu. Therefore, our data reveal that TB-DM is characterized by heightened cytokine responsiveness, indicating that chronic inflammation underlying Type 2 diabetes potentially contributes to increased immune pathology and poor control in TB infection. Cross-sectional analyses of levels of heme oxygenase-1 (HO-1), acute phase proteins, tissue metalloproteinases and their inhibitors (TIMPs) as well as a panel of cytokines and chemokines were performed in plasma samples from individuals with active pulmonary TB with or without DM. Compared to non-diabetic TB patients, those with DM exhibited increased bacillary loads in sputum. Plasma levels of HO-1 but not of other acute phase proteins were higher in TB-DM patients than in non-diabetics. Moreover, patients with TB-DM exhibited increased plasma levels of TIMP-4 and elevated peripheral blood neutrophil counts which along with HO-1 significantly influences TB-DM pathogenesis. Elevated plasma levels of HO-1 and TIMP-4 and peripheral blood neutrophil counts are reliable single and combined biomarkers of TB-DM.
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