In the past year, we have completed a number of studies on the pathogenesis of Strongyloidiasis and LF and the immunology of tuberculosis. We are also continuing our studies on TB - helminth co-infection and immune responses in TB and TB-diabetes. Strongyloidiasis and LF pathogenesis: A. Modulation of CD4+ and CD8+ T cell function and cytokine responses in Strongyloides stercoralis infection by IL-27 and IL-37: Strongyloides stercoralis (Ss) infection is associated with diminished antigen specific Th1- and Th17-associated responses and enhanced Th2-associated responses. IL-27 and IL-37 are 2 known anti-inflammatory cytokines that are highly expressed in Ss infection. We therefore wanted to examine the role of IL-27 and IL-37 in regulating CD4+ and CD8+ T cell responses in Ss infection. To this end, we examined the frequency of Th1/Tc1, Th2/Tc2, Th9/Tc9, Th17/Tc17, Th22/Tc22 and Tr1 cells in 15 Ss- infected individuals and 10 uninfected individuals, stimulated with parasite antigen following IL-27 or IL-37 neutralization. We also examined the production of prototypical Type 1, Type 2, Type 9, Type 17 and Type 22 cytokines in the whole blood supernatants. Our data reveal that IL-27 or IL-37 neutralization resulted in significantly enhanced frequencies of Th1/Tc1, Th2/Tc2, Th17/Tc17, Th9 and Th22 cells with parasite antigen stimulation. There was no induction of any T cell response in uninfected individuals following parasite antigen stimulation and IL-27 or IL-37 neutralization. Moreover, we also observed increased production of IFNg, IL-5, IL-9, IL-17 and IL-22 and decreased production of IL-10 following IL-27 and IL-37 neutralization and parasite antigen stimulation in whole blood cultures. Thus, we demonstrate that IL-27 and IL-37 limit the induction of particular T cell subsets along with cytokine responses in Ss infections and suggests the importance of IL-27 and IL-37 in immune modulation in a chronic helminth infection. B. Altered levels of memory T cell subsets and IL-7 and IL-15 cytokines in Strongyloides stercoralis infection and reversal following anthelmintic treatment: CD4+ and CD8+ T cells are central players in immunity to helminth infections. However, the role of T cell subsets in helminth infections is not well understood. In addition, the common g-chain cytokines, IL-7 and IL-15 play an important role in the maintenance of these CD4+ and CD8+ T cell subsets. We examined the phenotypic profile of the major T cell subsets as well as the plasma levels of IL-7 and IL-15 at baseline in a group of individuals with asymptomatic, Strongyloides stercoralis (Ss) infection (INF) and a group of uninfected controls (UN). We also examined this profile in INF individuals six months following anthelmintic treatment. Ss infection is characterized by significantly increased absolute numbers of nave CD4+ T cells and decreased absolute numbers of central memory and effector memory CD4+ T cells in comparison to UN individuals. No significant difference in the numbers of CD8+ T cell subsets was observed between the groups. The numbers of nave cells and central memory CD4+ T cells were significantly reversed after anthelmintic treatment. Circulating levels of IL-7 and IL-15 were significantly diminished in INF compared to UN individuals. Upon anthelminthic treatment, IL-7 and IL-15 levels were significantly increased. Our data also showed a significant positive correlation between the levels of IL-7 and the numbers of central memory and effector memory CD4+ T cells. Thus, Ss infection is characterized by alterations in the absolute numbers of CD4+ T cell subsets and diminished levels of common g-chain cytokines IL-7 and IL-15 each of which is reversed after anthelmintic treatment. Tuberculosis Studies: A. Heightened circulating levels of antimicrobial peptides in tuberculosis diabetes co-morbidity and reversal upon treatment: The role of antimicrobial peptides (AMPs) in tuberculosis (PTB) and tuberculosis diabetes comorbidity (PTB-DM) is not well understood. To study the association of AMPs with PTB-DM, we examined the systemic levels of cathelicidin (LL37), human beta defensin 2 (HBD2), human neutrophil peptides 1 3, (HNP1-3) and granulysin in individuals with either PTB-DM, PTB, latent TB (LTB) or no TB infection (NTB). Circulating levels of cathelicidin and HBD2 were significantly higher and granulysin levels were significantly lower in PTB-DM compared to PTB, LTB or NTB, while the levels of HNP1-3 were significantly higher in PTB-DM compared to LTB or NTB individuals. Moreover, the levels of cathelicidin and/or HBD2 were significantly higher in PTB-DM or PTB individuals with bilateral and cavitary disease and also exhibited a significant positive relationship with bacterial burden. Cathelidin, HBD2 and HNP1-3 levels exhibited a positive relationship with HbA1c and/or fasting blood glucose levels. Finally, anti-tuberculosis therapy resulted in significantly diminished levels of cathelicidin, HBD2, granulysin and significantly enhanced levels of HNP1-3 and granulysin in PTB-DM and/or PTB individuals. Therefore, our data demonstrate that PTB-DM is associated with markedly enhanced levels of AMPs and diminished levels of granulysin. B. Prediabetes is associated with the modulation of antigen-specific Th1/Tc1 and Th17/Tc17 responses in latent Mycobacterium tuberculosis infection: Type 2 diabetes mellitus (DM) is associated with the down modulation of Th1, Th2 and Th17 responses in latent Mycobacterium tuberculosis infection but the role of prediabetes (PDM) in this setting is not well understood. To examine the role of CD4+ and CD8+ T cell cytokines in latent tuberculosis (LTB) with coincident PDM, we studied the baseline, mycobacterial, control antigen and mitogen stimulated T cell cytokine responses in LTB individuals with (LTB-PDM; n=20) or without (LTB-NDM; n=20) concomitant prediabetes. LTB-PDM is characterized by diminished frequencies of mono and dual functional CD4+ Th1 and Th17 cells and mono-functional Th2 cells at baseline and/or following mycobacterial - antigen stimulation in comparison to LTB-NDM. LTB-PDM is also characterized by diminished frequencies of mono functional CD8+ Tc1, Tc2 and Tc17 cells at baseline and/or following mycobacterial antigen stimulation in comparison to LTB-NDM. LTB-PDM is therefore characterized by diminished frequencies of antigen specific Th1/Tc1 and Th17/Tc17 cells, indicating that PDM is associated with alterations of the immune response in latent TB associated with compromised CD4+ and CD8+ T cell function. C. Modulation of iron status biomarkers in tuberculosis-diabetes co-morbidity Tuberculosis (TB) and diabetes mellitus (DM) remain vital disease burdens in developing countries and the dual burden of DM and TB clearly signifies a growing global public health concern. While modulation of iron status biomarkers in TB is well described, very little is known about the association of these markers with TB-DM. To examine the association of circulating iron status biomarkers in TB disease, we examined the systemic levels of ferritin, hepcidin, soluble transferrin receptor (sTfR), transferrin, apotransferrin and hemopexin in pulmonary TB (PTB) individuals with DM (PTB-DM), without DM (PTB) and those with diabetes only (DM). Circulating levels of ferritin and hepcidin were significantly enhanced in PTB-DM and PTB compared to the DM group. On the other hand, the circulating levels of transferrin and apotransferrin were significantly diminished in PTB DM and PTB compared to the DM group. The levels of ferritin and hepcidin exhibited a significant positive relationship with HbA1c, whereas apotransferin exhibited negative relationship with HbA1c in PTB-DM and PTB. ROC analysis revealed that ferritin, hepcidin and transferrin are markers that can distinguish PTB from DM individuals.
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