In the past year, we have completed a number of sudies in the pathogenesis of lymphatic filarial disease and the immune responses in pulmonary and extra-pulmonary tuberculosis. In addition, we have begun two new studies to examine the influence of filarial infections on latent tuberculosis and the immunological responses in tuberculous lymphadenitis. Lymphatic filariasis (LF) pathogenesis: Lymphatic filariasis can be associated with development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Dysregulated host inflammatory responses leading to systemic immune activation are thought to play a central role in filarial disease pathogenesis. We measured the plasma levels of microbial translocation markers, acute phase proteins, and inflammatory cytokines in individuals with chronic filarial pathology with (CP Ag+) or without (CP Ag) active infection;with clinically asymptomatic infections (INF);and in those without infection (endemic normal EN). Comparisons between the two actively infected groups (CP Ag+ compared to INF) and those without active infection (CP Ag compared to EN) were considered to truly reflect markers of pathogenesis. Our data suggest that circulating levels of microbial translocation products (lipopolysaccharide and LPS-binding protein), acute-phase proteins (haptoglobin, a-2 macroglobulin, and serum amyloid protein-A), and inflammatory cytokines (IL-1b, IL-12, and TNF-a) are associated with pathogenesis of disease in lymphatic filarial infection and implicate an important role for circulating microbial products and acute phase proteins in establishing the chronic inflammatory milieu in this process. Matrix metalloproteinases (MMPs) are a family of enzymes governing extracellular remodeling by regulating cellular homeostasis, inflammation, and tissue reorganization, while tissue-inhibitors of metalloproteinases (TIMPs) are endogenous regulators of MMPs. Homeostatic as well as inflammation-induced balance between MMPs and TIMPs is considered critical in mediating tissue pathology. To elucidate the role of MMPs and TIMPs in filarial pathology, we compared the plasma levels of a panel of MMPs, TIMPs, other pro-fibrotic factors, and cytokines in individuals with chronic filarial pathology with (CP Ag+) or without (CP Ag) active infection to those with clinically asymptomatic infections (INF) and in those without infection (endemic normal EN). Our data reveal that an increase in circulating levels of MMPs and TIMPs is characteristic of the filarial disease process per se and not of active infection;however, filarial disease with active infection is specifically associated with increased ratios of MMP1/TIMP4 and MMP8/TIMP4 as well as with pro-fibrotic cytokines (IL-5, IL-13 and TGF-b). Our data therefore suggest that while filarial lymphatic disease is characterized by a non-specific increase in plasma MMPs and TIMPs, the balance between MMPs and TIMPs is an important factor in regulating tissue pathology during active infection. Lymphatic filarial disease is known to be associated with elevated Th1 and normal or diminished Th2 responses to parasite-specific antigens. The role of Th17 cells and T cells expressing the IL-10 family of cytokines, however, has not been well-defined nor has the contribution of CD8+ T cells to this cytokine response. To study the role of CD4+ and CD8+ Th1, Th2 and Th17 cell subsets as well as T cells expressing IL-10 family of cytokines in the development of lymphatic pathology, we examined the frequency of these cells in individuals with filarial lymphedema (CP) directly ex vivo and in response to parasite or non-parasite antigens;these frequencies were compared to those in clinically asymptomatic patently-infected individuals (INF). CP individuals exhibited a significant increase in the frequency of CD4+ T cells expressing IL-2, TNF-a, IFNg, IL-17 and IL-22 at baseline and in response to filarial antigens compared to INF individuals. In contrast, these same individuals exhibited a significant decrease in the frequency of CD4+ T cells expressing IL-4, IL-5, IL-9, IL-13 and IL-21. In addition, CP individuals displayed a significant decrease in the frequency of CD4+ T cells expressing IL-10, IL-19 and IL-24 but not IL-26. These differential frequencies between the 2 patient groups were also observed in CD8+ T cells, albeit to a much less extent. Our findings suggest that alterations in the frequencies of CD4+ and CD8+ Th1, Th2, Th17 cells and T cells expressing IL-10 family of cytokines are a characteristic feature underlying the pathogenesis of filarial lymphedema. Tuberculosis Studies Although Type 1 cytokine responses are known to play an important role in immunity to tuberculosis (TB), very little is know about the role of Type 2 and Type 17 cytokines in TB disease pathogenesis. To elucidate the immune responses important both in control of infection and in extrapulmonary dissemination, we examined mycobacteriaspecific cytokine responses in pulmonary TB (PTB) (n=26) and extrapulmonary TB (ETB) (n=24) and compared them with latently infected individuals (LTB) (n=24). Multiplex ELISA (IFNg, TNF-a, IL-2, IL-12, GM-CSF, IL-4, IL-5, IL-13, IL-6, IL-17, IL-17F, IL-22, IL-23, IL-10 and TGF-b) was carried out using culture supernatants of whole blood stimulated with TB antigens PPD, ESAT-6, CFP-10 and control anti-CD3. We observed significantly lower levels of Type 1 cytokines (IFNg, TNF-a, IL-2 and GM-CSF), 2 (IL-4), and 17 (IL-17A and IL-17F) -associated cytokines at baseline, as well as after stimulation with TB antigens in PTB compared to LTB, indicating that these cytokines play an important role in protection against disease. In addition, PTB showed markedly diminished production of Types 1 (IFNg, TNF-a, IL-2 and GM-CSF), 2 (IL-4), and 17 (IL-17A and IL-17F) -associated cytokines at baseline and following TB antigenic stimulation in comparison to ETB. This was associated with significantly altered production of IL-10 and TGF-b in PTB. Treatment and consequent bacteriological cure did not reverse the downregulation in cytokine responses. However, both IL-10 and TGF-b blockade was able to partially reverse the dimunition in cytokine responses, indicating an important regulatory role for these cytokines in PTB. Thus, Pulmonary TB is characterized by diminished Types 1, 2 and 17 cytokine responses, suggesting a crucial role for these cytokines in protection against TB disease development as well as extra-pulmonary dissemination. Th1 and Th17 responses are known to play an important role in immunity to pulmonary tuberculosis (PTB), although little is known about their role in extrapulmonary forms of tuberculosis (TB). To identify the role of Th1, Th17, and Th22 cells in multi-focal TB lymphadenitis (TBL), we examined mycobacteriaspecific immune responses in the whole blood of individuals with PTB (n = 20) and compared them with those with TBL (n = 25). Elevated frequencies of CD4+ T cells expressing IFNg, TNF-a, and IL-2 were present in individuals with TBL compared with those with PTB at homeostasis (baseline) as well as in response to mycobacterial antigens ESAT-6 and CFP-10. Similarly, increased frequencies of CD4+ T cells expressing IL-17A, IL-17F, and IFNg were also present in individuals with TBL at baseline and following ESAT-6 and CFP-10 stimulation although no significant difference in the Th1 (but not Th17) cells exhibited a significantly negative correlation with natural regulatory T cell frequencies at baseline. Multi-focal TB lymphadenitis is therefore, characterized by elevated frequencies of Th1 and Th17 cells, indicating that Th1 and Th17 responses in TB disease are probably correlates of disease severity rather than of protective immunity.
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