Failure of antigen-stimulated γδ T cells and CD4+ T cells from sensitized cattle to upregulate nitric oxide and mycobactericidal activity of autologous Mycobacterium avium subsp. paratuberculosis-infected macrophages

Abstract

The function of γδ T cells during ruminant paratuberculosis (Johne's disease) is presently unknown. An ex vivo system was used to test the hypothesis that γδ T cells are capable of activating Mycobacterium avium subsp. paratuberculosis-(M. paratuberculosis)-infected macrophages. Peripheral blood-derived macrophages were infected in vitro with live M. paratuberculosis, and autologous LN-derived γδ T cells or CD4+ T cells were co-cultured with infected macrophages for 48 hours, at which time bacterial survival as well as production of nitrites and IFN-γ were evaluated. Incubation of M. paratuberculosis-infected macrophages with autologous γδ T cells did not result in reduced intracellular bacterial viability compared to infected macrophage cultures without added T cells. IFN-γ production by-infected cultures containing added γδ T cells was not enhanced compared to that of infected macrophages alone. Although infection of macrophage cultures caused increased production of nitrites at both post-infection day (PID) 0 and PID 60, the addition of γδ T cells did not further increase nitrite production. In contrast, addition of PPD-stimulated CD4+ T cells obtained at PID 60 to M. paratuberculosis-infected macrophages resulted in significantly increased IFN-γ production compared to cultures without added T cells or cultures containing unstimulated CD4+ T cells or unstimulated or antigen-stimulated γδ T cells. However, the increased production of IFN-γ by co-cultures containing PPD-stimulated CD4+ T cells did not result in increased bacterial killing or increased production of nitrites compared to cultures without added T cells. In additional in vitro experiments, M. paratuberculosis-infected macrophages, but not uninfected macrophages, were unable to increase nitrite production when stimulated with recombinant IFN-γ. Taken together, the data suggest that (1) γδ T cells do not produce significant IFN-γ and do not significantly increase NO production from M. paratuberculosis-infected macrophages in vitro, (2) the production of significant IFN-γ by antigen-stimulated CD4+ T cells from infected calves is insufficient to enhance mycobacterial killing or nitrite production by infected macrophages, and (3) macrophages may have an impaired NO response following intracellular M. paratuberculosis infection, even in the presence of significant concentrations of IFN-γ.

The early lymphocyte proliferation response in sheep exposed to Mycobacterium avium subsp. paratuberculosis compared to infection status

“…Evidence from murine studies suggests that gd T cells play an important role in the development of granuloma in mycobacterial infection (D'Souza et al 1997;Saunders et al 1998) Tanaka, 2000 #515} rather than in the intracellular killing of the organism (Simutis et al 2007). The antigen specific proliferation of CD4 + and CD8 + T cells in blood remains enhanced from early to late stages of disease (4-13 months p.i.).…”

Mycobacterium avium Subspecies paratuberculosis Infection in Cases of Irritable Bowel Syndrome and Comparison with Crohn's Disease and Johne's Disease: Common Neural and Immune Pathogenicities

“…paratuberculosis-infected bovine macrophages (74), the hyporesponsiveness in ileal lymphocytes from Mycobacterium avium subsp. paratuberculosis-infected cows (75), abnormalities in the regulation of cytokine expression (9,13,16,30,31,65,73), the perturbation of macrophage activation and apoptosis (13,15), and the impairment of nitric oxide responsiveness (61 (33,35,68). Local and systemic immune dysfunctions in humans are well-described features of Crohn's disease (39,46).…”

“…Mycobacterium avium subsp. paratuberculosis infection in cases of Johne's disease is associated with a chronic enteric neuritis (6,29), together with immune activation and dysregulation (15,16,61,65,74,75).…”

LiveMycobacterium aviumsubsp.paratuberculosisand a Killed-Bacterium Vaccine Induce Distinct Subcutaneous Granulomas, with Unique Cellular and Cytokine Profiles

“…The M. avium subsp. paratuberculosis inoculum used in these studies was shown to be above 90% viable as checked via fluorescein diacetate staining and flow cytometry analysis (20,48). Two animals were given an injection of 5 ϫ 10 8 CFU live M. avium subsp.…”