Neither primary nor memory immunity to Mycobacterium tuberculosis infection is compromised in mice with chronic enteric helminth infection

Abstract

Previously we had reported that Nippostrongylus brasiliensis, a helminth with a lung migratory phase, affected host resistance against Mycobacterium tuberculosis infection through the induction of alternatively activated (M2) macrophages. Several helminth species do not have an obligatory lung migratory phase but establish chronic infections in the host that include potent immune downregulatory effects, in part mediated through induction of a FoxP3(+) T regulatory cell (Treg) response. Treg cells exhibit duality in their functions in host defense against M. tuberculosis infection since their depletion leads to enhanced priming of T cells in the lymph nodes and attendant improved control of M. tuberculosis infection, while their presence in the lung granuloma protects against excessive inflammation. Heligmosomoides polygyrus is a strictly murine enteric nematode that induces a strong FoxP3 Treg response in the host. Therefore, in this study we investigated whether host immunity to M. tuberculosis infection would be modulated in mice with chronic H. polygyrus infection. We report that neither primary nor memory immunity conferred by Mycobacterium bovis BCG vaccination was affected in mice with chronic enteric helminth infection, despite a systemic increase in FoxP3(+) T regulatory cells. The findings indicate that anti-M. tuberculosis immunity is not similarly affected by all helminth species and highlight the need to consider this inequality in human coinfection studies.

FIG 1

H. polygyrus causes induction of Tregs. Wild-type BALB/c mice were infected orally with 200 L3-stage larvae of H. polygyrus (HP). Four weeks postinfection, tissues were harvested and assessed for the percentage of FoxP3 expression by flow cytometry. Data are presented as means ± standard errors of the means (SEM): axillary lymph nodes (Ax.LN), P = 0.0023; mediastinal lymph nodes (MLN), P = 0.0033; mesenteric lymph nodes (GLN), P = 0.01; spleen, P = 0.0015. ns, not significant.

FIG 2

Prior enteric helminth infection does not affect progression of M. tuberculosis infection. Groups of BALB/c mice were infected orally with 200 L3-stage H. polygyrus larvae or remained uninfected. Four weeks post-H. polygyrus infection, both groups of mice were infected with a low aerosol dose of the Erdman strain of M. tuberculosis (Mtb). Mice were sacrificed at the indicated time points post-M. tuberculosis infection, and single-cell suspensions and lung homogenates were prepared for cellular analysis and to assess bacterial burden. (A) Percentage of FoxP3⁺ mediastinal lymph node (LN) cells as assessed by flow cytometry. (B) IFN-γ production by mediastinal lymph node cells. SFU, spot-forming units. (C) IFN-γ production by lung cells as assessed by ELISPOT assay. (D) Lung CFU. Data are presented as means ± SEM (n = 5/time point). **, P < 0.01; ns, not significant.

FIG 3

H. polygyrus coinfection does not alter the M. tuberculosis granuloma architecture and the accumulation of FoxP3⁺ Tregs in the lungs. Lungs from M. tuberculosis (Mtb)-coinfected animals were harvested at the indicated time points postinfection, and the percentages of FoxP3⁺ lung cells were assessed by flow cytometry (A). (B) Immunohistochemical analysis for FoxP3 staining (yellow arrows) from the lungs of M. tuberculosis-infected (bottom left panel) and M. tuberculosis-coinfected (bottom right panel) mice obtained at week 7 following M. tuberculosis infection. The top left and top right panels represent the respective isotype controls. The figure presents representative photomicrographs, and the images were captured with a 10× objective on a Nikon Eclipse 50i. (C) Histopathological evaluation of H&E-stained sections of formalin-fixed, paraffin-embedded tissues from the lungs of M. tuberculosis-infected (left panel) and M. tuberculosis-coinfected (right panel) mice obtained at week 7 following aerosol infection with M. tuberculosis. The figure presents representative photomicrographs, and the images were captured with a 10× objective on a Nikon Eclipse 50i.

FIG 4

Prior chronic enteric helminth infection does not affect BCG vaccine efficacy. Groups of BALB/c mice were infected orally with 200 L3-stage H. polygyrus larvae or remained uninfected. Four weeks post-H. polygyrus infection, both groups of mice were subcutaneously immunized with BCG vaccine. The different groups of mice were subsequently challenged with a low aerosol dose of the Erdman strain of M. tuberculosis. Mice were sacrificed at the indicated time points post-M. tuberculosis challenge, and single-cell suspensions and lung homogenates were prepared for identification of IFN-γ-producing cells in the lymph nodes (A) and lungs (B) and to assess bacterial burden (C). Data are presented as means ± SEM (n = 5 each/time point). **, P < 0.01; ****, P < 0.0001; ns, not significant.