Chronic helminth infection does not exacerbate Mycobacterium tuberculosis infection

Abstract

Background: Chronic helminth infections induce a Th2 immune shift and establish an immunoregulatory milieu. As both of these responses can suppress Th1 immunity, which is necessary for control of Mycobacterium tuberculosis (MTB) infection, we hypothesized that chronic helminth infections may exacerbate the course of MTB.

Methodology/principal findings: Co-infection studies were conducted in cotton rats as they are the natural host for the filarial nematode Litomosoides sigmodontis and are an excellent model for human MTB. Immunogical responses, histological studies, and quantitative mycobacterial cultures were assessed two months after MTB challenge in cotton rats with and without chronic L. sigmodontis infection. Spleen cell proliferation and interferon gamma production in response to purified protein derivative were similar between co-infected and MTB-only infected animals. In contrast to our hypothesis, MTB loads and occurrence and size of lung granulomas were not increased in co-infected animals.

Conclusions/significance: These findings suggest that chronic filaria infections do not exacerbate MTB infection in the cotton rat model. While these results suggest that filaria eradication programs may not facilitate MTB control, they indicate that it may be possible to develop worm-derived therapies for autoimmune diseases that do not substantially increase the risk for infections.

Figure 1. Chronic L. sigmodontis infection induces eosinophilia and a hyporesponsive milieu.

A, peripheral blood eosinophil counts from uninfected, 5, and 11 week-Litomosoides sigmodontis (L.s.) infected cotton rats. In vitro spleen cell proliferation (B, C, as proliferation index (OD of stimulated cells/baseline)) and IFNγ production (D, E) in response to L. sigmodontis antigen (LsAg) or Staphylococcal enterotoxin B (SEB) from cotton rats that were either uninfected or infected with L. sigmodontis for 5 or 11 weeks. Statistical significance between groups was analyzed by the Kruskal-Wallis test, followed by Dunn's post-hoc multiple comparisons. Single stars show significant differences compared to uninfected animals. *p<0.05.

Figure 2. Experimental setup.

Cotton rats were infected with 100 infectious Litomosoides sigmodontis L3 larvae and intranasally challenged with 5×10⁴ M. tuberculosis (MTB) bacteria 11 weeks later, a timepoint by which they had developed a patent (microfilaria (Mf)-releasing) filaria infection. At 20 weeks cotton rats were euthanized and immunological and histological studies performed. Numbers in brackets indicate the number of analyzed cotton rats in the first and second experiment.

Figure 3. Histological assessment of L. sigmodontis and M. tuberculosis infection at the 20 week timepoint.

A, lung with M. tuberculosis (MTB) granulomas obtained from a co-infected animal 9 weeks post MTB challenge (the 20 week timepoint). B, Lung granuloma (red arrow) with central necrosis (green arrow) observed in the lung of a cotton rat infected with MTB (H&E, 40×). C; Acid-fast stain of MTB bacteria in the lung (100×). D, L. sigmodontis microfilaria in peripheral blood (Eosin-Y Azure A Methylene Blue, 100×). E, H&E stained cross-section of lung tissue that shows a L. sigmodontis adult worm in the pleural space adjacent to the lung (40×).

Figure 4. M. tuberculosis infection has no consistent impact on L. sigmodontis worm burden.

A, total number of L. sigmodontis adult worms recovered from the pleural space and B, number of microfilaria per µl of peripheral blood of cotton rats that were infected with L. sigmodontis (L.s.) and M. tuberculosis (MTB) or L. sigmodontis alone (20 weeks post L. sigmodontis infection) from the first experiment. C, total number of L. sigmodontis adult worms recovered from the pleural space of the repeat experiment. Statistical significance was analyzed by the Mann-Whitney-U-test.

Figure 5. PPD-specific IFNγ production is not reduced by L. sigmodontis co-infection.

A, IFNγ production of spleen cells in response to M. tuberculosis PPD and (B), SEB. Cotton rats were infected with L. sigmodontis (L.s.) and/or M. tuberculosis (MTB), or were uninfected. Statistical significance between groups was analyzed by the Kruskal-Wallis test, followed by Dunn's post-hoc multiple comparisons. Shown are representative results from one of two experiments. Single stars show significant differences compared to the uninfected animals. **p<0.01, ***p<0.001.

Figure 6. PPD-specific proliferation is not impaired by L. sigmodontis co-infection.

A, Spleen cell proliferation in response to M. tuberculosis PPD and B, SEB. Cotton rats were infected with L. sigmodontis (L.s.) and/or M. tuberculosis (MTB), or were uninfected. Shown is the proliferation index (OD of stimulated cells/baseline). Statistical significance between groups were analyzed by the Kruskal-Wallis test, followed by Dunn's post-hoc multiple comparisons. Single stars show significant differences compared to the uninfected animals. Shown are representative results from one of two experiments. **p<0.01, ***p<0.001.

Figure 7. L. sigmodontis co-infection does not increase M. tuberculosis burden in lung or spleen and does not exacerbate lung granuloma formation.

A, Percentage of lung area covered with granulomas. B, M. tuberculosis colony-forming units (CFU) in lung and C, spleen. Cotton rats were infected with L. sigmodontis (L.s.) and/or M. tuberculosis (MTB), or were uninfected. Shown are representative results from one of two experiments. Statistical significance between co-infected and MTB-only infected groups was analyzed by the Mann-Whitney-U-test.