Suppression of airway inflammation by a natural acute infection of the intestinal epithelium

Abstract

Although chronic intestinal helminth infections may suppress allergen-induced airway pathology by inducing a combination of modified T-helper (Th) 2 and immunosuppressive cytokines, a similar capacity of natural acute intestinal infections has remained untested, despite their global prevalence. Here, we show that allergic airway phenotypes including eosinophilia, eotaxin mRNA, and Th2 cytokines are significantly suppressed in animals that were infected by and that have cleared the intestinal parasite Eimeria vermiformis. Unlike in helminth-infected animals, regulation requires temporal coincidence of infection with sensitization; depends on interferon-gamma; and is not associated with an enhanced antigen-specific immunoglobulin G1 response. Moreover, regulation was effective following allergen sensitization in different anatomical sites, and in young and adult mice. These data highlight a transient anatomical dissemination of "functional immunologic dominance" following infection of the gut mucosa. They strongly support the hypothesis that airway allergies are naturally suppressed by both acute and chronic mucosal pathogens, but by different mechanisms.

Figure 1

Prior infection with E. vermiformis inhibits airway reactions. (a) Main experimental protocol. Mice were infected at day 0 with oocysts by gavage. After 3 days, infected and control mice were sensitized intraperitoneally (IP) with 10 μg OVA in ALUM and this was repeated at day 17. Mice were challenged on day 29 with 100 μg OVA intranasally (i.n.) and culled 2 days later. Infected mice were housed separately and oocyst numbers determined from collected fecal pellets. (b) Parasite growth and clearance were comparable whether or not mice were simultaneously challenged with ova. Previously infected mice (diamonds, right hand side) or uninfected mice (diamonds, left hand side) subsequently sensitized and challenged with OVA or PBS from two independent experiments were assessed for total cell infiltration into the (c) BALF or (d) CCR3-positive cells (eosinophils) infiltrating into the BALF (normalized to 5,000 beads); levels of eotaxin and IL-5 mRNA in the lung from a representative experiment are shown in (e) with β-actin mRNA levels to indicate similar mRNA quantities; (f) protein levels in the BALF for IL-5 (cumulative data from three independent experiments, (g) IL-4 and (h) IL-6 (representative experiments) are also shown. The same outlier mouse (circled in g, h) showed the lowest oocyst count of the representative experiment shown. PowerPoint slide

Figure 2

Prior infection with E. vermiformis alters OVA-specific Ig responses. Levels of OVA-specific IgG2a, detected by ELISA, are significantly increased in the serum of mice previously infected compared to those that were uninfected (a: cumulative data from four independent experiments). Levels of OVA-specific IgG1 are decreased in the serum of mice previously infected compared to those that were uninfected as shown in a representative experiment in (b). The dotted line represents average levels of OVA-specific IgG1 detected in PBS-treated mice. The outlier mouse (circled) showed the lowest oocyst count of the experiment shown. PowerPoint slide

Figure 3

Prior infection with E. vermiformis inhibited allergy induced by epicutaneous sensitization. (a) The dosing regime. Eight mice were untreated or infected with 1,000 oocysts at day 0 and after shaving an occlusive patch was attached to the dorsal skin containing 100 μg OVA or PBS at day 3 after infection. Mice were then challenged with 50 μg OVA intranasally (i.n.) for 3 consecutive days before culling on day 28. Levels of (b) eotaxin-1 and (c) eotaxin-2 mRNA, as determined by quantitative real-time PCR, are significantly decreased in mice previously infected (right, diamonds) compared to uninfected (left diamonds). Levels are normalized to a control mouse sample. (d–f) Levels of IL-5, IL-6 and IL-4 (respectively) detected in the BALF of uninfected (diamonds) or previously infected (diamonds) mice. The outlier mouse (circled) showed the lowest oocyst count of the experiment. In each graph, average expression levels for PBS-sensitized animals are shown as a dotted line and median levels of expression indicated. PowerPoint slide

Figure 4

Infection of adult mice reduces allergic parameters. A total of 8 young mice (28 days old, left side) or 8 adult mice (8–10 weeks, right side) were infected with 100 oocysts at day 0 and then these mice and control mice were sensitized and challenged with OVA, as in Figure 1a. (a) Numbers of CCR3-positive cells infiltrating into the BALF, normalized to 5,000 beads, in infected young or adult mice as compared to the average levels of CCR3-positive cells seen in uninfected mice (diamond, middle). (b) Levels of IL-5 (pg/ml) in the BALF of infected young or adult mice in comparison to uninfected mice (diamond, center). (c) Levels of eotaxin-1 mRNA, as determined by quantitative PCR, in the lungs of young or old infected mice compared to uninfected mice (diamond, center). In each case, the P value asserts a significant difference between uninfected mice and previously infected adult mice, which conversely show no significant difference vs. previously infected young mice. PowerPoint slide

Figure 5

No protection is observed if mice are not sensitized while infected. (a) Experimental protocol for the delayed sensitization experiment where mice were infected at day 0 but then no sensitization was performed until day 24 and repeated at day 38. Mice were challenged with 100 μg OVA on day 50 and culled at day 52. (b) Number of CCR3-positive cells (normalized to beads) infiltrating into the BALF of challenged mice previously infected with E. vermiformis but not sensitized until after the infection had cleared (6 mice, diamonds, right) compared to uninfected mice (7 mice, diamonds, left). (c) IL-5 levels in the BALF, (d) eotaxin-2 mRNA in the lung, or (e) OVA-specific IgG2a in the serum are shown from mice infected with E. vermiformis but then not sensitized until D24PI. PowerPoint slide

Figure 6

Allergy suppression by infection depends on IFNγ but not on γδ T cells. (a) Levels of oocysts collected from the feces of wt adult mice infected with 100 oocysts (left) or IFNγ^−/− mice infected with 10 oocysts (right). A similar level of infection is seen in both. IFNγ^−/− mice were, therefore, infected with 10 oocysts and OVA-sensitized and challenged as in Figure 1a. (b) Levels of CCR3-positive cells infiltrating into the BALF of OVA or PBS-treated infected (diamonds, right) or uninfected (diamonds, left) IFNγ^−/− mice. Levels of eotaxin-2 mRNA in IFNγ^−/− mice infected (diamonds, right) or uninfected (diamonds, left) with E. vermiformis are shown in (c). Figure represents cumulative data from two independent experiments. TCRδ^−/− mice were infected, sensitized, and challenged as described in Figure 1a. Previously infected TCRδ^−/− mice (diamonds, right hand side) or uninfected TCRδ^−/− mice (diamonds, left hand side) subsequently sensitized and challenged with OVA or PBS were assessed for (d) IL-5 levels in the BALF, (e) CCR3-positive cell infiltration into the BALF, and (f) eotaxin-1 mRNA in the lung. All AAI parameters tested were induced by OVA sensitization and challenge in the TCRδ^−/− mice and all were reduced by previous infection with E. vermiformis. PowerPoint slide