Innate and adaptive type 2 immune cell responses in genetically controlled resistance to intestinal helminth infection

Abstract

The nematode Heligmosomoides polygyrus is an excellent model for intestinal helminth parasitism. Infection in mice persists for varying lengths of time in different inbred strains, with CBA and C57BL/6 mice being fully susceptible, BALB/c partially so and SJL able to expel worms within 2-3 weeks of infection. We find that resistance correlates not only with the adaptive Th2 response, including IL-10 but with activation of innate lymphoid cell and macrophage populations. In addition, the titer and specificity range of the serum antibody response is maximal in resistant mice. In susceptible strains, Th2 responses were found to be counterbalanced by IFN-γ-producing CD4(+) and CD8(+) cells, but these are not solely responsible for susceptibility as mice deficient in either CD8(+) T cells or IFN-γ remain unable to expel the parasites. Foxp3(+) Treg numbers were comparable in all strains, but in the most resistant SJL strain, this population does not upregulate CD103 in infection, and in the lamina propria the frequency of Foxp3(+)CD103(+) T cells is significantly lower than in susceptible mice. The more resistant SJL and BALB/c mice develop macrophage-rich IL-4Rα-dependent Type 2 granulomas around intestinal sites of larval invasion, and expression of alternative activation markers Arginase-1, Ch3L3 (Ym1) and RELM-α within the intestine and the peritoneal lavage was also strongly correlated with helminth elimination in these strains. Clodronate depletion of phagocytic cells compromises resistance of BALB/c mice and slows expulsion in the SJL strain. Thus, Type 2 immunity involves IL-4Rα-dependent innate cells including but not limited to a phagocyte population, the latter likely involving the action of specific antibodies.

Figure 1

Variation in susceptibility to primary infection with H. polygyrus manifests first with early differences in parasite fecundity, and subsequent loss of adult worms. Age-matched female SJL, BALB/c, C57BL/6 and CBA mice were infected with 200 H. polygyrus L3 larvae by gavage. Data presented are pooled from two independent experiments. Bars in a–d and h indicate means and standard errors of the mean. (a, b) Luminal adult parasites at day 14 and 28 of infection. (c, d) Fecal egg counts at day 14 and 28 of infection. (e, f) Representative images of d14 intestinal granulomas in different mouse strains. Scale bars show 5 mm. (g) Representative image of intestinal granuloma in a d14-infected BALB/c mouse, hemotoxylin and eosin-stained. Scale bar shows 200 μm. (h) Number of granulomas in small intestine in different strains of mice at day 14 of infection. (i) Negative relationship between egg numbers and granulomas at day 14 of infection. Statistically significant differences are indicated; ***P<0.001. A full colour version of this figure is available at the Immunology and Cell Biology journal online.

Figure 2

Profile of MLN and peritoneal cells during H. polygyrus infection of different mouse strains. MLNC from naive (Un) or day 7 H. polygyrus-infected female SJL, BALB/c, C57BL/6 and CBA were stimulated in vitro with medium alone (M) or anti-CD3 antibody and supernatants assayed for secreted cytokines (a–d), or stained directly for intracellular IFN-γ (e) after 4 h of stimulation with PMA, ionomycin and brefeldin. Also shown are the number of CD11b⁺F4/0⁺ macrophages and SiglecF⁺ eosinophils stained in the peritoneal lavage of day 7infected mice (f, g) and lineage-negative innate lymphoid cells from the MLN-expressing intracellular GATA-3 and surface CD127, ICOS and T1/ST2 (h) from day 10-infected mice, with total MLN cell number also shown (i). Data in a-e represent mean values±standard errors, from ⩾3 individual mice of each strain, from one of 4 independent experiments with similar results; data in f–i are pooled from two independent experiments with 3–6 mice in each group and show means with standard errors. Further data on T cell cytokine profiles are presented in Supplementary Figures 1 and 2 and on the gating of innate lymphoid cells in Supplementary Figure 3. (a–d) Polyclonal cytokine release by anti-CD3 stimulated MLNC—IL-4, IL-10, IL-13 and IFN-γ. (e)Intracellular staining for IFN-γ among CD8⁺ MLN T cells. (f) Macrophage (CD11b⁺F4/80⁺) numbers in the peritoneal lavage of naive and d7-infected mice. (g) SiglecF⁺ cell numbers in the same peritoneal lavage samples. (h) Number of ICOS⁺, T1/ST2⁺, CD127⁺, GATA-3⁺ lineage^- MLNC from naive and d10-infected mice. (i) Total MLN cell numbers from the same animals. Statistically significant differences are indicated; *P<0.05; **P<0.01; ***P<0.001.

Figure 3

Immunity to H. polygyrus is completely dependent on IL-4Rα-mediated signaling but is not significantly influenced by CD8 T cells and IFN-γ. (a) Ablation of intestinal granuloma formation in IL-4Rα-deficient and BALB/c wild-type mice. Data in this and other panels show means and standard errors. (b, c) Egg production and adult worm loads in IL-4Rα-deficient and BALB/c wild-type mice. (d, e) Effect of anti-CD8 antibody depletion on egg production and adult worm load following infection in C57BL/6 mice. (f, g) Adult worm loads and egg production and in IFN-γ-deficient and C57BL/6 wild-type mice. Statistically significant differences are indicated; **P<0.01; ***P<0.001.

Figure 4

Qualitative differences in CD4⁺FoxP3⁺ regulatory T cells from SJL mice. CD4⁺ T cells from naive or day 7 H. polygyrus-infected female SJL, BALB/c, C57BL/6 and CBA mice were stained for expression of Foxp3 together with Helios, CD103 and GATA3. Results are pooled from two experiments. Positive cells are expressed as the mean with standard error from 2–6 animals per group. Experiments in d and e show LP cells extracted from SJL and C57BL/6 mice before and after a 7-day infection, and are pooled results from two experiments. (a) Foxp3 expression as a percentage of CD4⁺ cells in MLN of naive and d7 infected mice. (b) CD103 expression as a percentage of CD4⁺Foxp3⁺ cells in the MLN of naïve and d7 infected mice. (c) GATA3 staining of CD4⁺Foxp3⁺ cells in the MLN of naïve or d7 infected mice. (d) Foxp3 expression of CD4⁺ LP cells. (e) CD103 expression as a percentage of CD4⁺Foxp3⁺ cells in the LP of naive and d7 infected mice. (f) Foxp3 expression as a percentage of CD4⁺ cells from splenocyte cultures for 72 h with media, 10 μg/ml HES or 10 ng/ml rhTGF-β. Statistically significant differences are indicated; *P<0.05; **P<0.01; ***P<0.001.

Figure 5

Expression of alternative activation genes in infected animals. All results are from two experiments pooled with 4–6 mice per group represented as mean values with standard error. (a, b) RELM-α and Chi3L3 (Ym1) expression by peritoneal macrophages of naive and d7 infected mice. (c) Expression of RELM-α protein in duodenal tissue homogenate of naïve and d7 infected mice by ELISA. (d–f) Quantification of RELM-α, Arginase-1 and RELMβ in duodenal tissue of naïve and d7-infected mice by real-time PCR, relative to the housekeeping gene GAPDH. Statistically significant differences are indicated; *P<0.05; **P<0.01; ***P<0.001.

Figure 6

Primary resistance to H. polygyrus infection is associated with granuloma-like formation in the gut wall expressing Chi3L3. Histological sections of granulomas and normal intestinal tissue from H. polygyrus-infected mice. All images in (a–c) were captured and are presented at the same magnification. (Row a) Hematoxylin and eosin staining of sections from mice 14 days following infection; note presence of trapped larvae in granuloma from SJL mouse. Scale bars show 200 μm. (Rows b, c) Staining with anti-Chi3L3 and control antibodies in sections from the same mice. Scale bars show 200 μm. (Row d) Staining with anti-Chi3L3 in SJL and BALB/c mice; note both staining of diffuse protein within the granuloma and within large mononuclear cells (indicated by arrows). Scale bars show 200 μm. A full colour version of this figure is available at the Immunology and Cell Biology journal online.

Figure 7

Clodronate depletion of macrophages in BALB/c and SJL mice infected with H. polygyrus. All results are pooled from two experiments and bars represent the mean and standard error. Data showing the efficacy of clodronate depletion are presented in Supplementary Figure 5. (a) Adult worm burdens from female BALB/c mice treated i.v. with either PBS or clodronate liposomes and infected for 28 days with H. polygyrus. (b) Egg burden in feces from female BALB/c mice treated i.v. with either PBS or clodronate liposomes, at day 14 and 28 after infection with H. polygyrus. (c) Intestinal granuloma counts from female BALB/c mice treated i.v. with either PBS or clodronate liposomes and infected for 28 days with H. polygyrus. (d) Intestinal granuloma counts from female SJL mice treated i.v. with either PBS or clodronate liposomes and infected for 28 days with H. polygyrus. (e) Egg burden in feces from female SJL mice treated i.v. with either PBS or clodronate liposomes, at day 14 and 28 after infection with H. polygyrus. (f) Adult worm burdens from female SJL mice treated i.v. with either PBS or clodronate liposomes and infected for 14 or 28 days with H. polygyrus. Statistically significant differences are indicated; *P<0.05; **P<0.01; ***P<0.001.