Ccl24 neutralization reduced expression of early type-2 memory responses, correlating with a loss of resistance to <i>H</i>. <i>polygyrus</i> in <i>Map3k8</i>^{<i>–/–</i>}mice.

<p>A) <i>Arg1</i> expression; B) <i>Retnla</i> expression; C) <i>Chil3</i> expression; D) <i>Ear11</i> expression from small intestinal duodenal tissue of D5 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice treated with Ccl24 neutralizing antibody or isotype control antibody. E) Adult luminal worms in the D21 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice treated with Ccl24 neutralizing antibody or isotype control. Data represents 8–11 mice/genotype/group, pooled from 2 independent experiments. * denotes p≤0.05 using Mann-Whitney test.</p

<i>Map3k8</i>^{<i>–/–</i>}mice are resistant to <i>H</i>. <i>polygyrus</i> infection.

<p>A) WT and <i>Map3k8</i>^{<i>–/–</i>}mice were infected with 200 L3 stage <i>H</i>. <i>polygyrus</i> larvae. Adult luminal worms from the intestinal tissue were counted on days 14 (D14) and 28 (D28). B) Fecal egg burden in <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice at D14 and D28. C) ATP levels of adult <i>H</i>. <i>polygyrus</i> worms harvested from the duodenal tissue of WT and <i>Map3k8</i>^{<i>–/–</i>}mice at day 14 post infection. D) Representative FACS plots from the mesenteric lymph nodes (mLNs) and frequency of CD4⁺ <i>Il4</i>^GFP+ and CD4⁺ <i>FoxP3</i>^RFP+ T cells from the spleen, mLNs and Peyer’s patches (PP) of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. E) Total number of cells from the spleen, mLNs and PP of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. F) Arbitrary units (a.u.) of <i>H</i>. <i>polygyrus</i> adult worm extract (HEX)-specific IgG1 in the serum of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. G) HEX-specific IL-5 and IL-13 in mLN cell culture supernatants of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. Data from readouts in A) and B) represent 7–8 mice/genotype pooled from 2 independent experiments. Data from C)—G) is representative of 2–3 independent experiments with 3–4 mice/genotype and 4 adult worms/mouse. * and ** denote p≤0.05 using a two-tailed Mann-Whitney test.</p

Enhanced <i>Ccl24</i>, myeloid cells and <i>Il4</i>^<i>GFP+</i> Th2 cell infiltration in <i>Map3k8</i>^{<i>–/–</i>}mice.

<p>A) Representative FACS profile for CD11c⁺ CD11b⁺ cells from the intestinal LP of D5 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. B) <i>Ccl24</i> and <i>Ear11</i> expression from CD11c⁺ CD11b⁺ cells D5 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. C-H) Frequency and number of (C) eosinophils (live/CD45⁺/SiglecF⁺/SSChi); (D) neutrophils (live/CD45⁺/SiglecF^-/CD11b⁺/Ly6G⁺); (E) monocytes (live/CD45⁺/SiglecF^-/CD11b⁺/Ly6G^-/Ly6C⁺); (F) <i>Il4</i>^<i>GFP+</i> Th2 cells (live/CD45⁺/CD4⁺/CD3⁺/TCRβ⁺/<i>Il4</i>^<i>GFP+</i>) in the intestinal LP of D5 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. Data from B) is representative of 2 independent experiments with 3 biological replicates per group. Data from C-H) is representative of 2 independent experiments with 5–6 mice/group. * and ** denotes p≤0.05 using Mann-Whitney test.</p

Accelerated expulsion in <i>Map3k8</i>^{<i>–/–</i>}mice not due to alterations in the intestinal microbiota.

<p>A) Schematic representation of co-housing, infection and fecal sample collection from two different groups of WT and <i>Map3k8</i>^{<i>–/–</i>}mice. Group 1 mice were co-housed together for 14 days and then separated into the different genotypes for the next 14 days. Group 2 mice included the two genotypes housed individually for 14 days following which they were cohoused together for 14 days. Both groups of mice were infected with 200 L3 stage <i>H</i>. <i>polygyrus</i> larvae and fecal samples (S) were collected at the indicated times points. B) Changes in fecal microbiota composition over time (represented as a percentage of total bacterial content) in the two different groups of WT and <i>Map3k8</i>^{<i>–/–</i>}mice infected with <i>H</i>. <i>polygyrus</i>. Data represents 5 mice/genotype. Shaded grey sections indicate the time points after infection with <i>H</i>. <i>polygyrus</i>. C) Adult luminal worm burden in the two different groups of WT and <i>Map3k8</i>^{<i>–/–</i>}mice infected with <i>H</i>. <i>polygyrus</i> at day 28. D) Fecal egg burden in the two different groups of WT and <i>Map3k8</i>^{<i>–/–</i>}mice infected with <i>H</i>. <i>polygyrus</i> at day 28. Data represents 6 mice/group pooled from 2 independent experiments. * denotes p≤0.05 using Mann-Whitney test.</p

Elevated Ccl24 from <i>Map3k8</i>^{<i>–/–</i>}mice mediates resistance to <i>H</i>. <i>polygyrus</i> infection.

<p>A) Ratios of ratio plot depicting differentially expressed genes in the duodenal tissue of D5 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. The x axis represents fold change for gene differentially expressed in infected <i>Map3k8</i>^{<i>–/–</i>}mice over infected WT mice and the y axis represents fold change for gene differentially expressed in infected WT or <i>Map3k8</i>^{<i>–/–</i>}mice relative to their respective naïve uninfected controls. B) Top 10 gene regulatory pathways upregulated in D5 <i>H</i>. <i>polygyrus</i> infected <i>Map3k8</i>^{<i>–/–</i>}/ WT mice gut tissue relative to naive control mice as predicted by ingenuity pathway analysis (IPA). The microarray analysis was performed on duodenal tissue from 3 biological replicate samples/genotype for each group.</p

Top 10 upregulated genes in D5 <i>H</i>. <i>polygyrus</i> infected <i>Map3k8</i>^{<i>–/–</i>}mice relative to WT mice.

<p>Top 10 upregulated genes in D5 <i>H</i>. <i>polygyrus</i> infected <i>Map3k8</i>^{<i>–/–</i>}mice relative to WT mice.</p

CD11c⁺ cells deficient in TPL-2 but not Villin⁺ cells, LysM⁺ cells or CD4⁺ cells mediate enhanced expulsion of <i>H</i>. <i>polygyrus</i>.

<p>A) Adult luminal worm burden in D21 <i>H</i>. <i>polygyrus</i> infected WT, <i>Map3k8</i>^{<i>–/–</i>}, <i>Villin</i>^<i>Cre</i><i>Map3k8</i>^<i>fl/fl</i> and <i>Cd11c</i>^<i>Cre</i><i>Map3k8</i>^<i>fl/ko</i> mice. B) Adult luminal worm burden in D21 <i>H</i>. <i>polygyrus</i> infected WT, <i>Map3k8</i>^{<i>–/–</i>}, <i>LysM</i>^<i>Cre</i><i>Map3k8</i>^<i>fl/wt</i> and <i>LysM</i>^<i>Cre</i><i>Map3k8</i>^<i>fl/ko</i> mice. C) Adult luminal worm burden in D21 <i>H</i>. <i>polygyrus</i> infected WT, <i>Map3k8</i>^{<i>–/–</i>}, <i>Cd4</i>^<i>Cre</i><i>Map3k8</i>^<i>wt/wt</i> and <i>Cd4</i>^<i>Cre</i><i>Map3k8</i>^<i>fl/ko</i> mice. Data from A) is representative of 2 independent experiments with 4–5 mice/genotype and data from B) and C) is 8–13 mice/genotype pooled from 2 independent experiments. * and ** denotes p≤0.05 using Mann-Whitney test.</p

<i>H</i>. <i>polygyrus/ P</i>. <i>chabaudi</i> co-infection leads to impaired Th2 responses.

<p>A-C). Triple reporter mice were orally infected with 200 <i>H</i>. <i>polygyrus</i> larvae. 6 days post-infection, mice were infected i.p. with 10⁵<i>P</i>. <i>chabaudi</i>. At day 8 of <i>P</i>. <i>chabaudi</i> infection (d14 <i>H</i>. <i>polygyrus</i>), mice were harvested. B). Total numbers of CD4⁺CD44^hi<i>Il4</i>^gfp+ cells in the mesenteric lymph nodes. Data are representative of 5 independent experiments with 2–4 mice per group. C). IgE measured in the serum by ELISA from 3 pooled experiments. D and E). C57BL/6 mice were infected with 200 <i>H</i>. <i>polygyrus</i> larvae, treated on 2 consecutive days (days 14–15) with pyrantel pamoate (5 mg), infected with 10⁵<i>P</i>. <i>chabaudi</i>, and re-infected with <i>H</i>. <i>polygyrus</i>. Adult worms in intestine were counted on day 51. Data are representative of 4 independent experiments with 6–7 mice per group. * denotes P<0.05.</p

Blockade of IL-12 and IFNγ during co-infection preserves Th2 responses.

<p>A-C). C57BL/6 mice were orally infected with 200 <i>H</i>. <i>polygyrus</i> larvae. 6 days post-infection, mice were infected with 10⁵<i>P</i>. <i>chabaudi</i>. At day 8-post infection with <i>P</i>. <i>chabaudi</i> (d14 <i>H</i>. <i>polygyrus</i>), mice were harvested. Mice were treated with 0.5 mg of anti-IL-12 and anti-IFNγ i.p. at days 0, 5, and 11. B). Total numbers of CD4⁺CD44^hi<i>Il4</i>^gfp+ cells in the mesenteric lymph nodes. C). IgE measured in the serum by ELISA. Data are representative of 2 independent experiments with 6 mice per group. * denotes P<0.05.</p

<i>In vitro</i> Th2 cells produce IFNγ and protect <i>Rag1</i>^{<i>–/–</i>}mice during <i>Plasmodium</i> infection.

<p>A). Experimental set-up: 2-week <i>in vitro</i> polarized Th2 cells were FACS sorted as CD4⁺<i>Il4</i>^<i>gfp+</i><i>Ifng</i>^{<i>yfp–</i>}<i>Il17a</i>^{<i>FP635–</i>} and transferred i.v. to <i>Rag1</i>^{<i>–/–</i>}mice. As a control, a group of <i>Rag1</i>^{<i>–/–</i>}mice received naïve CD4⁺ T cells. A second control group received no T cells. Recipient mice were infected with 10⁵<i>P</i>. <i>chabaudi</i> i.p. on day 14 post-transfer and harvested at day 8 post-infection. B). Percent and total number of CD4⁺<i>Il4</i>^<i>gfp+</i> and <i>Ifng</i>^<i>yfp+</i> cells in the spleen, as determined by FACS. C). Serum IFNγ levels determined by ELISA. D). Percent parasitemia was determined by blinded counting of Giemsa-stained blood smears. E and F). Hemoglobin and eHred blood cell counts were measured in peripheral blood by Vetscan. Data is representative of at least 3 independent experiments, with 3–5 mice per group. G). Converted CD4⁺TCRβ⁺<i>Ifng</i>^<i>yfp+</i><i>Il4</i>^{<i>gfp–</i>}<i>Il17a</i>^{<i>FP635–</i>} cells were sorted from pooled spleens of 3 recipient <i>Rag1</i>^{<i>–/–</i>}mice at day 8 post-<i>P</i>. <i>chabaudi</i> infection. H). Sorted <i>Ifng</i>^<i>yfp+</i> cells were cultured <i>in vitro</i> in Th2 conditions for 5 days. ELISAs for IFNγ (H), IL-5 and IL-13 (I) were run on cell supernatants. Error bars represent technical replicates. Data are representative of 3 independent experiments. * denotes P<0.05.</p