Memory T(H)2 cells induce alternatively activated macrophages to mediate protection against nematode parasites

Abstract

Although primary and memory responses against bacteria and viruses have been studied extensively, T helper type 2 (T(H)2) effector mechanisms leading to host protection against helminthic parasites remain elusive. Examination of the intestinal epithelial submucosa of mice after primary and secondary infections by a natural gastrointestinal parasite revealed a distinct immune-cell infiltrate after challenge, featuring interleukin-4-expressing memory CD4(+) T cells that induced IL-4 receptor(hi) (IL-4R(hi)) CD206(+) alternatively activated macrophages. In turn, these alternatively activated macrophages (AAMacs) functioned as important effector cells of the protective memory response contributing to parasite elimination, demonstrating a previously unknown mechanism for host protection against intestinal helminths.

Figure 1

CD4⁺ T cell–dependent protective mechanisms occur at early stages after Hp challenge inoculation. (a) Anti-CD4 was administered to challenged mice at days 0 and 7⁽ⁱ⁾, 7⁽ⁱⁱ⁾, 9⁽ⁱⁱⁱ⁾ or 11^(iv) after inoculation. Treatment group number indicated in parentheses. (b) Worm and egg burdens determined 14 d after challenge in mice receiving anti-CD4 treatments. Mean and s.e.m. of five mice per treatment group are shown and are representative of three separate experiments. *P < 0.05 by Kruskal-Wallis test followed by Dunn post hoc test. (c) Parasite larvae recovered from the small intestines of primed or challenged mice using a Baermann apparatus 4 (black bars) and 7 d (gray bars) after infection. Mean and s.e.m. of four or five mice per treatment group are shown and are representative of three separate experiments. * P < 0.001 by Student t-test. (d) Fluorescence immunohistochemistry of the host-parasite interface 4 d after challenge, showing IL-4Rα^hi (red) cells encircling the parasite, Gr-1⁺ (green) cells accumulating adjacent to the parasite and CD4⁺ T (blue) cells surrounding both the parasite and the Gr-1⁺ population (M, muscularis; Lp, lamina propria; Hp, H. polygyrus). (e) Cytokine mRNA expression levels determined by real-time RT-PCR for CD4⁺, Gr-1⁺ and treated lamina propria (CD4⁻ Gr-1⁻) samples obtained from the host-parasite interface using IF-LCM. Mean and s.e.m. from five mice per treatment group are plotted on a log scale and are representative of two independent experiments. (f) IL-4 protein (green) was detected in situ in the region occupied by CD4⁺ T cells (blue), which surround Gr-1⁺ neutrophils (red). (g) Rat IgG1–Alexa488 isotype control antibody showed minimal nonspecific binding in a serial section of the tissue shown in f. (h) F4/80⁺ macrophages (blue) costained for the IL-4Rα (red), as demonstrated by the ring of purple cells around the parasite. (i) F4/80⁺ macrophages (red) accumulating at the host-parasite interface expressed CD206 (green), resulting in a yellow-orange stain indicative of their alternatively activated state.

Figure 2

AAMac accumulation 4 d after H. polygyrus challenge is Stat6 dependent. (a,b) Four days after primary infection, Gr-1⁺ neutrophils (green) accumulated adjacent to the parasite, with substantially fewer CD4⁺ T cells (blue), IL-4R^hi cells (red), F4/80⁺ macrophages (b, red) and CD206⁺ cells (green) compared to what is seen after secondary infection (see Fig. 1d). (c) F4/80⁺ cells dissected from the host-parasite interface by IF-LCM 4 d after challenge showed gene expression profiles characteristic of AAMacs, including undetectable iNOS (left) and high levels of arginase, Fizz1 (right), Ym1 and AMCase. Mean and s.e.m. of four or five mice per treatment group is plotted on log scales. (d–g) CD4⁺ T cells transferred from H. polygyrus–primed mice induced alternatively activated macrophages in wild-type (d,e) but not Stat6^-/- recipients (f,g). Gr-1⁺ neutrophils (red) and CD4⁺ T cells (blue) accumulated around the parasite 4 d after infection following adoptive transfer of T_H2 memory cells into wild-type (d) and Stat6^-/- (f) recipients. Serial tissue sections show staining for macrophages (F4/80, red) and macrophage mannose receptor (CD206, green), showing that wild-type recipients (e) developed alternatively activated macrophages (yellow), whereas Stat6^-/- recipients (g) did not. Lp, lamina propria; m, muscularis; Hp, H. polygyrus.

Figure 3

Macrophage depletion abrogates a protective T_H2 memory response. (a) Parasite and egg burdens determined 14 d after challenge in mice treated with Cl₂MDP or PBS liposomes. (b) Larvae collected using a Baermann apparatus from intestines of challenged mice receiving clodronate or PBS liposome treatment at day 4 and 7 after inoculation. Mean and s.e.m. of five mice per groups are shown; results are representative of three separate experiments. *P < 0.01 by ANOVA followed by Tukey post hoc test. (c–f) Immunofluorescence of the host-parasite interface 4 d after challenge showed that neither clodronate (d) nor PBS liposome (c) treatment affected infiltration of Gr-1⁺ (red), CD4⁺ (blue) or CD11c⁺ (green) cells at the host-parasite interface. F4/80⁺CD206⁺ macrophages were specifically depleted in the CL₂MDP liposome–treated (f) as compared to the PBS liposome–treated group (e). Cytokine gene expression in mesenteric lymph node (MLN; g, left) and proximal small intestine (right) samples were collected 7 d after infection, as determined by real-time RT-PCR and plotted on log scales. Lp, lamina propria; m, muscularis; Hp, H. polygyrus.

Figure 4

Arginase inhibition abrogates protective memory response to H. polygyrus. (a,b) Shown are parasite and egg burdens determined 14 d after inoculation (a), and larvae recovered from infected small intestines 4 (b, left) or 7 d (right) after infection using a Baermann apparatus, in challenged mice that were given 0.2% BEC (2′HpD14 + BEC) in their drinking water. Bars represent mean and s.e.m. of five mice per group. **P < 0.001 and *P < 0.01 by ANOVA followed by Tukey post hoc test. (c–e) Cytochrome oxidase (black granules) staining of parasite cross-sections 7 d after primary infection (c), secondary infection (d) and secondary infection with BEC treatment (e). Staining intensity within parasite cross-sections was scored blindly (1–4, lightest to darkest). (f) Box plots of scores from four mice per treatment group are plotted. *P < 0.05 by Kruskal-Wallis test followed by Dunn post hoc test.