Type 2 innate immunity in helminth infection is induced redundantly and acts autonomously following CD11c(+) cell depletion

Abstract

Infection with gastrointestinal helminths generates a dominant type 2 response among both adaptive (Th2) and innate (macrophage, eosinophil, and innate lymphoid) immune cell types. Two additional innate cell types, CD11c(high) dendritic cells (DCs) and basophils, have been implicated in the genesis of type 2 immunity. Investigating the type 2 response to intestinal nematode parasites, including Heligmosomoides polygyrus and Nippostrongylus brasiliensis, we first confirmed the requirement for DCs in stimulating Th2 adaptive immunity against these helminths through depletion of CD11c(high) cells by administration of diphtheria toxin to CD11c.DOG mice. In contrast, responsiveness was intact in mice depleted of basophils by antibody treatment. Th2 responses can be induced by adoptive transfer of DCs, but not basophils, exposed to soluble excretory-secretory products from these helminths. However, innate type 2 responses arose equally strongly in the presence or absence of CD11c(high) cells or basophils; thus, in CD11c.DOG mice, the alternative activation of macrophages, as measured by expression of arginase-1, RELM-α, and Ym-1 (Chi3L3) in the intestine following H. polygyrus infection or in the lung following N. brasiliensis infection, was unaltered by depletion of CD11c-expressing DCs and alveolar macrophages or by antibody-mediated basophil depletion. Similarly, goblet cell-associated RELM-β in lung and intestinal tissues, lung eosinophilia, and expansion of innate lymphoid ("nuocyte") populations all proceeded irrespective of depletion of CD11c(high) cells or basophils. Thus, while CD11c(high) DCs initiate helminth-specific adaptive immunity, innate type 2 cells are able to mount an autonomous response to the challenge of parasite infection.

Fig 1

CD11c⁺ cell depletion ablates adaptive Th2 responsiveness. CD11c.DOG mice were given 8 ng/g DTx (or PBS as a control) daily from day 1 to 6 of mouse-adapted N. brasiliensis (Nb) infection, and MLNC were harvested at day 7 postinfection. (A and B) DC depletion in the mesenteric lymph node (MLN) as staining for CD11c and MHC class II, shown as a representative bivariant plot scaled to 210,000 events (A) and a graphical summary of all data (B). (C) Intracellular IL-4 expression by MLN CD4⁺ T cells in PBS- and DTx-treated uninfected and infected (Nb) mice. (D to I) Cytokine release by cultured MLNC stimulated with NES (black bars) or medium (open bars), in PBS- and DTx-treated uninfected and infected (Nb) mice, assayed for IL-4 (D), IL-10 (E), IL-5 (F), IL-13 (G), IFN-γ (H), and IL-17 (I). Data presented are means ± standard errors (SE) for LNC from 3 to 5 individual mice per group and are representative of two similar experiments. Statistically significant differences are shown as * (P < 0.05), ** (P < 0.01), or *** (P < 0.001).

Fig 2

Basophils do not initiate or contribute to adaptive or innate type 2-associated parameters following primary N. brasiliensis infection. (A to D) Antigen-specific and polyclonal cytokine responses of popliteal LN at 5 days following transfer of bone marrow-derived DCs or basophils, pulsed with medium alone or with N. brasiliensis ES (NES) antigen. Cells were challenged in vitro with medium alone (open bars), 5 μg/ml NES (black bars), or 1 μg/ml anti-CD3 (gray bars) and supernatants collected after 72 h. (A) NES-specific IL-4 responses or medium-alone controls; (B) polyclonal IL-4 responses (NES responses are shown for comparison); (C) NES-specific IL-10 responses; (D) NES-specific IL-17 responses. (E and F) Depletion of peripheral blood CD3⁻ CD19⁻ IgE⁺CD49b⁺ c-kit⁻ basophils in naïve and 4-day N. brasiliensis-infected mice treated with isotype control or MAR-1 antibody. (G and H) NES-specific IL-4 and IL-10 production in response to medium (open bars) or NES (black bars) by MLNC from naïve, isotype-treated, and MAR-1-treated N. brasiliensis-infected (Nb) mice. (I and J) RT-PCR for arginase-1 and Ym-1 expression from lung homogenates from naïve, isotype-treated, and MAR-1-treated N. brasiliensis-infected (Nb) mice. (K) Intracellular RELM-α staining of CD11b⁺ F4/80⁺ SiglecF⁻ AAMs from naïve, isotype-treated, and MAR-1-treated N. brasiliensis-infected (Nb) mice. (L) Eosinophil populations identified as CD11b⁺ Siglec F⁺ cells in the lungs of naïve, isotype-treated, and MAR-1-treated N. brasiliensis-infected (Nb) mice. For transfer experiments, data presented are means ± SE for LNC from 4 individual mice and are representative of two similar experiments. For infection experiments, MLNC and lungs were harvested at 7 days following infection. Data presented are means from 4 or 5 individual mice and are representative of two similar experiments. Statistically significant differences are shown as * (P < 0.05), ** (P < 0.01), or *** (P < 0.001).

Fig 3

Basophil depletion does not diminish adaptive or innate type 2-associated parameters following H. polygyrus infection. (A and B) HES-specific IL-4 and IL-10 production in response to medium (open bars) or HES (black bars) by MLNC from naïve, isotype-treated, and MAR-1-treated H. polygyrus-infected (Hp) mice. (C and D) RT-PCR for arginase-1 and RELM-β expression in intestinal tissue from naïve, isotype-treated, and MAR-1-treated H. polygyrus-infected (Hp) mice. (E) Eosinophil populations in the mesenteric lymph nodes of isotype- and MAR-1-treated H. polygyrus-infected (Hp) mice. Mesenteric LNC and lungs were harvested at 7 days following infection. Data presented are means from 4 or 5 individual mice and are representative of two similar experiments. Statistically significant differences are shown as ** (P < 0.01) or *** (P < 0.001).

Fig 4

Depletion of CD11c⁺ cells does not diminish type 2 innate responder populations. (A) Expression of Siglec F and CD11b by innate immune cells in lung homogenates from naïve and N. brasiliensis-infected CD11c.DOG mice given PBS or DTx. Alveolar macrophages (Siglec F⁺ CD11b^int CD11c⁺ F4/80⁺) are shown as light gray, eosinophils (Siglec F^+t CD11b⁺ CD11cⁱⁿ F4/80^int SSC^hi) as dark gray, and nonalveolar macrophages (Siglec F⁻ CD11b⁺ F4/80⁺) as black. A total of 20,000 events are shown for each analysis. (B and C) Lung eosinophils (B) and alveolar macrophages (C) in PBS- and DTx-treated uninfected and infected (Nb) mice. (D) F4/80 expression on alveolar (light gray line) and nonalveolar (black line) macrophages compared to an isotype control (filled light gray) in PBS- and DTx-treated N. brasiliensis-infected mice. (E) RELM-α expression by CD11b⁺ Siglec F⁻ F4/80⁺ macrophages in PBS-treated naïve (filled dark gray histogram with black line) and N. brasiliensis-infected (filled light gray histogram with light gray line) mice along with DTx-treated groups (open histograms with corresponding lines) (left panel). The associated interstitial macrophage RELM-α staining intensity (right panel) is shown for all samples. (F and G) RT-PCR for Ym-1 (Chi3L3) (F) and RELM-α (G) mRNA expression in lung homogenates from PBS- and DTx-treated uninfected and infected (Nb) mice. Lungs were harvested at 7 days following infection. Data presented are means from 4 or 5 individual mice and are representative of two similar experiments. Statistically significant differences are shown as * (P < 0.05), ** (P < 0.01), or *** (P < 0.001).

Fig 5

Innate type 2 responsiveness in the intestinal tract is not compromised by CD11c⁺ cell depletion. (A and B) RT-PCR for Ym-1 (Chi3L3) (A) and RELM-β (B) mRNA expression in intestinal homogenates from PBS- and DTx-treated uninfected and infected (Nb) mice. (C) Eosinophilia (SSC^high cells) in the MLNs of PBS- and DTx-treated uninfected and infected (Nb) mice. (D and E) Intracellular IL-4 and IL-13 expression by CD3⁻ CD19⁻ NBNT cells in MLNs of PBS- and DTx-treated uninfected and infected (Nb) mice. (F) Surface phenotype of CD3⁻ CD19⁻ IL-13⁺ NBNT cells in N. brasiliensis-infected mice given PBS (filled black) or DTx (filled dark gray) compared to a positive control (black line) or an isotype control (filled light gray). Data presented are means from 4 or 5 individual mice/group and are representative of two similar experiments; tissues from infected mice were harvested at day 7 postinfection. Statistically significant differences are shown as * (P < 0.05), ** (P < 0.01), or *** (P < 0.001).