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. 2020 Oct 23:11:592325.
doi: 10.3389/fimmu.2020.592325. eCollection 2020.

Defined Intestinal Regions Are Drained by Specific Lymph Nodes That Mount Distinct Th1 and Th2 Responses Against Schistosoma mansoni Eggs

Johannes U Mayer  1   2 , Sheila L Brown  3 , Andrew S MacDonald  3 , Simon W Milling  1
Affiliations

Defined Intestinal Regions Are Drained by Specific Lymph Nodes That Mount Distinct Th1 and Th2 Responses Against Schistosoma mansoni Eggs

Johannes U Mayer et al. Front Immunol. .

Abstract

The balance of type 1 and type 2 immune responses plays a crucial role in anti-helminth immunity and can either support chronic infection or drive type 2 mediated expulsion of the parasite. Helminth antigens and secreted molecules directly influence this balance and induce a favorable immunological environment for the parasite's survival. However, less is known if the site of infection also influences the balance of type 1 and type 2 immunity. Here, we report that tissue-specific immune responses are mounted against helminth antigens, which elicited strong IL-4 responses when injected into the skin, while the same antigen, delivered into the intestinal subserosa, induced increased IFN-γ and reduced Th2 responses. Immune responses in individual mesenteric lymph nodes that drain defined regions of the intestine furthermore displayed a site-specific pattern of type 1 and type 2 immunity after Schistosoma mansoni or Heligmosomoides polygyrus infection. S. mansoni egg-specific Th2 responses were detectable in all mesenteric lymph nodes but Th1 responses were only present in those draining the colon, while H. polygyrus infection elicited mixed Th1 and Th2 responses in the lymph nodes associated with the site of infection. Similar site-specific type 1 and type 2 immune responses were observed in the draining lymph nodes after the controlled delivery of S. mansoni eggs into different segments of the small and large intestine using microsurgical techniques. Different subsets of intestinal dendritic cells were hereby responsible for the uptake and priming of Th1 and Th2 responses against helminth antigens. Migratory CD11b+CD103- and especially CD11b+CD103+ DC2s transported S. mansoni egg antigens to the draining lymph nodes to induce Th1 and Th2 responses, while CD103+ DC1s induced only IFN-γ responses. In contrast, H. polygyrus antigens were predominantly transported by CD11b+CD103- DC2s and CD103+ DC1s and all DC subsets induced similar Th1 but weaker Th2 responses, compared to S. mansoni egg antigens. The development of adaptive anti-helminth immune responses is therefore influenced by the antigen itself, the uptake and priming characteristics of antigen-positive dendritic cell subsets and the site of infection, which shape the level of Th1 and Th2 responses in order to create a favorable immunological environment for the parasite.

Keywords: Heligmosomoides polygyrus bakeri; Schistosoma mansoni; dendritic cells; helminth antigen; mesenteric lymph node; microsurgery; mucosal immunology; th1/th2 balance.

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Figures

Figure 1
Figure 1
Schistosoma mansoni eggs induce increased IFN-γ and decreased Th2 responses when injected into the intestine compared to the footpad. (A, B) Lymph node IFN-γ+ and IL-4+ CD4+ T cell responses in naïve mice or 5 days after the injection of 2,500 S. mansoni eggs or PBS in the footpad or intestinal subserosa (n = 5 mice per group, combined data from three independent experiments; mean ± SEM; unpaired t-tests compare LN responses within each experimental group; ***p ≤ 0.001). (C) Ratio of IFN-γ+ and IL-4+ CD4+ T cell responses after S. mansoni egg injection from data shown in (B) (mean ± SEM; unpaired t-test compares LN responses; **p ≤ 0.01). (D) From the same experiments, lymph node cells were restimulated with soluble egg antigen (SEA) or media for 3 days in vitro and Th1 and Th2 cytokines were measured by ELISA (mean ± SEM; unpaired t-tests compare LN cytokine responses within each treatment group; ***p ≤ 0.001). (E) Ratio of IFN-γ and Th2 cytokine levels after SEA restimulation from data shown in (D) (mean ± SEM; unpaired t-test; **p ≤ 0.01, ***p ≤ 0.001).
Figure 2
Figure 2
Th1 and Th2 responses are detected in individual mesenteric lymph nodes after Schistosoma mansoni infection and egg immunization. (A) Schematic and photograph of the murine intestine illustrate the different segments and indicate the position and nomenclature of the individual draining lymph nodes. (B) Total cell counts of pooled or individual mesenteric lymph nodes (MLNs) seven weeks after S. mansoni (Sm) infection (n = 5 mice per group, representative of two independent experiments; mean ± SEM; unpaired t-tests compare cell numbers between naïve and infected groups; **p ≤ 0.01). (C) From the same experiments, pooled or individual lymph node cells were restimulated with SEA or media for 3 days in vitro and Th1 and Th2 cytokines were measured by ELISA (mean ± SEM; two-way ANOVA followed by Dunnett’s multiple comparisons test compare samples to pooled infected MLNs within each restimulation group; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (D) 1,000 S. mansoni eggs were injected into the ileal subserosa and pooled or individual lymph nodes were collected after 5 days. Cells were restimulated with SEA or media for 3 days in vitro and Th1 and Th2 cytokines were measured by ELISA (n = 3 mice per group, combined data from three independent experiments; mean ± SEM; two-way ANOVA followed by Dunnett’s multiple comparisons test compare samples to pooled infected MLNs within each restimulation group; ***p ≤ 0.001).
Figure 3
Figure 3
Heligmosomoides polygyrus antigens induce stronger Th1 and reduced Th2 responses in the intestinal draining lymph nodes compared to the footpad. (A) 20 µg of H. polygyrus ES antigen (HES) was injected in the footpad or ileal subserosa. IFN-γ+ and IL-4+ CD4+ T cell responses were analyzed 5 days after injection in the popliteal (pLN) or mesenteric (MLN) lymph nodes (n = 5 mice per group, combined data from two independent experiments; mean ± SEM; unpaired t-tests compare LN responses within each experimental group; ***p ≤ 0.001). (B) Ratio of IFN-γ+ and IL-4+ CD4+ T cell responses after HES injection as shown in (A) (mean ± SEM; unpaired t-test compares LN responses; **p ≤ 0.01). (C) From the same experiments, LN cells were restimulated with HES or media for 3 days in vitro and Th1 and Th2 cytokines were measured by ELISA (mean ± SEM; unpaired t-tests compare LN responses within each experimental group; *p ≤ 0.05, ***p ≤ 0.001). (D) Ratio of IFN-γ and IL-4 cytokine levels after HES restimulation as shown in (C) (mean ± SEM; unpaired t-test compares LN responses; **p ≤ 0.01, ***p ≤ 0.001). (E) Worm counts from the upper half of the small intestine (upper SI), lower half of the small intestine (lower SI) and the colon 17 days after infection with 200 L3 H. polygyrus larvae (n = 4 mice, representative of two independent experiments; mean ± SEM; unpaired t-tests compare worm counts to upper SI numbers; ***p ≤ 0.001). (F, G) Mice were infected with 200 L3 H. polygyrus larvae by oral gavage and individual MLNs were collected after 17 days. Frequency and number of IFN-γ, IL-4, and IL-13 producing CD4+ T cells in the individual MLNs are shown (n = 5 mice per group, representative of two independent experiments; mean ± SEM; ordinary one-way ANOVA followed by Holm–Sidak’s multiple comparisons test compare LN responses to naïve controls; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
Figure 4
Figure 4
Subserosal dye injection along the murine intestine identifies individual segment draining lymph nodes. Anesthetized animals were injected with 10 µl of black calligraphy ink into different segments of the small (A) or large intestine (B). Photographs were taken 1 min after dye injection. Schematics indicate injection site, labeled lymphatics and individual draining lymph nodes as seen in each photograph.
Figure 5
Figure 5
Subserosal injection of Schistosoma mansoni eggs induces segment-specific Th1 and Th2 responses in the individual MLNs. (A) 1,000 S. mansoni eggs were injected into different segments of the small intestine. Individual MLNs were collected five days after injection; LN cells were restimulated with SEA in vitro and IFN-γ, and IL-4 were measured by ELISA. Schematics indicate injection site and responding LNs (n = 3 mice per group, combined data from three independent experiments; mean ± SEM; ordinary one-way ANOVA followed by Holm–Sidak’s multiple comparisons test compare LN responses to naïve controls; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (B) Similar to (A), 1,000 Schistosoma mansoni eggs were injected into the different segments of the large intestine. After 5 days LNs were collected, restimulated in vitro, and assessed for antigen-specific cytokines by ELISA (n = 3 mice per group, combined data from three independent experiments; mean ± SEM; ordinary one-way ANOVA followed by Holm–Sidak’s multiple comparisons test compare LN responses to naïve controls; ***p ≤ 0.001). (C) Schematic of the murine intestine indicating the draining pattern of the different intestinal segments to their individual MLNs. (D) Segment-specific ratio of IFN-γ and Th2 cytokine levels after SEA restimulation from experiments described in (A, B) and Supplementary Figure 3 (mean ± SEM; ordinary one-way ANOVA followed by Holm–Sidak’s multiple comparisons test compare samples to sMLN1 ratios; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
Figure 6
Figure 6
Soluble helminth antigens are transported to the MLN by distinct subsets of intestinal dendritic cells that prime Th1 and Th2 responses. 15 µg of AF660-labeled antigens were injected into the intestinal serosa and antigen-positive cells were assessed 24 h after injection. (A) Representative dot plot of viable single MLN cells and frequency of SEA-AF660+ cells in pooled MLNs 24 h after ileal injection (n = 4 mice per group, representative of two independent experiments; mean ± SEM; unpaired t-tests compare cell frequencies to monocytes; ***p ≤ 0.001). (B) Frequency of SEA-AF660+ dendritic cells in pooled or individual MLNs 24 h after ileal injection (n = 4 mice per group, representative of two independent experiments; mean ± SEM; ordinary one-way ANOVA followed by Holm–Sidak’s multiple comparisons test compares DC frequencies to naïve controls; unpaired t-test compares pooled MLN to sMLN4 responses; **p ≤ 0.01, ***p ≤ 0.001). (C) DC subset distribution (left and middle) and frequency (right) of SEA-AF660+ lymph DCs collected over 18 h after ileal injection of SEA-AF660 (n = 2–3 mice per group, combined data from two independent experiments; mean ± SEM; unpaired t-tests compare cell distribution and frequencies to CD11b+CD103− DCs; ***p ≤ 0.001). (D) Lymph DC subsets were collected from naïve mice over 18 h, sorted into subsets and incubated with or without 1 mg/ml SEA for 18 h. 30,000 cells were then transferred under the MLN capsule of naïve mice. After 5 days LNs were collected, restimulated with SEA in vitro, and assessed for antigen-specific cytokines by ELISA (n = 2–3 mice per group, combined data from two independent experiments; mean ± SEM; unpaired t-tests compare cytokine responses from transferred SEA-treated DC subsets to untreated DCs; *p ≤ 0.05, **p ≤ 0.01). (E) DC subset distribution (left and middle) and frequency (right) of HES-AF660+ lymph DCs collected over 18 h after ileal injection of HES-AF660 (n = 2–3 mice per group, combined data from two independent experiments; mean ± SEM; unpaired t-tests compare cell distribution and frequencies to CD11b+CD103− DCs; *p ≤ 0.05, ***p ≤ 0.001). (F) Lymph DC subsets were collected from naïve mice over 18 h, sorted into subsets and incubated with or without 1 mg/ml HES for 18 h. 30,000 cells were then transferred into the MLN subcapsule of naïve mice. After 5 days LNs were collected, restimulated with HES in vitro, and assessed for antigen-specific cytokines by ELISA (n = 2–3 mice per group, combined data from two independent experiments; mean ± SEM; unpaired t-tests compare cytokine responses from transferred HES-treated DC subsets to untreated DCs; *p ≤ 0.05, **p ≤ 0.01).
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