Immunosuppressive Ability of Trichinella spiralis Adults Can Ameliorate Type 2 Inflammation in a Murine Allergy Model

Abstract

Background: There is an increase in the global incidence of allergies. The hygiene hypothesis and the old friend hypothesis reveal that helminths are associated with the prevalence of allergic diseases. The therapeutic potential of Trichinella spiralis is recognized; however, the stage at which it exerts its immunomodulatory effect is unclear.

Methods: We evaluated the differentiation of bone marrow-derived macrophages stimulated with T spiralis excretory-secretory products. Based on an ovalbumin-induced murine model, T spiralis was introduced during 3 allergy phases. Cytokine levels and immune cell subsets in the lung, spleen, and peritoneal cavity were assessed.

Results: We found that T spiralis infection reduced lung inflammation, increased anti-inflammatory cytokines, and decreased Th2 cytokines and alarms. Recruitment of eosinophils, CD11b+ dendritic cells, and interstitial macrophages to the lung was significantly suppressed, whereas Treg cells and alternatively activated macrophages increased in T spiralis infection groups vs the ovalbumin group. Notably, when T spiralis was infected prior to ovalbumin challenge, intestinal adults promoted proportions of CD103+ dendritic cells and alveolar macrophages.

Conclusions: T spiralis strongly suppressed type 2 inflammation, and adults maintained lung immune homeostasis.

Keywords: Trichinella spiralis; allergic asthma; alternatively activated macrophages; immunosuppressive response; ovalbumin.

Graphical Abstract

Figure 1.

Immunoregulation of BMDMs by ESPs at various Trichinella spiralis stages. A, BMDMs were exposed to 20/50 μg of ESPs, and flow cytometry analysis revealed CD206⁺ macrophage levels. B, Proportion of CD206⁺ macrophages. C, Concentrations of interleukins 1β, 10, and 12p70 (IL-1β, IL-10, and IL-12p70) and transforming growth factor β1 (TGF-β1) in the cell culture supernatant. Data represent the mean ± SD from 5 independent experiments. ns, not significant. *P < .05. **P < .01. ***P < .001. One-way analysis of variance, followed by a Dunnett multiple-comparisons test. BMDM, bone marrow–derived macrophage; ESP, excretory-secretory product.

Figure 2.

Trichinella spiralis mitigates allergic inflammation in the lungs. A, Experimental design. Mice underwent sensitization with OVA/alum via intraperitoneal injection on days 0, 7, and 14, followed by aerosolized OVA challenge on days 28, 29, and 30. A total of 350 muscular larvae were orally infected on days −3, 25, and 31, whereas 40 000 newborn larvae were introduced via tail vein injection on day 15. Mice were euthanized and analyzed on day 37. B, Lung sections were prepared and stained with hematoxylin and eosin (H-E). Inflammatory cell infiltration and bronchiolar hyperplasia were semiquantitatively assessed on a scale from absent (0) to minimal (1), mild (2), moderate (3), marked (4), and massive (5). C, Lung sections were stained with periodic acid–Schiff (PAS), and mucus production and goblet cell hyperplasia were also evaluated. D, Total cells, macrophages, lymphocytes, eosinophils, and neutrophils in BALF were stained with DiffQuik and analyzed. Data are expressed as the mean ± SD from 5 independent experiments. ***P < .001. B and C, One-way analysis of variance, followed by a Dunnett multiple-comparisons test. D, Two-way analysis of variance, followed by a Dunnett multiple-comparisons test. BALF, bronchoalveolar lavage fluid; OVA, ovalbumin.

Figure 3.

Regulation of OVA-specific antibodies and inflammatory cytokines by Trichinella spiralis adults. A, Serum levels of OVA-specific IgG, IgG1, and IgE. B, Lung mRNA expression of IL-5, IL-25, IL-33, and TSLP assessed via real-time quantitative polymerase chain reaction. C, Levels of inflammatory-related cytokines in BALF or serum. Data are expressed as the mean ± SD from 5 independent experiments. ns, not significant. *P < .05. **P < .01. ***P < .001. One-way analysis of variance, followed by a Dunnett multiple-comparisons test. BALF, bronchoalveolar lavage fluid; IFN-γ, interferon γ; Ig, immunoglobulin; IL, interleukin; OD, optical density; OVA, ovalbumin; TGF-β1, transforming growth factor β1; TSLP, thymic stromal lymphopoietin.

Figure 4.

The immunoregulation of immune cells in the lung by Trichinella spiralis adults. A and B, Representative dot plots of Siglec-F⁺ CD11b⁺ eosinophils and Ly-6G⁺ CD11b⁺ neutrophils in the lungs. C, Representative dot plots of IMs, AMs, CD103⁺ DCs, and CD11b⁺ DCs in the lungs. D, Proportions of eosinophils, neutrophils, IMs, AMs, CD103⁺ DCs, and CD11b⁺ DCs in single cells. Data are expressed as the mean ± SD from 5 independent experiments. ns, not significant. *P < .05. **P < .01. ***P < .001. One-way analysis of variance, followed by a Dunnett multiple-comparisons test. AM, alveolar macrophage; DC, dendritic cell; IM, interstitial macrophage; OVA, ovalbumin; SSC-W, side scatter width.

Figure 5.

Immunofluorescence of CD4⁺ T cells and macrophages in lung. A, Immunostained lung sections show F4/80, CD4, and Hoechst. CD4⁺ T cells and F4/80⁺ macrophages are indicated by arrows. B, The number of CD4⁺ T cells per square millimeter was counted. Data are expressed as the mean ± SD from 5 independent experiments. ***P < .001. One-way analysis of variance, followed by a Dunnett multiple-comparisons test. OVA, ovalbumin.

Figure 6.

Suppression of immune cell proliferation and promotion of Treg cell differentiation by Trichinella spiralis adults in the spleen. Flow cytometry analysis of (A) CD45⁺ immune cells, (B) B220⁺ B cells and CD4⁺ T cells, and (C) Foxp3⁺ Treg cells in the spleen. D, Proportion of CD45⁺ immune cells in single cells, B cells and CD4⁺ T cells in CD45⁺ cells, and Treg cells in CD4⁺ T cells. Data are expressed as the mean ± SD from 5 independent experiments. ns, not significant. *P < .05. **P < .01. ***P < .001. One-way analysis of variance, followed by a Dunnett multiple-comparisons test. OVA, ovalbumin; SSC-A, side scatter area.

Figure 7.

Induction of alternative activation in peritoneal macrophages by Trichinella spiralis adults. A and B, Representative dot plots of F4/80⁺ macrophages and CD16/32⁺ CD206⁺ cells. C, Proportion of F4/80⁺ macrophages in single cells and CD16/32⁺ CD206⁺ cells in F4/80⁺ macrophages. Data are expressed as the mean ± SD from 5 independent experiments. ns, not significant. *P < .05. **P < .01. ***P < .001. One-way analysis of variance, followed by a Dunnett multiple-comparisons test. OVA, ovalbumin; SSC-A, side scatter area.