Itaconate suppresses house dust mite-induced allergic airways disease and Th2 cell differentiation

Abstract

Itaconate was initially identified as an antimicrobial compound produced by myeloid cells. Beyond its antimicrobial role, itaconate may also serve as a crucial metabolic and immune modulator. We therefore examined the roles of aconitate decarboxylase 1 (Acod1) and itaconate in house dust mite (HDM)-sensitized and -challenged mice, a model of T helper 2 (Th2)-driven allergic airways disease. HDM treatment induced lung Acod1 mRNA expression and bronchoalveolar lavage (BAL) itaconate levels in wild-type C57BL/6 mice. Acod1 knockout mice (Acod1-KO) with negligible BAL itaconate showed heightened HDM-induced type 2 cytokine expression, increased serum IgE, and enhanced recruitment of Th2 cells in the lung, indicating a shift towards a more pronounced Th2 immune response. Acod1-KO mice also showed increased eosinophilic airway inflammation and hyperresponsiveness. Experiments in chimeric mice demonstrated that bone marrow from Acod1-KO mice is sufficient to increase type 2 cytokine expression in wild-type mice, and that restitution of bone marrow from wild type mice attenuates mRNA expression of Th2 cytokines in Acod1-KO mice. Specific deletion of Acod1 in lysozyme-secreting macrophages (LysM-cre⁺Acod1^flox/flox) recapitulated the exaggerated phenotype observed in whole-body Acod1-KO mice. Adoptive transfer of Acod1-KO bone marrow-derived macrophages also increased lung mRNA expression of Th2 cytokines. In addition, treatment of Th2-polarized CD4 cells with itaconate impeded Th2 cell differentiation, as shown by reduced expression of Gata3 and decreased release of IL-5 and IL-13. Finally, public datasets of human samples show lower Acod1 expression in subjects with allergic asthma, consistent with a protective role of itaconate in asthma pathogenesis. Together, these data suggest that itaconate plays a protective, immunomodulatory role in limiting airway type 2 inflammation after allergen challenge by attenuating T cell responses.

Keywords: Aconitate decarboxylase 1; Allergen; Asthma; House dust mite; Itaconate; Macrophage.

Fig. 1.. Itaconate deficiency increased HDM-induced type 2 cytokines and Th2 cells.

A. PBS or HDM was administered to WT or Acod1-KO mice. Mice were sensitized on days 1 and 4 and then challenged on days 8, 10 and 12. Lungs were harvested at day 14. B. mRNA expression of Acod1 in wild-type and Acod1-KO mice (n = 8 per group from 2 independent experiments). C. BAL itaconate concentration (n = 5 per group from a single experiment). D. Expression of mRNAs encoding the inflammatory cytokines IL-4, IL-5, IL-13, CCL11, IL-1β, TNF-α, IFN- γ and IL-17α, and the mucin gene Muc5ac (n = 3 for PBS treated groups, n = 8 for HDM treated groups from 2 different experiments). E. Flow cytometric analysis of live CD45⁺CD3e⁺CD4⁺CD8⁻ T helper cells from HDM treated wild-type and Acod1-KO mice. F. Quantification of Th1, Th2, and Th17 cells (n = 4 per group from a single experiment). Data shown are mean ± SD and were analyzed by one-way ANOVA (panels B, C, D) or unpaired t-test (panel F). For ANOVA, group differences were pinpointed by the Tukey multiple comparison test.

Fig. 2.. Itaconate deficiency exacerbates HDM-induced allergic airway disease.

A. Serum HDM-specific IgE and IgG1 levels in WT and Acod1-KO mice (n = 3 for PBS treated groups, n = 10 for HDM treated groups from 2 different experiments). B. The number of BAL inflammatory cells (monocytes, neutrophils, lymphocytes and eosinophils) from HDM-treated wild-type and Acod1-KO mice were compared. BAL cells were stained with hematoxylin and eosin (n = 10 for PBS treated groups, n = 9–10 from 2 different experiments). D. Lung sections were stained with PAS. The fraction of epithelium stained positively for PAS was quantified using QuPath software. Three airways were examined for each mouse (n = 3 mice from 2 independent experiments). D. mRNA expression of M2 macrophage markers Arg1, Chil3, Retnla and CD206 (n = 6 for each group from 2 different experiments). E. Airway resistance was measured in anesthetized, intubated and ventilated mice before and after administration of methacholine (n = 4 per group from a single experiment). Data shown are mean ± SD; data were analyzed by unpaired t test (panels B, C, D) or one-way ANOVA (panels A and E). Group differences were pinpointed by the Tukey multiple comparison test.

Fig. 3.. Bone marrow-derived itaconate attenuates allergic airway inflammation.

mRNA expression of type 2 cytokines Il4, Il5 and Il3 were measured in HDM-treated (A) wild-type mice transplanted with wild-type (WT to WT) or Acod1-KO (KO to WT) bone marrow and (B) Acod1-KO mice transplanted with wild-type (WT to KO) or Acod-KO (KO to KO) bone marrow. Data shown are mean ± SD (n = 3–5 per group from a single experiment). Data were analyzed by unpaired t-test.

Fig. 4.. Macrophage-derived itaconate attenuates HDM-induced allergic airway inflammation.

A. mRNA expression of Acod1 in wild-type bone marrow-derived and alveolar macrophages treated with PBS or HDM (n = 3 per group from a single experiment). Data were analyzed by unpaired t-test. B. Protein levels of Acod1 in wild-type bone marrow-derived macrophages treated with PBS or HDM. C. Expression of mRNAs encoding IL-4, IL-5, IL-13, Muc5ac and CCL11 from HDM-treated cre⁻Acod1^fl/fl, LysM-cre⁺Acod1^fl/fl, CD11c-cre⁺Acod1^fl/fl, CD4-cre⁺Acod1^fl/fl mice (n = 9 per group from two experiments). Data shown are mean ± SD and were analyzed by one-way ANOVA. Group differences were pinpointed by the Tukey multiple comparison test.

Fig. 5.. Adoptive transfer of bone marrow-derived macrophages from wild-type and Acod1-KO mice.

Acod1-KO mice were sensitized on days 1 and 4 with 100 μg HDM. Bone marrow-derived macrophages from wild-type or Acod1-KO mice were intranasally transferred to Acod1-KO mice at day 8. Mice were challenged with PBS or 100 μg HDM on day 10 and lungs collected 4 h later. Expression of mRNAs encoding the Th2 cytokines IL-4, IL-5 and IL-13 (n = 2 for PBS-treated groups and n = 7 for HDM-treated groups from two experiments). Data shown are mean ± SD and were analyzed by one-way ANOVA. Group differences were pinpointed by the Tukey multiple comparison test.

Fig. 6.. Exogenous itaconate inhibits Th2 cell differentiation.

A. Spleen T cells were isolated by CD4 selection and differentiated to Th2 cells. B. mRNA expression of Gata3 from undifferentiated T cells (Th0 cells) and T cells differentiated to Th2 in the presence or absence of exogenous itaconate (0.1, 1 or 5 mM). C. Protein levels of Gata3 from Th0 and Th2 cells treated with 5 mM exogenous itaconate. D. Effect of itaconate on protein abundance of IL-5 and IL-13 (n = 6 per group from two experiments). E. Effect of itaconate on cell death (n = 5 per group from two experiments). Data shown are mean ± SD and were analyzed by one-way ANOVA (B, E) or unpaired t test (D).