Aspirin-triggered resolvin D1 modulates pulmonary and neurological inflammation in an IL-22 knock-out organic dust exposure mouse model

Abstract

Agriculture dust contains many organic immunogenic compounds, and organic dust exposure is strongly associated with the development of immune-mediated chronic pulmonary diseases such as chronic obstructive pulmonary disease (COPD). Chronic organic dust exposure from agriculture sources induces chronic lung inflammatory diseases and organic dust exposure has recently been linked to an increased risk of developing dementia. The cytokine interleukin-22 (IL-22) has been established as an important mediator in the resolution and repair of lung tissues. The omega-3 fatty acid metabolite aspirin-triggered Resolvin D1 (AT-RvD1) has shown efficacy in modulating the immune response in both pulmonary and neurological inflammation but has not been explored as a therapeutic in organic dust exposure-induced neuroinflammation. Investigating the link between IL-22 and AT-RvD1 may help in developing effective therapies for these immune-mediated diseases. We aimed to investigate the link between organic dust exposure and neuroinflammation, the role of IL-22 in the pulmonary and neurological immune response to organic dust exposure, and the immune-modulating therapeutic applications of AT-RvD1 in an IL-22 knock-out mouse model of organic dust exposure. C57BL/6J (WT) and IL-22 knock-out (KO) mice were repetitively exposed to aqueous agriculture organic dust extract (DE) 5 days per week for 3 weeks (15 total instillations) and treated with AT-RvD1 either once per week (3 total injections) or 5 times per week (15 total injections) for 3 weeks and allowed to recover for 3 days. We observed a significant pulmonary and neurological immune response to DE characterized by the development of inducible bronchus associated lymphoid tissue in the lung and gliosis in the frontal areas of the brain. We also observed that IL-22 knock-out increased pulmonary and neurological inflammation severity. Animals exposed to DE and treated with AT-RvD1 displayed reduced lung pathology severity and gliosis. Our data demonstrate that DE exposure contributes to neurological inflammation and that IL-22 is crucial to effective tissue repair processes. Our data further suggest that AT-RvD1 may have potential as a novel therapeutic for organic dust exposure-induced, immune-mediated pulmonary and neurological inflammation, improving outcomes of those with these diseases.

Keywords: AT-RvD1; SPM; agriculture dust; aspirin-triggered resolvin D1; lung inflammation; neuroinflammation; omega-3 fatty acids.

Figure 1

AT-RvD1 dosing strategies in WT and IL-22 KO mice. (A) once weekly AT-RvD1 dosing strategy timeline; WT and KO animals were instilled i.n. with 12.5% DE 5 days/week for 3 weeks and treated with 250 ng AT-RvD1 i.p. once/week for 3 weeks, (B) Once daily AT-RvD1 dosing strategy timeline; WT and KO animals were instilled i.n. with 12.5% DE 5 days/week for 3 weeks and treated with 250 ng AT-RvD1 i.p. 5 days/week for 3 weeks. Created with BioRender.com.

Figure 2

Impacts of DE and AT-RvD1 on BALF immune cellular infiltrates in WT and IL-22 KO animals. WT and KO animals were instilled i.n. with 12.5% DE 5 days/week for 3 weeks and treated with 250 ng AT-RvD1 i.p. once/week for 3 weeks. (A) total cell counts, (B) macrophages, (C) neutrophils, (D) eosinophils, (E) lymphocytes. 3 way ANOVA with Benjamini, Krieger and Yekutieli post-hoc analysis, error bars = SEM; * = p ≤ 0.05; ** = p ≤ 0.01; **** = p ≤ 0.0001. Sample sizes: WT Saline i.n. + Saline i.p (3 female/3 male), WT Saline i.n. + AT-RvD1 i.p (3 female/3 male), WT Dust i.n. + Saline i.p (5 female/5 male), WT Dust i.n. + AT-RvD1 i.p (4 female/4 male), KO Saline i.n. + Saline i.p (3 female/3 male), KO Saline i.n. + AT-RvD1 i.p (3 female/3 male), KO Dust i.n. + Saline i.p (5 female/4 male), KO Dust i.n. + AT-RvD1 i.p (6 female/5 male).

Figure 3

AT-RvD1 administration once weekly does not improve lung pathology. WT and KO animals were instilled i.n. with 12.5% DE 5 days/week for 3 weeks and treated with 250 ng AT-RvD1 i.p. once/week for 3 weeks. (A) representative images at 20X magnification, (B) mean inflammatory score for iBALT, (C) mean inflammatory score for peribronchiolar inflammation, (D) mean inflammatory score for alveolar inflammation. Scale bar = 50 μm. 3 way ANOVA with Benjamini, Krieger and Yekutieli post-hoc analysis, error bars = SEM; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001. Sample sizes: WT Saline i.n. + Saline i.p (2 female/2 male), WT Saline i.n. + AT-RvD1 i.p (2 female/2 male), WT Dust i.n. + Saline i.p (2 female/3 male), WT Dust i.n. + AT-RvD1 i.p (3 female/3 male), KO Saline i.n. + Saline i.p (3 female/3 male), KO Saline i.n. + AT-RvD1 i.p (2 female/2 male), KO Dust i.n. + Saline i.p (3 female/3 male), KO Dust i.n. + AT-RvD1 i.p (3 female/3 male).

Figure 4

AT-RvD1 treatment daily reduces iBALT in animals exposed to DE. WT and KO animals were instilled i.n. with 12.5% DE 5 days/week for 3 weeks and treated with 250 ng AT-RvD1 i.p. 5 days/week for 3 weeks. (A) representative images at 4X and 10X magnification, (B) iBALT percentage in all animals, (C) iBALT percentage in WT animals by sex, (D) iBALT percentage in KO animals by sex, (E) combined peribronchiolar and perivascular inflammation percentage in all animals, (F) bronchiolar epithelium area, (G) alveolar space percentage, (H) total alveolar nuceli. Scale bar = 50 μm. 3 way ANOVA with Benjamini, Krieger and Yekutieli post-hoc analysis, error bars = SEM; * = p ≤ 0.05; ** = p ≤ 0.01; **** = p ≤ 0.0001. Sample sizes: WT Saline i.n. + Saline i.p (3 female/3 male), WT Saline i.n. + AT-RvD1 i.p (3 female/3 male), WT Dust i.n. + Saline i.p (6 female/6 male), WT Dust i.n. + AT-RvD1 i.p (6 female/6 male), KO Saline i.n. + Saline i.p (3 female/3 male), KO Saline i.n. + AT-RvD1 i.p (3 female/3 male), KO Dust i.n. + Saline i.p (7 female/7 male), KO Dust i.n. + AT-RvD1 i.p (7 female/8 male).

Figure 5

Effects of repetitive dust exposure and RvD1 treatment on bronchoalveolar lavage fluid (BALF) and lung tissue homogenate cytokines. WT and KO animals were instilled i.n. with 12.5% DE 5 days/week for 3 weeks and treated with 250 ng AT-RvD1 i.p. 5 days/week for 3 weeks. (A) amphiregulin (AREG) concentrations in BALF, (B) interleukin-10 (IL-10) concentrations in BALF, (C) transforming growth factor-β (TGF-β) concentrations in BALF, (D) AREG concentrations in lung tissue, (E) IL-10 concentrations in lung tissue, and (F) TGFβ concentrations in lung tissue. 3 way ANOVA with Benjamini, Krieger and Yekutieli post-hoc analysis, error bars = SEM; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. BALF sample sizes: WT Saline i.n. + Saline i.p (3 female/3 male), WT Saline i.n. + AT-RvD1 i.p (3 female/3 male), WT Dust i.n. + Saline i.p (6 female/6 male), WT Dust i.n. + AT-RvD1 i.p (6 female/6 male), KO Saline i.n. + Saline i.p (3 female/3 male), KO Saline i.n. + AT-RvD1 i.p (3 female/3 male), KO Dust i.n. + Saline i.p (7 female/7 male), KO Dust i.n. + AT-RvD1 i.p (7 female/8 male). Lung tissue sample sizes: WT Saline i.n. + Saline i.p (3 female/3 male), WT Saline i.n. + AT-RvD1 i.p (3 female/3 male), WT Dust i.n. + Saline i.p (3 female/3 male), WT Dust i.n. + AT-RvD1 i.p (3 female/3 male), KO Saline i.n. + Saline i.p (3 female/3 male), KO Saline i.n. + AT-RvD1 i.p (3 female/3 male), KO Dust i.n. + Saline i.p (3 female/3 male), KO Dust i.n. + AT-RvD1 i.p (3 female/3 male).

Figure 6

AT-RvD1 treatment reduces cxcl10 mRNA expression in WT animals exposed to DE. WT and KO animals were instilled i.n. with 12.5% DE 5 days/week for 3 weeks and treated with 250 ng AT-RvD1 i.p. 5 days/week for 3 weeks. (A) representative images of the alveolar compartment at 40X magnification, (B) areg expression, (C) il10 expression, (D) cxcl10 expression, (E) representative images of the airway compartment at 40X magnification, (F) areg expression, (G) il10 expression, (H) cxcl10 expression. Scale bar = 50 μm. 3 way ANOVA with Benjamini, Krieger and Yekutieli post-hoc analysis, error bars = SEM; * = p ≤ 0.05; ** = p ≤ 0.01. Sample sizes: WT Saline i.n. + Saline i.p (3 female/3 male), WT Saline i.n. + AT-RvD1 i.p (3 female/3 male), WT Dust i.n. + Saline i.p (4 female/3 male), WT Dust i.n. + AT-RvD1 i.p (3 female/3 male), KO Saline i.n. + Saline i.p (3 female/3 male), KO Saline i.n. + AT-RvD1 i.p (3 female/3 male), KO Dust i.n. + Saline i.p (3 female/3 male), KO Dust i.n. + AT-RvD1 i.p (3 female/3 male).

Figure 7

Gliosis is associated with agriculture dust exposure. WT and KO animals were instilled i.n. with 12.5% DE 5 days/week for 3 weeks and treated with 250 ng AT-RvD1 i.p. 5 days/week for 3 weeks. (A) representative images of the olfactory bulb at 40X magnification, (B) microglia counts in the olfactory bulb, (C) representative images of the frontal cortex at 40X magnification, (D) microglia counts in the frontal cortex, (E) representative images of the isocortex at 40X magnification, (F) microglia counts in the isocortex, (G) representative images of the hippocampus at 40X magnification, (H) microglia counts in the hippocampus, (I) representative images of the cerebellum at 40X magnification, (J) microglia counts in the cerebellum, (K) representative images of the hindbrain at 40X magnification, (L) microglia counts in the hindbrain. Scale bar = 50 μm and 5 μm. 3 way ANOVA with Benjamini, Krieger and Yekutieli post-hoc analysis, error bars = SEM; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. WT Saline i.n. + Saline i.p (3 female/3 male), WT Saline i.n. + AT-RvD1 i.p (3 female/3 male), WT Dust i.n. + Saline i.p (6 female/6 male), WT Dust i.n. + AT-RvD1 i.p (8 female/6 male), KO Saline i.n. + Saline i.p (3 female/3 male), KO Saline i.n. + AT-RvD1 i.p (4 female/4 male), KO Dust i.n. + Saline i.p (7 female/6 male), KO Dust i.n. + AT-RvD1 i.p (6 female/7 male).

Figure 8

IL-22 knock-out alters brain mRNA transcripts in the presence of AT-RvD1 and DE. WT and KO animals were instilled i.n. with 12.5% DE 5 days/week for 3 weeks and treated with 250 ng AT-RvD1 i.p. 5 days/week for 3 weeks. (A) representative images of the olfactory bulb at 40X magnification, (B) tgfb expression in the olfactory bulb, (C) il10 expression in the olfactory bulb, (D) il1b expression in the olfactory bulb, (E) representative images of the frontal cortex at 40X magnification, (F) tgfb expression in the frontal cortex, (G) il10 expression in the frontal cortex, (H) il1b expression in the frontal cortex, (I) representative images of the isocortex at 40X magnification, (J) tgfb expression in the isocortex, (K) il10 expression in the isocortex, (L) il1b expression in the isocortex, (M) representative images of the hippocampus at 40X magnification, (N) tgfb expression in the hippocampus, (O) il10 expression in the hippocampus, (P) il1b expression in the hippocampus, (Q) representative images of the cerebellum at 40X magnification, (R) tgfb expression in the cerebellum, (S) il10 expression in the cerebellum, (T) il1b expression in the cerebellum, (U) representative images of the hindbrain at 40X magnification, (V) tgfb expression in the hindbrain, (W) il10 expression in the hindbrain, (X) il1b expression in the hindbrain. Scale bar = 50 μm. 3 way ANOVA with Benjamini, Krieger and Yekutieli post-hoc analysis, error bars = SEM; * = p ≤ 0.05; ** = p ≤ 0.01. Sample sizes: WT Saline i.n. + Saline i.p (3 female/3 male), WT Saline i.n. + AT-RvD1 i.p (2 female/2 male), WT Dust i.n. + Saline i.p (3 female/3 male), WT Dust i.n. + AT-RvD1 i.p (3 female/3 male), KO Saline i.n. + Saline i.p (3 female/3 male), KO Saline i.n. + AT-RvD1 i.p (2 female/2 male), KO Dust i.n. + Saline i.p (2 female/2 male), KO Dust i.n. + AT-RvD1 i.p. (3 female/3 male).