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. 2021 Jun;11(6):e427.
doi: 10.1002/ctm2.427.

Dual role of the miR-146 family in rhinovirus-induced airway inflammation and allergic asthma exacerbation

Anet Laanesoo  1 , Egon Urgard  1 , Kapilraj Periyasamy  1 , Martti Laan  1 , Yury A Bochkov  2 , Alar Aab  1 , Nathaniel Magilnick  3 , Margus Pooga  4 , James E Gern  2 , Sebastian L Johnston  5   6 , Jonathan M Coquet  7 , Mark P Boldin  3 , Jesper Wengel  8 , Alan Altraja  9   10 , Grazyna Bochenek  11 , Bogdan Jakiela  11 , Ana Rebane  1
Affiliations

Dual role of the miR-146 family in rhinovirus-induced airway inflammation and allergic asthma exacerbation

Anet Laanesoo et al. Clin Transl Med. 2021 Jun.

Abstract

Rhinovirus (RV) infections are associated with asthma exacerbations. MicroRNA-146a and microRNA-146b (miR-146a/b) are anti-inflammatory miRNAs that suppress signaling through the nuclear factor kappa B (NF-κB) pathway and inhibit pro-inflammatory chemokine production in primary human bronchial epithelial cells (HBECs). In the current study, we aimed to explore whether miR-146a/b could regulate cellular responses to RVs in HBECs and airways during RV-induced asthma exacerbation. We demonstrated that expression of miR-146a/b and pro-inflammatory chemokines was increased in HBECs and mouse airways during RV infection. However, transfection with cell-penetrating peptide (CPP)-miR-146a nanocomplexes before infection with RV significantly reduced the expression of the pro-inflammatory chemokines CCL5, IL-8 and CXCL1, increased interferon-λ production, and attenuated infection with the green fluorescent protein (GFP)-expressing RV-A16 in HBECs. Concordantly, compared to wild-type (wt) mice, Mir146a/b-/- mice exhibited more severe airway neutrophilia and increased T helper (Th)1 and Th17 cell infiltration in response to RV-A1b infection and a stronger Th17 response with a less prominent Th2 response in house dust mite extract (HDM)-induced allergic airway inflammation and RV-induced exacerbation models. Interestingly, intranasal administration of CPP-miR-146a nanocomplexes reduced HDM-induced allergic airway inflammation without a significant effect on the Th2/Th1/Th17 balance in wild-type mice. In conclusion, the overexpression of miR-146a has a strong anti-inflammatory effect on RV infection in HBECs and a mouse model of allergic airway inflammation, while a lack of miR-146a/b leads to attenuated type 2 cell responses in mouse models of allergic airway inflammation and RV-induced exacerbation of allergic airway inflammation. Furthermore, our data indicate that the application of CPP-miR-146a nanocomplexes has therapeutic potential for targeting airway inflammation.

Keywords: asthma; bronchial epithelial cell; house dust mite; microRNA; neutrophils; noncoding RNA; viral infection.

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Conflict of interest statement

Ana Rebane and Margus Pooga are board members of RNAexact OÜ. All other authors declare no conflict of interest regarding this manuscript.

Figures

FIGURE 1
FIGURE 1
MiR‐146a/b inhibit expression of pro‐inflammatory cytokines and induce interferon response genes in HBECs during RV infection. (A) HBECs were stimulated with RVs for 24 h or 48 h. Relative expression of miR‐146a/b in HBECs was measured by RT‐qPCR and is shown in comparison to corresponding mock‐stimulated cells. (B‐F) HBECs were transfected with miR‐146a or control mimics and stimulated 24 h later with mock or the indicated RVs for 48 h. (B‐E) mRNA expression of the indicated genes in primary HBECs was measured by RT‐qPCR and compared to expression levels of the mock‐stimulated control group mean (= 1). (F) Protein expression of selected genes was measured by ELISA. (A‐F) Data represent mean ± SEM. Unpaired t‐test, *p < 0.05, **p < 0.01, ***p < 0.001. One representative of three independent experiments in HBECs from two different donors is shown
FIGURE 2
FIGURE 2
MiR‐146a inhibits RV infection and neutrophil migration. HBECs were transfected with miRNA mimics for 24 h and stimulated with the indicated RVs for 48 h or stimulated with only RV or mock. (A) Representative flow cytometry plots of HBECs infected with RV‐A16‐GFP (green) and (B) corresponding quantification graph. (C‐D) Chemotaxis assay of human neutrophils toward supernatants of primary HBECs stimulated with RVs (C) or toward supernatants of HBECs transfected with the indicated miRNA mimics and stimulated with RVs (D). (C‐D) Data from two independent experiments in HBECs from two different donors are shown. Data represent mean ± SEM. Unpaired t‐test, *p < 0.05, **p < 0.01, ***p < 0.001
FIGURE 3
FIGURE 3
Mir146a/b−/− mice exhibit increased airway neutrophilia in a mouse model of RV‐induced airway inflammation. (A) RV‐A1b or PBS was administered intranasally (i.n.) to wt or Mir146a/b−/− mice. Twenty‐four hours after RV‐A1b infection, BAL was analyzed. (B‐C) BAL cells were counted using a hemocytometer and then subjected to FACS analysis, and according to dot plots, the total numbers of immune cells were calculated. Data represent mean ± SEM. Unpaired t‐test, *p < 0.05, ***p < 0.001. (C) One representative FACS dot plot of BAL fluid from three PBS‐treated and five RV‐A1b‐treated wt and Mir146a/b−/− mice. Eosinophils, macrophages, DCs, and neutrophils (upper and middle panel) were analyzed as a percentage of the granulocyte population, and T cells and B cells (bottom panel) were analyzed as a percentage of the lymphocyte population
FIGURE 4
FIGURE 4
Gene expression of wt and Mir146a/b−/− mouse lungs in an RV‐induced mouse model of airway inflammation. Twenty‐four hours after i.n. administration of PBS or RV‐A1b, mouse lung lobes were collected. (A) Relative miRNA expression and (B‐E) mRNA expression in lung lobes was measured with RT‐qPCR and are shown compared to the PBS wt group mean (= 1). (F) Protein content in BAL measured with ELISA. Data from two independent experiments. Data represent mean ± SEM. Unpaired t‐test, *p < 0.05, **p < 0.01, ***p < 0.001
FIGURE 5
FIGURE 5
Lack of miR‐146a/b in mice results in an altered immune response in allergic airway inflammation and RV‐induced exacerbation of allergic airway inflammation models. (A) Mice were sensitized (D0) and challenged (D7‐11) with house dust mite extract (HDM) and, when indicated, infected with RV‐A1b (D21) for 24 h. Control mice received only PBS. (B) BAL cells were counted with a hemocytometer and then subjected to FACS analysis. From the dot plot percentages, the total numbers of immune cells were calculated. Data represent mean ± SEM. One‐way ANOVA with Tukey's multiple comparisons test and adjusted p‐values are shown. + p < 0.05, ++ p < 0.01 HDM compared to the same mouse line with PBS; ## p < 0.01 HDM+RV‐A1b compared to same mouse line with HDM; *p < 0.05, **p < 0.01, ***p < 0.001 wt compared to Mir146a/b−/− mice for the same treatment. (C) One representative FACS dot plot of BAL fluid from five HDM‐treated or HDM+RV‐A1b‐treated wt and Mir146a/b−/− mice. Data from one representative of three independent experiments are shown
FIGURE 6
FIGURE 6
Mir146a/b−/− mice display differential gene expression in their lungs, splenocytes, and DCs in response to HDM or RV‐A1b. Relative miRNA (A) and mRNA expression in (B‐C) mouse lungs subjected to HDM‐induced airway inflammation and RV‐induced exacerbation of allergic airway inflammation models, (D) in splenocytes and (E) in BMDCs as measured by RT‐qPCR. Data are presented as the mean ± SEM and are compared to mean values of the wt PBS group (= 1) using one‐way ANOVA with Tukey's multiple comparisons test. Adjusted p‐values are shown. (A) **p < 0.01 wt PBS compared to wt HDM, ***p < 0.001 wt HDM compared to wt HDM+RV‐A1b. Data from one representative of three independent experiments are shown. (B‐C) #p < 0.05, ##0.01, ###p < 0.001 HDM+RV‐A1b compared to the same HDM mouse line; *p < 0.05 wt compared to Mir146a/b−/− mice during the same treatment. (D) Splenocytes from wt and Mir146a/b−/− mice were stimulated for 48 h; *p < 0.05, **p < 0.01, ***p < 0.001 wt compared to Mir146a/b−/− mice during the same treatment. (E) BMDCs from wt and Mir146a/b−/− mice were stimulated for 24 h; $ p < 0.05, $$$ p < 0.001 RV‐A1b compared to the same line mock; +++ p < 0.001 HDM compared to the same line US; ***p < 0.001 wt compared to Mir146a/b−/− during same treatment
FIGURE 7
FIGURE 7
Administration of CPP‐miR‐146a nanocomplexes inhibits the accumulation of eosinophils, T cells, and B cells in an allergic airway inflammation model. Wt mice were sensitized i.n. at day 0 with 1 μg and challenged at days 7–11 daily with 10 μg of HDM. CPP‐miR‐146a (miR‐146a) and CPP‐miRNA control (control) nanocomplexes containing 60 pmol of corresponding miRNA mimic were applied 2 h after each challenge. On day 15, BAL cells and lung lobes for RNA were harvested. PBS group was left untransfected (UT). (A) Schematic of the experimental setup. (B and C) BAL fluid cells were counted with a hemocytometer, then subjected to FACS analysis. From the dot plots, the total numbers of immune cells were calculated. (B) One representative FACS dot‐plot of BAL macrophages, eosinophils, CD3+ T cells, and B220+ B cells. (C) Data are represented as mean ± SEM from five mice in study groups and three mice in UT PBS group, unpaired t‐test, *p < 0.05. Data from one representative of two independent experiments are shown. (C‐E) BAL cells were counted using a hemocytometer and then subjected to FACS analysis. From the dot plots, the total numbers of immune cells were calculated. Data are represented as the mean ± SEM, unpaired t‐test, *p < 0.05. Data from one representative of two independent experiments are shown
FIGURE 8
FIGURE 8
CPP‐miR‐146a nanocomplexes inhibit the expression of pro‐inflammatory genes in an HDM‐induced airway inflammation model. (A) Localization of labeled Cy5‐miR‐146a mimics (red) in lung lobes counterstained with DAPI (blue). Scale bar = 100 μm. (B) Relative mRNA expression levels of the indicated genes were compared to the PBS‐treated untransfected (UT) group (= 1) or HDM‐treated and miR‐146a mimic‐ or control‐transfected mouse lung lobes measured by RT‐qPCR. Data are represented as the mean ± SEM. Unpaired t‐test, *p < 0.05, **p < 0.01, ***p < 0.001. Data from one representative of two independent experiments are shown
FIGURE 9
FIGURE 9
miR‐146a/b‐deficient mice develop more severe airway neutrophilia during RV infection and a less prominent Th2 cell mediated immune response in HDM‐induced allergic airway inflammation and RV‐induced exacerbation of allergic airway inflammation when compared with wild‐type mice. Application of cell‐penetrating peptide (CPP)‐miR‐146a nanocomplexes into the airways has an anti‐inflammatory effect in the HDM‐induced allergic airway inflammation model in wild‐type mice.
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