Intranasal administration of Acinetobacter lwoffii in a murine model of asthma induces IL-6-mediated protection associated with cecal microbiota changes

Abstract

Background: Early-life exposure to certain environmental bacteria including Acinetobacter lwoffii (AL) has been implicated in protection from chronic inflammatory diseases including asthma later in life. However, the underlying mechanisms at the immune-microbe interface remain largely unknown.

Methods: The effects of repeated intranasal AL exposure on local and systemic innate immune responses were investigated in wild-type and Il6^-/- , Il10^-/- , and Il17^-/- mice exposed to ovalbumin-induced allergic airway inflammation. Those investigations were expanded by microbiome analyses. To assess for AL-associated changes in gene expression, the picture arising from animal data was supplemented by in vitro experiments of macrophage and T-cell responses, yielding expression and epigenetic data.

Results: The asthma preventive effect of AL was confirmed in the lung. Repeated intranasal AL administration triggered a proinflammatory immune response particularly characterized by elevated levels of IL-6, and consequently, IL-6 induced IL-10 production in CD4⁺ T-cells. Both IL-6 and IL-10, but not IL-17, were required for asthma protection. AL had a profound impact on the gene regulatory landscape of CD4⁺ T-cells which could be largely recapitulated by recombinant IL-6. AL administration also induced marked changes in the gastrointestinal microbiome but not in the lung microbiome. By comparing the effects on the microbiota according to mouse genotype and AL-treatment status, we have identified microbial taxa that were associated with either disease protection or activity.

Conclusion: These experiments provide a novel mechanism of Acinetobacter lwoffii-induced asthma protection operating through IL-6-mediated epigenetic activation of IL-10 production and with associated effects on the intestinal microbiome.

Keywords: adaptive immunity; asthma; epigenomics; innate immunity; microbiota.

Figure 1.. Repeated intranasal administration of Acinetobacter lwoffii (AL) stimulates chronic IL-6-dependent inflammation in the lung and systematically.

(A) Protocol of repeated AL exposures followed by induction of experimental allergic asthma to ovalbumin (OVA). Control groups received intranasal Phosphate-Buffered Saline (PBS) instead of AL or PBS instead of OVA sensitization. (B and C) Bronchoalveolar lavage fluid (BAL) and serum were collected from 2 to 4 mice at 4-, 12-, and 24-hours following AL exposure and IL-6, TNF-α, and IL-1β were measured in cell-free samples. Shown are mean ± s.e.m for each time point. (D) Means of peak cytokine productions from 1^st to 4^th application versus 5^th to 12^th application for BAL IL-6, TNF-α, and IL-1β production after AL administration. Shown are mean ± s.e.m computed from peak values measured after 4 hours of each AL administration for the respective time frame. Data from D are representative of one experiment with different time points (n=10 biologically independent samples for the 1^st to 4^th application group and n=8 biologically independent samples for the 5^th to 12^th application group). Significant differences were evaluated using two-tailed unpaired t-test. *, P < 0.05. (E) Bone marrow (BM) was collected from naïve WT mice and differentiated in vitro to BM-Derived Macrophages (BMDMs), Myeloid Dendritic Cells (MDCs), and plasmacytoid Dendritic Cells (pDCs). Differentiated cells were subsequently stimulated in vitro for 24 hours with either LPS (10 ng/ml), 1×10⁶ CFU of AL, or with medium alone as a negative control, supernatants collected, and cytokine levels were measured. Data from E are representative of two pooled independent experiments (n=6 biologically independent samples/group). Shown are mean ± s.e.m. Significant differences were tested using unpaired two-tailed t-tests. *, P < 0.05; **, P < 0.01, *** P < 0.001, *** P < 0.0001.

Figure 2.. Il6^−/− mice are resistant to the asthma-protective effects provided by AL.

Il6^−/− mice and their WT littermates were exposed to AL as shown in Figure 1A. (A) Differential cell counts were measured in BAL fluids: Total BAL leukocytes, eosinophils, and lymphocytes. Quantitation of mucus-producing goblet cells expressed/millimeter of basement membrane and inflammation score were measured in periodic acid–Schiff (PAS)-stained lung tissue sections as described in Methods. (B) Concentrations of IL-5 and IL-13 and IL-10 were measured in cell-free BAL fluids. (C) Representative PAS-stained lung tissues showing mucus-producing goblet cells in airway inflammation. Tissue samples were taken on day 67. Data in A of total BAL leukocytes, eosinophils, and lymphocytes are representative of three-pooled independent experiments (n=15 biologically independent samples/group except for the PBS Il6^−/− controls with no OVA which are from two independent experiments with n≥8 biologically independent samples per group). Data of the Goblet cells and Inflammation score in A are from two-pooled independent experiments (n=8 biologically independent samples/group). Data in B are from three-pooled independent experiments for the IL-5 and IL-13 (n≥15 biologically independent samples/group) for the IL-10 measurement data are from two-pooled independent experiments (n≥10 biologically independent samples/group). only significant differences of the following comparisons between groups were shown (WT-OVA-AL vs. WT-OVA-no AL), (IL-6 KO-OVA-AL vs. IL-6 KO-OVA-no AL), (WT-OVA-no AL vs. IL-6 KO-OVA-no AL), (WT-OVA-AL vs. IL-6 KO-OVA-AL), and (WT-PBS-no AL vs. WT-OVA-no AL). Shown in A and B are mean ± s.e.m. Significant differences in A and B were calculated using unpaired two-tailed t-test. *, P < 0.05; **, P < 0.01; *** P < 0.001, **** P < 0.0001; PBS: Phosphate-buffered saline; OVA: ovalbumin; AL: Acinetobacter lwoffii; +: pretreated with AL; - pretreated with PBS.

Figure 3.. The AL-triggered allergic airways protective effect is mediated through IL-6 and IL-10, but not IL-17.

(A) Naïve splenic CD4⁺ T-cells derived from WT mice were co-stimulated with anti-CD3/anti-CD28 monoclonal antibodies and cultured with AL-conditioned peritoneal macrophage supernatant or recombinant IL-6 (rIL-6), as described in the Methods section. Medium alone was used as a negative control. Cytokines were measured in culture supernatants after 5 days. (B) Relative gene expression of the Il10 mRNA normalized to the housekeeping gene Rpl32, and IL-10 protein production levels of the CD4⁺ T-cell cultured with AL-conditioned or unconditioned macrophage supernatants alone or pre-incubated with anti-IL-6 antibody, recombinant IL-6 alone, or medium, used as a negative control. (C,D) Il10^−/− and Il17^−/− mice and their WT littermates were exposed to AL as shown in Figure 1A. Differential cell counts were measured in BAL fluids, with total BAL leukocytes and eosinophils presented. Data in A are representative of two-pooled independent experiments (n=6 biologically independent samples/group). Data in B are representative of one experiment (n=4 biologically independent samples/group). Data in C are from two-pooled independent experiments (n=12 biologically independent samples/group for WT and n≥8 biologically independent samples/group for Il10^−/−). Data in D are from three-pooled independent experiments (n=19 biologically independent samples/group for WT, and from two-pooled independent experiments, n=8 biologically independent samples/group for Il17^−/−). For c and d only significant differences of the following comparisons between groups were shown (WT-OVA-AL vs. WT-OVA-no AL), (IL-6 KO-OVA-AL vs. IL-6 KO-OVA-no AL), (WT-OVA-no AL vs. IL-6 KO-OVA-no AL), (WT-OVA-AL vs. IL-6 KO-OVA-AL), and (WT-PBS-no AL vs. WT-OVA-no A). Shown in A-D are Mean ± s.e.m. Significant differences in A-D were calculated using two tailed unpaired two-tailed t-tests. *, P < 0.05; **, P < 0.01; *** P < 0.001, **** P < 0.0001. PBS: Phosphate-buffered saline; OVA: ovalbumin; AL: Acinetobacter lwoffii; +: pretreated with AL; - pretreated with PBS.

Figure 4.. AL alters the landscape of histone modifications in CD4⁺ T-cells mainly via IL-6.

(A-C) Venn Diagram showing the numbers of overlapped gene-annotated peaks for the active histone mark H3K27ac in CD4⁺ T-cells treated with (A) medium compared to AL-conditioned macrophage supernatant or (B) medium compared to rIL-6 or (C) AL-conditioned macrophage supernatant pretreated with anti-IL-6 antibody compared to medium. (D) Venn Diagram showing the numbers of overlapped of the unique-gained gene-annotated peaks of the AL-conditioned macrophage supernatant and rIL-6. Peaks are associated with genes based on the AnnotatR database. (E) Representative images of the Integrative Genomics Viewer (IGV) of H3K27ac peaks in CD4⁺ T-cells incubated with AL-conditioned macrophage supernatant (top line), rIL-6 (middle line) and AL-conditioned macrophage supernatant pretreated with anti-IL-6 antibody (bottom line), after subtracting H3K27ac modification profile of the medium from all conditions. Images depict the histone modifications at the promoter regions of the Il10 pathway-related genes Mapk1, Mapk3, Stat3 and c-MAF and the Il10 gene. (F) Functional molecular pathway enrichment analysis of the histone mark H3K27ac in CD4⁺ T-cells using KEGG_2019 and WikiPathways_2019 databases for mice. Pathways depicted found to be enriched following incubation with AL-conditioned macrophage supernatant or rIL-6 each compared to medium. (G) Pathway network analysis was performed as described in the Methods section. The sample processing and numbers are as described in the Methods section.

Figure 5.. Heatmap representing genus level taxa in the KO mouse samples from the IL-6 and IL-10 experiments that are significantly different between treatment groups.

(A) Significant associations in the Il6^−/− mice. (B) Significant associations in the Il17^−/− mice. Each row represents a different taxon and each column a different treatment time point. Log₁₀ taxon relative abundance is shown using red-blue scales. Significantly different taxa were inferred by MaAsLin2 analysis. All comparisons shown are of animals not pretreated with Acinetobacter lwoffii (AL) and not subjected to ovalbumin (OVA)-model versus the specified treatment group. +AL: pretreated with AL; -AL: not pretreated with AL; +OVA: subjected to OVA-model; -OVA: not subjected to OVA-model.