Altered ontogeny and transcriptomic signatures of tissue-resident pulmonary interstitial macrophages ameliorate allergic airway hyperresponsiveness

Abstract

Introduction: Environmental exposures and experimental manipulations can alter the ontogenetic composition of tissue-resident macrophages. However, the impact of these alterations on subsequent immune responses, particularly in allergic airway diseases, remains poorly understood. This study aims to elucidate the significance of modified macrophage ontogeny resulting from environmental exposures on allergic airway responses to house dust mite (HDM) allergen.

Methods: We utilized embryonic lineage labeling to delineate the ontogenetic profile of tissue-resident macrophages at baseline and following the resolution of repeated lipopolysaccharide (LPS)-induced lung injury. We investigated differences in house dust mite (HDM)-induced allergy to assess the influence of macrophage ontogeny on allergic airway responses. Additionally, we employed single-cell RNA sequencing (scRNAseq) and immunofluorescent staining to characterize the pulmonary macrophage composition, associated pathways, and tissue localization.

Results: Our findings demonstrate that the ontogeny of homeostatic alveolar and interstitial macrophages is altered after the resolution from repeated LPS-induced lung injury, leading to the replacement of embryonic-derived by bone marrow-derived macrophages. This shift in macrophage ontogeny is associated with reduced HDM-induced allergic airway responses. Through scRNAseq and immunofluorescent staining, we identified a distinct subset of resident-derived interstitial macrophages expressing genes associated with allergic airway diseases, localized adjacent to terminal bronchi, and diminished by prior LPS exposure.

Discussion: These results suggest a pivotal role for pulmonary macrophage ontogeny in modulating allergic airway responses. Moreover, our findings highlight the implications of prior environmental exposures in shaping future immune responses and influencing the development of allergies. By elucidating the mechanisms underlying these phenomena, this study provides valuable insights into potential therapeutic targets for allergic airway diseases and avenues for further research into immune modulation and allergic disease prevention.

Keywords: asthma; house dust mite; macrophages; ontogeny; single-cell RNA-seq.

Figure 1

Time-mated lineage labeling of lung macrophages supports that interstitial macrophages (IMØ) are Yolk Sac-derived and persist into adulthood. (A) Overview of experimental design for time-mated lineage labeling in Cx3cr1cre-er;tdTomato mice induced with tamoxifen at E7.5 to label yolk sac progenitors or at E13.5 to label yolk sac and fetal monocytes. (B) Flow cytometry of whole lung tissue in 7-week-old mice following embryonic lineage labeling cells in the alveolar macrophage (AMØ) and IMØ compartments based on time of lineage labeling (E7.5 versus E13.5). Live, CD45⁺, Ly6G^-, and CD64⁺ cells were then assessed for TdTomato expression versus CD11b to define AMØ (CD11b^-) and IMØ (CD11b⁺). Histograms define representative percentages for TdTomato⁺ AMØ or IMØ in adult mice following induction. (C) Extent of lineage labeling in IMØ, defined as TdTomato⁺ cells in IMØ/microglia, was determined based on the time point of lineage induction, the tamoxifen dose, the sex of mice, and the time point of harvest after induction. (D) Microarray of sorted AMØ, IMØ, Ly6C^- and Ly6C⁺ monocytes, yolk sac (YS), and microglia reveals clustering of AMØ with monocytes and not IMØ that have greater overlap with YS. N=3–10 mice/group, experiments repeated >3 times, **p<0.005, ns=non-significant using ANOVA with post-hoc testing.

Figure 2

Replacement of embryonic-derived macrophages with bone marrow-derived macrophages following LPS exposure and recovery attenuates house dust mite (HDM) allergic airway responses. (A) Overview of experimental design to assess the impact of macrophage turnover on allergic airway responses. Mice underwent intranasal (i.n) LPS exposure (12.5µg) every other week for 3 doses. They were then allowed to recover for > 8 weeks. Following recovery, mice underwent acute HDM exposure and were assessed for phenotypic allergic airway responses. (B) Cx3cr1^CreER/tdTomato mice underwent lineage reporter induction with tamoxifen at E13.5 and were allowed to age until 6 weeks. At 6 weeks of age, i.n. LPS or PBS exposure, as described above, was performed. Following >8 weeks of recovery from the exposure, lung tissues were harvested and processed for flow cytometry to define tdTomato expression in AMØ and IMØs. LPS causes the turnover of embryonic-derived macrophages to bone marrow-derived macrophages as determined by a reduction in the percentage of TdTomato+ cells. (C-F) LPS-mediated turnover reduces HDM-mediated allergic airway responses, including reduced airway hyperresponsiveness to increasing doses of methacholine (C, R_rs-resistance, E_rs-elastance), reduced periodic acid–Schiff (PAS) staining (a measure of mucus) without a difference in hematoxylin and eosin (H+E) or trichrome staining (D), decreased lung tissue (as a % of CD45+ cells or total cells) and bronchoalveolar lavage fluid (BALF) eosinophils (E), and decreased BALF cytokines including IL-4, IL-13, IL-1β, TNF-α, IL-5, IL-10, KC and IL-6 (F). n=10 mice/group in the PBS/PBS and LPS/PBS groups and n=30 mice/group in the PBS/HDM and LPS/HDM groups. *p<0.05 when compared to PBS control or ^#p<0.05 when compared between PBS-HDM and LPS-HDM groups by 2-way ANOVA with testing for multiple comparisons. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005 for other comparisons by 1-way ANOVA or Students T-test, n.s., non-significant.

Figure 3

Single cell RNA sequencing of immune cells allows segregation of alveolar and interstitial macrophages and identifies unique clustering based on exposure conditions. (A) Bone marrow chimeras were generated from lineage labeled Cx3cr1^CreER/tdTomato mice by injecting CD45.1 donor cells into CD45.2 recipients to define resident (CD45.2) and recruited (CD45.1) cell populations. Mice were exposed in the following groupings PBS_PBS, PBS_HDM, LPS_PBS, and LPS_HDM. Following exposures, lungs were harvested and processed for sorting. Live, Ly6G^-, B220^- cells were segregated based on CD45.1 from CD45.2 expression and processed for single cell RNA-seq. (B) Clustering of immune cells based on defined markers identifies macrophages (pink), dendritic cells (DC, yellow), monocytes (orange), and T cells (blue). (C) Sub-clustering of macrophages segregates interstitial (IM, blue) from alveolar macrophages (AM, green) based on annotation using established markers. (D) Display of sub-clustering of macrophages based on exposure condition and whether the cells are recruited (CD45.1) or tissue-resident (CD45.2) identifies distinct clustering based on exposure and origin. The bar graph reveals that IMs increase as a percentage in response to exposures compared to the PBS_PBS or LPS_PBS groups. Following the flow sorting, data were pooled from individual sorted mouse lung samples (n=3 mice per exposure group).

Figure 4

Alveolar macrophage sub-clustering reveals unique clusters with distinct gene expression patterns without evidence of distinct clustering based on ontogeny. (A) Alveolar macrophages (AM) are sub-clustered, identifying 5 unique clusters (AM1–5) defined by individual gene expression patterns (B). (C) An overlay of the clusters based on exposure and CD45.1 (bone marrow-derived) or CD45.2 (tissue-resident) group in the UMAP plot, as well as a bar graph depicting or percentage contribution by each sample to individual AM subclusters. Data do not support clear cluster segregation based on ontogeny. Data were pooled from individual sorted mouse lung samples (n=3 mice per exposure group).

Figure 5

Interstitial macrophage sub-clustering reveals a unique IMØ subcluster that segregates by exposure condition and ontogeny and is defined by expression of Chi313. (A) Sub-clustering of interstitial macrophages (IM), defined by individual cellular markers, identifies seven unique clusters (IM1–7). (B) Overlay of IM clusters on UMAP and bar graph based on exposure condition and cell ontogeny identifies that IM2 cluster segregates based on CD45.2 (resident origins) and HDM exposure, (C) Pie chart demonstrating the distribution of the IM2 cluster based on the exposure condition and cell origins. This highlights that tissue-resident CD45.2 IMØ from the PBS_HDM exposed mice as the predominant constituent of the IM2 cluster. (D) Top gene expression for each cluster highlights that cluster IM2 is defined by gene expression for Chi313. Following the flow sorting, data were pooled from individual sorted mouse lung samples (n=3 mice per exposure group).

Figure 6

Tissue-resident interstitial macrophages from the IM2 cluster are enriched with allergic genes following HDM exposure and abrogated by LPS pre-HDM exposure. (A) Top 72 differentially regulated genes from the IM2 cluster from the following exposure conditions and macrophage origin: LPS_HDM in CD45.1 cells (LPS_HDM_CD45.1, red bar), PBS_HDM in CD45.1 cells (PBS_HDM_CD45.1, light blue bar), LPS_HDM in CD45.2 cells (LPS_HDM_CD45.2, orange bar) and PBS_HDM in CD45.2 cells (PBS_HDM_CD45.2, blue bar) expressed as a heatmap. (B) Ingenuity pathway analysis (Z score >2 or <2) of IM2 from CD45.1 (recruited) and CD45.2 cells (tissue-resident) based on exposure condition reveals that the IM2 cluster in CD45.2 (tissue-resident) cells is enriched for eosinophilic signaling (red bar), inflammation (yellow bar), vascular-associated signaling (green bar), remodeling (blue bar) and metabolism/cell death (purple bar). In the LPS_HDM exposed CD45.2 cluster, these pathways are reduced compared to the PBS_HDM group.

Figure 7

Immunofluorescence staining of IM2 cluster markers defines a subset of interstitial macrophages that localizes to the lung parenchyma adjacent to terminal bronchioles in PBS_HDM exposed mice. (A) Evaluation of gene expression patterns across macrophage clusters for pan-macrophage markers revealed broad expression of CD206 (mrc1). Cathepsin K (Ctsk) was widely expressed in alveolar macrophages, but in interstitial macrophages, Ctsk was identified as a marker primarily restricted to the CD45.2 IM2 cluster in the PBS_HDM exposed mice. (B) This was confirmed with violin plots of the IM clusters identifying broad Mrc1 expression but Ctsk expression unique to the IM2 cluster. (C) Immunofluorescence staining on lung tissue sections from PBS_PBS, PBS_HDM, LPS_PBS, and LPS_HDM exposed was performed for CD206 (pink, pan-macrophage marker), DAPI (blue, to identify nuclei) and Ctsk (green, CD45.2 IM2 cluster). In all exposure groups, CD206 staining was noted in airspace and parenchyma macrophages. In the PBS_HDM mice, CD206⁺ Ctsk⁺ and DAPI⁺ (white stained cells identified by arrowheads) cells were located in the lung parenchyma adjacent to terminal airways consistent with resident IM2 cluster cells. Co-localization of CD206 and Ctsk in macrophages was not appreciated in the PBS_PBS, LPS_PBS, or LPS_HDM groups supporting that these macrophages were unique to the HDM-exposed groups.