Identification of a nerve-associated, lung-resident interstitial macrophage subset with distinct localization and immunoregulatory properties

Abstract

Tissue-resident macrophages are a diverse population of cells that perform specialized functions including sustaining tissue homeostasis and tissue surveillance. Here, we report an interstitial subset of CD169⁺ lung-resident macrophages that are transcriptionally and developmentally distinct from alveolar macrophages (AMs). They are primarily localized around the airways and are found in close proximity to the sympathetic nerves in the bronchovascular bundle. These nerve- and airway-associated macrophages (NAMs) are tissue resident, yolk sac derived, self-renewing, and do not require CCR2⁺ monocytes for development or maintenance. Unlike AMs, the development of NAMs requires CSF1 but not GM-CSF. Bulk population and single-cell transcriptome analysis indicated that NAMs are distinct from other lung-resident macrophage subsets and highly express immunoregulatory genes under steady-state and inflammatory conditions. NAMs proliferated robustly after influenza infection and activation with the TLR3 ligand poly(I:C), and in their absence, the inflammatory response was augmented, resulting in excessive production of inflammatory cytokines and innate immune cell infiltration. Overall, our study provides insights into a distinct subset of airway-associated pulmonary macrophages that function to maintain immune and tissue homeostasis.

Fig 1.. Airway associated interstitial macrophages display distinct morphology, and localization.

(A) Stitched (left) and single (right) confocal images of C57BL/6 naïve lungs immunostained for EpCAM (blue), CD11c (green), and CD169 (red). AMs (CD11c⁺CD169⁺) (purple arrows) and NAMs (CD169⁺CD11c⁻) (white arrows) depicting two distinct populations of CD169⁺ macrophages. Scale bar: 400 μm (stitched images), 80 um (single image).(B) Zeiss Light Sheet Z.1 image of clarified CD169-tdTomato lung. Scale bar: 300 μm.(C) Healthy human lungs immunostained with CD169 (red) and CD11c (green). AMs (CD11c⁺CD169⁺) (purple arrows) NAMs (CD169⁺CD11c⁻) (white arrows)(D) CX3CR1-eGFP lungs immunostained with CD11c (green) and CD169 (red). NAMs (CD169⁺CD11c⁻CX3CR1⁺) (white arrows), AMs (CD169⁺CD11c⁺CX3CR1⁻) (purple arrows). AW: Airways. Scale bar: 80 μm. Data are representative of two-three independent experiments (n = 3 to 5).

Fig 2.. Airway associated interstitial macrophages interact closely with nerves.

Confocal images of C57BL/6 naïve lungs immunostained for EpCAM (purple), beta 3 tubulin (green), CD11c (cyan), and CD169 (red). Magnified views of the boxed areas in subsequent isosurfaced images (Left to Right). Scale bar: 100, 50, and 20 μm (left to right). Data are representative of 3–4 independent experiments (n = 3).

Fig 3.. Seurat analysis of unbiased single cell RNA sequencing of pulmonary macrophages.

(A) t-SNE plot identifying 5 clusters of MerTK⁺CD64⁺ pulmonary macrophage subsets with distinct gene expression profiles(B and C) Heat maps of hierarchical cluster analysis of cluster 1 through cluster 5.(B) Top 42 DEGs between cluster 1, 2, and 3.(C) Top 78 DEGs between cluster 3 and 5.(D) t-SNE plots of specific genes highly enriched in cluster 3 (NAMs)(E) Violin plots showing expression pattern of additional genes that were enriched in cluster 3 (NAMs) compared to all other clusters.(F) Violin plots showing expression pattern of genes that were enriched in cluster 4 compared to all other clusters.

Fig 4.. NAMs are yolk sac derived and require CSF1-CSF1R axis for development.

(A) Immunofluorescence of E19.5 (left) and 6 weeks old (right) E8.5 tamoxifen treatedCx3cr1-CreEr-Rosa26-tdTomato lungs labeled by tdTomato⁺ (blue), CD169 (red), and CD11c (green).(B-E) Immunostaining of CD169 (red) and CD11c (green) of lung(B and D) and flow cytometry analysis of NAMs, CD169⁻IMs, and AMs(C and E) ofCsf2^−/− (left) andCsf1^op/op (right) mice. AW: Airways. Scale Bar: 80 μm. Data are representative of two independent experiments. Data shown as mean ± SEM (n = 4 – 5, ** P<0.01, ****P<0.0001, Student’s t-test).

Fig. 5.. NAMs are self-maintaining resident macrophages.

(A) Chimerism of lung AMs (F4/80⁺, CD169⁺, CD11c⁺), NAMs (F4/80⁺, CD169⁺, CD11c⁻, Ly6C⁻), and CD169⁻IMs (F4/80⁺, CD169⁻, CD11c⁺) (left) and splenic T and B cells (right) from CD45.1-CD45.2 parabionts at 5 (top) and 12 (bottom) weeks after chimerism.(B) Immunostaining of CD169 (red), Ki-67 (green), and CD11c (cyan) in naïve C57BL/6 lungs reveal active proliferation (yellow arrows). Scale bar: 80 μm (left) and 40 μm (right). Data representative of two experiments. Data shown as mean ± SEM (n = 3 – 6, ** P<0.01, ****P<0.0001 paired Student’s t-test).

Fig. 6.. NAMs are maintained independent of CCR2⁺ bone marrow monocytes

(A) CD169 (red) and CD11c (green) immunostaining (left) and flow cytometric analysis (right) depicting similar localization and frequency of NAMs (F4/80⁺, CD169⁺, CX3CR1⁺, CD11c⁻, Ly6C⁻) between WT and CCR2^−/− mice.(B) CD169 (red) and CD11c (green) immunostaining (left) and flow cytometric analysis (right) of Pdzk1ip1^CreERT2-tdTtomato lungs. AMs (CD169⁺CD11c⁺) (yellow arrows), NAMs (CD169⁺CD11c⁻), and HSCs originating cells (tdTomato⁺).(C) Confocal image (left) and flow cytometric analysis (right) of WT and CCR2^−/− bone marrow cells engrafted to DT treated and irradiated CD169-DTR mice at 3 d.p. irrad. Control (left), CCR2^−/− BM (center), and WT BM (right). AW: Airways. Scale Bar: 80 μm. All data are representative of two-three independent experiments. Data shown as mean ± SEM (n = 4 – 5, ***P<0.001, Student’s t-test).

Fig. 7.. NAMs regulate the inflammatory response and react robustly to influenza infection.

(A) Foci formation unit (FFU) of C57BL/6, CD169-DTR, AM-DTR, and NAM-DTR lungs 3 dpi with 100 EID50 of PR8.(B) Confocal images of C57BL/6 lungs 24 and 48 hpi with NS1-GFP expressing PR8 and counterstained with CD11c (blue) and CD169 (red). Scale bar: 50–20 μm.(C) Weight loss (left) and survival (center) curve of C57BL/6 and CD169-DTR mice and weight loss (right) of C57BL/6 and NAM-DTR mice following PR8 intranasal infection.(D) Confocal imaging of EpCAM (purple), CD169 (red), and CD11c (blue) in IL10-GFP mice following PR8 infection. Co-localization of CD169 and IL-10 pseudo-colored in white (left) and zoomed (right). AW: Airways. Scale bar: 20–80 μm. Data representative of two independent experiments. Data shown as mean ± SEM (n = 3 – 5, ** P<0.01, *** P<0.001, 2-way ANOVA with Bonferroni’s post-test and Student’s t-test).