Neutrophil Heterogeneity Is Modified during Acute Lung Inflammation in Apoa1-/- Mice

Abstract

Neutrophils play important roles in inflammatory airway diseases. In this study, we assessed whether apolipoprotein A-I modifies neutrophil heterogeneity as part of the mechanism by which it attenuates acute airway inflammation. Neutrophilic airway inflammation was induced by daily intranasal administration of LPS plus house dust mite (LPS+HDM) to Apoa1-/- and Apoa1+/+ mice for 3 d. Single-cell RNA sequencing was performed on cells recovered from bronchoalveolar lavage fluid on day 4. Unsupervised profiling identified 10 clusters of neutrophils in bronchoalveolar lavage fluid from Apoa1-/- and Apoa1+/+ mice. LPS+HDM-challenged Apoa1-/- mice had an increased proportion of the Neu4 neutrophil cluster that expressed S100a8, S100a9, and Mmp8 and had high maturation, aggregation, and TLR4 binding scores. There was also an increase in the Neu6 cluster of immature neutrophils, whereas neutrophil clusters expressing IFN-stimulated genes were decreased. An unsupervised trajectory analysis showed that Neu4 represented a distinct lineage in Apoa1-/- mice. LPS+HDM-challenged Apoa1-/- mice also had an increased proportion of recruited airspace macrophages, which was associated with a reciprocal reduction in resident airspace macrophages. Increased expression of a common set of proinflammatory genes, S100a8, S100a9, and Lcn2, was present in all neutrophils and airspace macrophages from LPS+HDM-challenged Apoa1-/- mice. These findings show that Apoa1-/- mice have increases in specific neutrophil and macrophage clusters in the lung during acute inflammation mediated by LPS+HDM, as well as enhanced expression of a common set of proinflammatory genes. This suggests that modifications in neutrophil and macrophage heterogeneity contribute to the mechanism by which apolipoprotein A-I attenuates acute airway inflammation.

Figure 1.. Neutrophilic inflammation is increased in the lungs of Apoa1^−/− mice following intranasal administration of lipopolysaccharide plus house dust mite (LPS+HDM).

A. Murine model of neutrophilic airway inflammation induced by intranasal administration of LPS+HDM(15). Apoa1^+/+ and Apoa1^−/− mice received intranasal administration of either HDM (25 μg), LPS (1 μg), or LPS (1 μg) plus HDM (25 μg), daily, for 3 consecutive days, and bronchoalveolar lavage fluid (BALF) was collected on day 4. B. Total number of BALF inflammatory cells. C - E. BALF neutrophils, macrophages, and lymphocytes. F. A representative cytospin of BALF cells from an LPS+HDM-challenged Apoa1^−/− mouse. The scale bar indicates 100 μm. BALF experiments represent pooled data from 2 separate experiments with a total of 10 mice per group, except for the HDM-challenged Apoa1^+/+ mice that had a total of 9 mice. Differences between Apoa1^+/+ and Apoa1^−/− mice were analyzed using a one-way ANOVA with Sidak’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 2.. Inflammatory mediators are increased in the lungs of Apoa1^−/− mice following intranasal administration of lipopolysaccharide plus house dust mite (LPS+HDM).

Apoa1^+/+ and Apoa1^−/− mice received intranasal administration of either HDM (25 μg), LPS (1 μg), or LPS (1 μg) plus HDM (25 μg), daily, for 3 consecutive days, and bronchoalveolar lavage fluid (BALF) was collected on day 4. A. BALF neutrophil elastase (ng/ml). B. BALF myeloperoxidase (ng/ml). C. BALF cell-free DNA (μg/ml). D - F. BALF IL-1β (pg/ml), TNF-α (pg/ml), IL-6 (pg/ml), CXCL1 (pg/ml) and G-CSF (pg/ml). BALF experiments represent pooled data from 2 separate experiments with a total of 10 mice per group. Differences between Apoa1^+/+ and Apoa1^−/− mice were analyzed using a one-way ANOVA with Sidak’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 3.. Bone marrow-derived neutrophils from Apoa1^−/− mice secrete increased amounts of Il-1β after stimulation with lipopolysaccharide plus house dust mite (LPS+HDM).

Neutrophils isolated from the bone marrows of wild type (WT) and Apoa1^−/− mice were stimulated with LPS (100 ng/ml) for 4 h, as signal 1, and HDM (10 μg/ml) for 3 h, as signal 2, for activation of the NLRP3 inflammasome, or media (control). A. The quantity of IL-1β secreted into the culture medium was quantified by ELISA. B. LDH release (ng/ml) from bone marrow-derived neutrophils stimulated with LPS (100 ng/ml) for 4 h, followed by HDM (10 μg/ml) for 3 h. Bone marrow-derived neutrophil experiments represent pooled data from 3 separate experiments with a total of 12 mice per group. Differences between WT and Apoa1^−/− groups were analyzed using the Mann-Whitney test. * P < 0.05.

Figure 4.. Intranasal administration of the 5A apoA-I mimetic peptide suppresses neutrophilic inflammation in wild type (WT) mice.

A. WT mice received intranasal administration of the 5A apoA-I mimetic peptide (1 mg/kg), an irrelevant scrambled control peptide (1 mg/kg), or PBS, as a control, every morning for 3 consecutive days, and LPS (1 μg) plus HDM (25 μg) or PBS, as a control, every afternoon for 3 consecutive days. Bronchoalveolar lavage fluid (BALF) was collected on day 4. B. Total number of BALF inflammatory cells. C. BALF neutrophils. D. BALF macrophages. E. BALF lymphocytes. BALF experiments represent pooled data from 2 separate experiments with a total of 10 mice per group. Differences between groups were analyzed using a one-way ANOVA with Sidak’s multiple comparisons test. * P < 0.05, **** P < 0.0001.

Figure 5.. Intranasal administration of the 5A apoA-I mimetic peptide suppresses levels of inflammatory mediators in bronchoalveolar lavage fluid from wild type (WT) mice.

WT mice received intranasal administration of the 5A apoA-I mimetic peptide (1 mg/kg), an irrelevant scrambled control peptide (1 mg/kg), or PBS, as a control, every morning for 3 consecutive days, and LPS (1 μg) plus HDM (25 μg) or PBS, as a control, every afternoon for 3 consecutive days. Bronchoalveolar lavage fluid (BALF) was collected on day 4. A. BALF neutrophil elastase (ng/ml). B. BALF myeloperoxidase (ng/ml). C. BALF cell-free DNA (μg/ml). D - H. BALF IL-1β (pg/ml), TNF-α (pg/ml), IL-6 (pg/ml), CXCL1 (pg/ml) and G-CSF (pg/ml). BALF experiments represent pooled data from 2 separate experiments with a total of 10 mice per group. Differences between groups were analyzed using a one-way ANOVA with Sidak’s multiple comparisons test. * P < 0.05, *** P < 0.001, **** P < 0.0001.

Figure 6.. ApoA-I modifies netosis and caspase 1 activation in lung neutrophils.

Apoa1^−/− and wild type (WT) mice received intranasal administration of LPS (1 μg) plus HDM (25 μg), daily, for 3 consecutive days. Alternatively, WT mice received intranasal administration of the 5A APOA1 mimetic peptide or an irrelevant scrambled control peptide (1 mg/kg) every morning for 3 consecutive days, and LPS (1 μg) plus HDM (25 μg), or PBS, as a control, every afternoon for 3 consecutive days. Bronchoalveolar lavage fluid (BALF) was collected on day 4. A and C. % BALF cytoplasts. B and D. Caspase 1 activation in CD45⁺/CD11b⁺/Ly-6G⁺ BALF neutrophils was assessed by quantifying the mean fluorescence intensity (MFI) of FAM-YVAD-FMK binding. BALF experiments represent pooled data from 2 separate experiments with a total of 10 mice per group. Differences between groups were analyzed using a Mann-Whitney test * P < 0.05, ** P < 0.01, **** P < 0.0001.

Figure 7.. Single cell RNA-sequencing analysis of neutrophils in bronchoalveolar lavage fluid (BALF) from Apoa1^−/− and Apoa1^+/+ mice following intranasal administration of lipopolysaccharide plus house dust mite (LPS+HDM).

Apoa1^−/− and Apoa1^+/+ mice received intranasal administration of LPS (1 μg) plus HDM (25 μg) daily, for 3 consecutive days. Bronchoalveolar lavage fluid (BALF) was collected on day 4 and single cell RNA sequencing was performed on the recovered cells. A. UMAP plot showing 10 neutrophil clusters in the total population of neutrophils present in BALF from Apoa1^−/− and Apoa1^+/+ mice. B. UMAP plots and pie chart showing the relative proportion of each of the 10 neutrophil clusters in BALF from Apoa1^−/− and Apoa1^+/+ mice. C. Dot plot showing the scaled expression of signature genes for each of the 10 neutrophil clusters. D. Heat map showing differentially expressed genes in Apoa1^−/− as compared to Apoa1^+/+ mice for each neutrophil cluster. E. Violin plots showing the expression of selected genes by the 10 neutrophil clusters from Apoa1^−/− and Apoa1^+/+ mice. F. Gene ontology pathways showing differential expression in Apoa1^−/− as compared to Apoa1^+/+ mice for each of the 10 neutrophil clusters.

Figure 8.. Intranasal administration of the 5A apoA-I mimetic peptide attenuates S100a8, S100a9, and Lcn2 expression in neutrophils isolated from the lungs of wild type (WT) mice.

WT mice received intranasal administration of the 5A apoA-I mimetic peptide (1 mg/kg), an irrelevant scrambled control peptide (1 mg/kg), or PBS, as a control, every morning for 3 consecutive days, and LPS (1 μg) plus HDM (25 μg) or PBS, as a control, every afternoon for 3 consecutive days. Neutrophils were purified from the lungs on day 4 and mRNAs encoding S100a8, S100a9, and Lcn2 were quantified by qPCR. Data are presented as relative quantification (RQ) as compared to expression of 18S rRNA. Each group was comprised of 16 mice and differences between groups were analyzed by ANOVA with a Sidak’s multiple comparisons test. ** P = 0.0023, *** P = 0.0006, **** P < 0.0001.

Figure 9.. The Neu4 cluster is a distinct lineage in Apoa1^−/− mice following intranasal administration of lipopolysaccharide plus house dust mite (LPS+HDM).

A. Unsupervised trajectory analyses of neutrophil populations from Apoa1^−/− and Apoa1^+/+ mice. B - D. Comparisons of functional scores for neutrophil maturation, neutrophil aggregation, and TLR4 binding for each of the 10 neutrophil clusters between Apoa1^−/− and Apoa1^+/+ mice. *** P < 0.001, Student’s t-Test.

Figure 10.. Single cell RNA-sequencing analysis of macrophages in bronchoalveolar lavage fluid (BALF) from Apoa1^−/− mice following intranasal administration of lipopolysaccharide plus house dust mite (LPS+HDM).

A. UMAP plot showing resident airspace macrophages (RAM) and recruited airspace macrophages (RecAM) in BALF from Apoa1^−/− and Apoa1^+/+ mice. B. The percentage of RAMs and RecAMs in BALF from Apoa1^−/− and Apoa1^+/+ mice. C. Heat map showing differentially expressed genes in Apoa1^−/− as compared to Apoa1^+/+ mice for RecAMs and RAMs. D. Dot plot showing scaled expression of selected genes expressed by RecAMs and RAMs in BALF from Apoa1^−/− and Apoa1^+/+ mice.