Tissue-Resident Alveolar Macrophages Reduce Ozone-induced Inflammation via MerTK-mediated Efferocytosis

Abstract

Lung inflammation, caused by acute exposure to ozone (O₃), one of the six criteria air pollutants, is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung, and their number increases after O₃ exposure. However, the role of AMØs in promoting or limiting O₃-induced lung inflammation has not been clearly defined. In this study, we used a mouse model of acute O₃ exposure, lineage tracing, genetic knockouts, and data from O₃-exposed human volunteers to define the role and ontogeny of AMØs during acute O₃ exposure. Lineage-tracing experiments showed that 12, 24, and 72 hours after exposure to O₃ (2 ppm) for 3 hours, all AMØs were of tissue-resident origin. Similarly, in humans exposed to filtered air and O₃ (200 ppb) for 135 minutes, we did not observe at ∼21 hours postexposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O₃ exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØs demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK (MER proto-oncogene, tyrosine kinase), a key receptor involved in efferocytosis, also resulted in impaired clearance of apoptotic neutrophils after O₃ exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O₃-induced inflammation via MerTK-mediated efferocytosis.

Keywords: MerTK; efferocytosis; macrophages; ozone.

Figure 1.

Monocyte-derived alveolar macrophages (AMØs) are not recruited after acute ozone (O₃) exposure. (A) Cx3cr1^ERCre × zsGreen mouse model was used. Circulating blood monocytes and monocyte-derived AMØs express Cx3cr1 and after tamoxifen induction are GFP⁺, whereas tissue-resident AMØs remain unlabeled. Lineage label was induced with tamoxifen 24 hours before exposure with filtered air (FA) or O₃ (2 ppm) for 3 hours. Twelve, 24, or 72 hours after exposure, whole lungs were processed and analyzed by flow cytometry. Schematic made using BioRender.com. (B) Bar plots represent individual mouse GFP⁻ (tissue-resident AMØs, gray) and GFP⁺ (monocyte-derived AMØs, white) populations. ANOVA was conducted using a post hoc Tukey’s honestly significant difference test. Data points are represented with SEM, showing significant increase of tissue-resident AMØs from FA to 12 and 72 hours after O₃ exposure. *P < 0.05. (C) Acute O₃ exposure does not induce recruitment/differentiation of monocyte-derived AMØs. Representative flow cytometry plots of AMØs were negative for zsGreen/GFP-positive cells, supporting no evidence of monocyte-derived AMØ recruitment. The red boxes indicate where GFP⁺ cells (monocyte-derived AMØs) would be observed if they were present in these samples. The percentage of GFP⁺ is indicated within the respective box in red. Full flow cytometry gating strategy and antibodies can be found in the data supplement (Figure E1). Flow plots are representative of n = 3–6 mice per time point and replicated once. Mice used included four females at 12 hours, with all other mice being male. SSC = side scatter.

Figure 2.

Monocyte-derived AMØs are not recruited after acute O₃ exposure in humans. (A) Human subjects were exposed to FA and O₃ (2 ppm) for 3 hours in separate visits. They then underwent a bronchoscopy in which BAL fluid was collected and processed and analyzed by flow cytometry. Demographic information is listed for all human subjects, including sex, age, and ethnicity. (B) Representative flow cytometry plots of the gating strategy used to identify AMØs were generated, demonstrating populations of CD206^Lo, monocyte-derived AMØ, and CD206^Hi, tissue-resident AMØ populations. The CD206^Lo population did not increase after acute O₃ exposure, supporting no evidence of monocyte-derived AMØ function in this context. The red boxes indicate where GFP⁺ monocyte-derived AMØs would be observed if they were present in these samples. (C) Acute O₃ exposure does not induce recruitment of monocyte-derived AMØs. Bar plots represent human cellular response to FA (white) and O₃ (gray) populations. Paired t test analysis was conducted, and data points are represented with SEM. No significant difference is seen in tissue-resident AMØ, monocyte-derived AMØ, and monocyte populations after O₃ exposure. (D) Hierarchical clustering of the flow cytometry data from BAL samples. Column headers are color coded by the exposure type (FA or O₃) and subject. Samples were clustered by Euclidean distance using average linkage method. Flow plots are representative of n = 12 human subjects.

Figure 3.

Airspace administration of clodronate (CL) depletes tissue-resident AMØs. (A) C57BL/6J female mice were dosed with CL or PBS (control) and were harvested 72 hours after dosing. The whole-lung tissue samples were perfused, digested, and stained to identify the individual cells via flow cytometry. Bars shaded green indicate PBS/vehicle control, whereas blue indicates CL administration. Data are from n = 4 mice per group; P value is as identified. (B) Cx3cr1^ERCre × zsGreen mice were dosed with CL 72 hours before harvest and then underwent lineage labeling with tamoxifen 48 hours after dosing. They were then harvested and processed for flow cytometry. The lack of GPF⁺ AMØs (red box) suggests a lack of Cx3cr1^ERCre × GFP⁺ monocyte-derived AMØs as a result of CL depletion of tissue-resident AMØs. The percentage of GFP⁺ is indicated within the respective box in red. Flow plot is a representative sample of n = 4. (C) Bar plot represents individual mouse GFP⁻ (tissue-resident AMØs, gray) and GFP⁺ (monocyte-derived AMØs, white) populations, indicating no recruitment of monocyte-derived AMØs after CL depletion. Full flow cytometry gating strategy and antibodies can be found in the data supplement (Figure E1).

Figure 4.

Tissue-resident AMØ depletion leads to persistent O₃-induced BAL neutrophilia. (A) Male mice were dosed with 50 μl of 5 mg/ml clodronate liposomes via oropharyngeal aspiration. Seventy-two hours after CL depletion of tissue-resident AMØs, mice were exposed to FA or O₃ (2 ppm) for 3 hours. Mice were then harvested 12 or 24 hours after exposure, and the samples were processed for cell differentials and other measures of inflammation. Schematic made with BioRender.com. (B) BAL total cells, (C) macrophages, and (D) neutrophils were enumerated after PBS or CL administration and FA or O₃ exposure. Bars shaded green indicate PBS/vehicle control, and blue indicates CL administration. By total cell numbers, CL-exposed mice exhibited a reduction of macrophages at 12 hours and increased neutrophils at 24 hours. n = 4–10 mice per group/exposure/time point. *P < 0.05. ANOVA was conducted using a post hoc Tukey’s honestly significant difference test.

Figure 5.

Increased BAL cytokines associated with neutrophil recruitment in O₃-exposed tissue-resident AMØ-depleted mice. BAL fluid from PBS- or CL- and FA- or O₃ (2 ppm)-exposed male mice was assessed for cytokine expression by multiplex ELISA for inflammatory cytokines and known neutrophilic chemotactic factors. Bars shaded green indicate PBS/vehicle control, whereas blue indicates CL administration. n = 4–10 mice per group/exposure/time point. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. ANOVA was conducted using a post hoc Tukey’s honestly significant difference test. ND indicates samples below the assay limit of detection. G-CSF = granulocyte colony-stimulating factor; LIF = leukemia inhibitory factor; MCP = monocyte chemoattractant protein; MIP = macrophage inflammatory protein.

Figure 6.

Tissue-resident AMØ depletion increased the proportion of apoptotic neutrophils after O₃ exposure. (A) The gating strategy to delineate neutrophil populations. Flow plots are a representative example of n = 5 samples per condition. (B) PBS or CL was administered to male C57BL/6J mice 72 hours before O₃ (2 ppm) for 3 hours. At 24 hours after exposure, BAL was collected, stained with Ly6G, 7-aminoactinomycin (7AAD), and annexin V, and analyzed by flow cytometry. Neutrophils were defined as Ly6G⁺ cells and then assessed for 7AAD and annexin V staining. “Live” neutrophils were defined as Ly6G⁺, 7AAD⁻, annexin V⁻; “early apoptosis” neutrophils were defined as Ly6G⁺, 7AAD⁻, annexin V⁺; “late apoptosis” neutrophils were defined as Ly6G⁺, 7AAD⁺, annexin V⁺; and “dead” neutrophils were categorized as Ly6G⁺, 7AAD⁺, annexin V⁻. **P < 0.005 and ***P < 0.0005.

Figure 7.

Tissue-resident AMØ depletion decreased efferocytosis. (A) C57BL/6J male mice were dosed with CL or PBS. The mice were then instilled with apoptotic immortalized T cells, Jurkat cells, via oropharyngeal aspiration 22.5 hours after exposure for 1.5 hours immediately before the harvest. During the harvest, 24 hours after exposure, BAL was collected and prepared for use in flow cytometry. Schematic made with BioRender.com. (B) Apoptotic cell clearance was defined using calcein AM, with positive cells indicating live or early apoptotic cells. Healthy cells would not be engulfed by AMØs; thus, decreased calcein AM–positive cells indicate clearance of apoptotic cells (efferocytosis). The graphs depict the ratio of counted recovered versus instilled cells from the collected BAL. Bars shaded green indicate PBS/vehicle control, and blue indicates CL administration. An unpaired t test was conducted with n = 10 mice per group/exposure/time point. ****P < 0.0001. UV = ultraviolet.

Figure 8.

MER proto-oncogene, tyrosine kinase–null (MerTK^−/−) mice have increased O₃-induced airspace inflammation. (A) C57BL/6J male mice were exposed to O₃ (2 ppm) for 3 hours and harvested 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 7 days later. mRNA expression of MerTK was assessed using the collected BAL cells. n = 5 mice per time point. (B) MerTK^−/− mice and C57BL/6J mice (wild type) were exposed to FA or O₃ (2 ppm) for 3 hours. The mice were harvested 24 hours after exposure, and the BAL was collected and processed for total cell counts and differentials. Green bars indicate the C57BL/6J mice, and the orange indicates the MerTK^−/− genotype. n = 9–19 mice per group. *P < 0.05, **P < 0.01 and ***P < 0.001. ANOVA was conducted using a post hoc Tukey’s honestly significant difference test. ND indicates samples below the limit of assay detection.

Figure 9.

Increased BAL cytokines associated with neutrophil recruitment in O₃-exposed MerTK^−/− mice. BAL fluid from C57BL/6J and MerTK^−/− mice. FA- or O₃ (2 ppm)-exposed mice were assessed for cytokine expression by multiplex ELISA for inflammatory cytokines and known neutrophilic chemotactic factors. Green bars indicate the C57BL/6J mice, and the orange indicates the MerTK^−/− genotype. n = 8–19 mice per group/exposure/time point. ***P < 0.001 and ****P < 0.0001. ANOVA was conducted using a post hoc Tukey’s honestly significant difference test. ND indicates samples below the limit of assay detection.