The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation

Abstract

Fine control of macrophage activation is needed to prevent inflammatory disease, particularly at barrier sites such as the lungs. However, the dominant mechanisms that regulate the activation of pulmonary macrophages during inflammation are poorly understood. We found that alveolar macrophages (AlvMs) were much less able to respond to the canonical type 2 cytokine IL-4, which underpins allergic disease and parasitic worm infections, than macrophages from lung tissue or the peritoneal cavity. We found that the hyporesponsiveness of AlvMs to IL-4 depended upon the lung environment but was independent of the host microbiota or the lung extracellular matrix components surfactant protein D (SP-D) and mucin 5b (Muc5b). AlvMs showed severely dysregulated metabolism relative to that of cavity macrophages. After removal from the lungs, AlvMs regained responsiveness to IL-4 in a glycolysis-dependent manner. Thus, impaired glycolysis in the pulmonary niche regulates AlvM responsiveness during type 2 inflammation.

Fig. 1 |. AlvMs are unresponsive to IL-4.

a, Flow cytometry plots identifying AlvMs and IntMs from BAL fluid or lung tissue of naïve mice. Data representative of eight independent experiments. b, Imaging cytometry of AlvMs and IntMs from lung tissue of naïve mice (scale bar, 10 μm). c, Histograms of expression of F4/80, CD11c, Ym1 and eGFP by Cx3cr1^eGFP/+ mice by AlvMs and IntMs from lung tissue of naïve mice. d, Flow cytometry plots of AlvMs, IntMs and monocytes from lung tissue of naïve mice following CD45 i.n. and i.v. administration. b–d, Representative data from three independent experiments. e, Numbers of lung tissue AlvMs and IntMs, or PECMs, on day 4 following i.p. PBS or IL-4c administration on day 0 and day 2. Data representative of seven to nine independent experiments, n = 26 (AlvM PBS, AlvM IL-4c, IntM PBS, IntM IL-4c), n = 18 (PECM PBS), n = 13 (PECM IL-4c) mice per group. f, RELMα and Ki67 expression, or EdU incorporation, by lung tissue AlvMs and IntMs, or PECMs, on day 4 following i.p. PBS or IL-4c administration on day 0 and day 2, and EdU injection i.p. 3 h before tissue collection. Graphs show individual replicate mice, data pooled from five to nine independent experiments. RELMα: n = 29 (AlvM PBS, AlvM IL-4c, IntM PBS, IntM IL-4c), n = 24 (PECM PBS), n = 23 (PECM IL-4c) mice per group. Ki67: n = 24 (AlvM PBS, IntM PBS), n = 23 (AlvM IL-4c, IntM IL-4c), n = 20 (PECM PBS, PECM IL-4c) mice per group. EdU: n = 22 (AlvM PBS, AlvM IL-4c, IntM PBS), n = 23 (IntM IL-4c), n = 14 (PECM PBS), n = 17 (PECM IL-4c) mice per group. g, Percentage of RELMα⁺ PECMs, PLECMs, Kupffer cells, IntMs and AlvMs on day 4 following i.p. PBS or IL-4c administration on day 0 and day 2. Data representative of two to five independent experiments, n = 5 (PBS PECMs, Kupffer cells, Colon Ms, IntMs and AlvMs), n = 4 (IL-4c PLECMs), n = 3 (PBS PLECMs, colon Ms and IL-4c PECMs, Kupffer cells, colon Ms, IntMs, AlvMs) mice per group. e–g, Data analyzed by two-way analysis of variance (ANOVA) with Tukey’s post-test for multiple comparisons, displayed as mean ± s.e.m., *P <0.05, ***P <0.001 and ****P <0.0001.

Fig. 2 |. AlvMs are less responsive than IntMs to IL-4c administered directly into the airways.

a, IL-4Rα expression by AlvMs, IntMs or PECMs from mice injected with PBS or IL-4c i.p. on day 0 and day 2, and lung tissue and PEC collected on day 4. Histograms representative of two independent experiments. b, ELISA of IL-4 amounts in BAL or PEC fluids 6, 12, 24 or 48 h after i.p. injection of PBS or IL-4c. Data representative of two independent experiments, n = 2 (PBS, 24 and 48 h), n = 3 (6 and 12 h) mice per group. c, Flow cytometry plots of RELMα expression in lung tissue AlvMs and IntMs on day 4 following i.n. PBS or IL-4c administration on day 0 and day 2 (left) and quantification of the percentage of RELMα⁺ cells (right). Data representative of three independent experiments, n = 2 (5 μg AlvM), n = 3 (AlvM: PBS, 0.05 and 0.5 μg. IntM: PBS, 0.05, 0.5 and 5 μg) mice per group. d, Histograms of pSTAT6 amounts in lung tissue AlvMs or PECMs 15 min after PBS or rIL-4 administered i.n. or i.p. to wild-type (WT) or Il4ra^−/− mice (left), and quantification of AlvM and PECM pSTAT6 expression (right). Data representative of two independent experiments, n = 3 mice per group. Data analyzed by one-way ANOVA with Tukey’s post-test for multiple comparisons, displayed as mean ± s.e.m., *P <0.05, ***P <0.001 and ****P <0.0001.

Fig. 3 |. AlvMs are less responsive than IntMs during helminth infection.

a, Eosinophil numbers from lung tissue of naïve mice or on day 2, day 4 and day 7 following infection s.c. with 500 L3 N. brasiliensis larvae. Graphs show individual replicate mice, data pooled from four independent experiments, n = 18 (naïve), n = 17 (day 2), n = 9 (day 4), n = 5 (day 7) mice per group. b, ELISA of RELMα amounts in BAL fluid from naïve or infected mice. Data pooled from two independent experiments, n = 8 (naïve), n = 7 (day 2), n = 4 (day 4), n = 3 (day 7) mice per group. c, Numbers of lung tissue AlvMs and IntMs from naïve or infected mice. Data pooled from four independent experiments, n = 18 (naïve), n = 17 (day 2), n = 9 (day 4), n = 8 (day 7) mice per group. d, Flow cytometry plots identifying lung tissue AlvMs and IntMs from naïve or infected mice. Data representative of four independent experiments. e, Quantification of the percentage of RELMα⁺ and Ki67⁺ lung tissue AlvMs and IntMs from naïve or infected mice. Data pooled from four independent experiments, n = 18 (Naïve RELMα), n = 17 (day 2 RELMα), n = 9 (day 4 RELMα), n = 8 (day 7 RELMα), n = 13 (Naïve Ki67), n = 12 (day 2 Ki67), n = 5 (day 4 Ki67), n = 3 (day 7 Ki67) mice per group. Data analyzed by one-wayANOVA with Tukey’s post-test for multiple comparisons, displayed as mean ± s.e.m., *P <0.05, **P <0.01, ***P <0.001 and ****P <0.0001.

Fig. 4 |. The pulmonary niche regulates AlvM responsiveness to IL-4 independently of host commensals.

a, mRNA expression by quantitative PCR (qPCR) of lung tissue AlvMs from naïve mice following culture for 12, 24 or 48 h in media alone or with rIL-4 (20 ng ml⁻¹). AU, arbitrary units. Data representative of four independent experiments, n = 2 (media), n = 3 (rIL-4) wells per group, each group pooled cells from eight mice. b–e, Donor (CD45.2⁺) and host (CD45.1⁺) macrophage populations identified by flow cytometry in lung tissue or PEC from host mice on day 5 after i.n. PBS or donor PECM transfer on day 0, then injection with IL-4c i.p. on day 1 and day 3. b, Flow cytometry plots identifying donor PECMs in host lung tissue. Data representative of four independent experiments. c, Quantification of the percentage of RELMα⁺ and Ki67⁺ host and donor macrophages isolated from host lung tissue or PEC. Data representative of four independent experiments, n = 6 mice per group. d, IL-4Rα expression by donor PECMs isolated from host lung tissue. Histogram representative of two independent experiments. e, RELMα and Ki67 expression by lung tissue AlvMs and IntMs, or PECMs, from specific pathogen-free (SPF) or germ-free (GF) mice on day 4 following i.p. PBS or IL-4c administration on day 0 and day 2. Data representative of three independent experiments, n = 3 mice per group. Data analyzed by one-way ANOVA with Tukey’s post-test for multiple comparisons, displayed as mean ± s.e.m., **P <0.01, ***P <0.001 and ****P <0.0001.

Fig. 5 |. AlvMs and PECMs display different metabolic gene profiles.

a, mRNA expression profiles (volcano plots) as determined by RNA-seq of PECMs, IntMs or AlvMs isolated from PEC or lung tissue by flow cytometry on day 4 following i.p. PBS or IL-4c administration on day 0 and day 2. Dashed lines represent P <0.01, and about two-fold change, IL-4c relative to PBS. b, Heatmaps of selected mRNA transcripts of genes that have been previously described as M(IL-4) markers, *indicates significance between IL-4c vs PBS of at least P <0.01.c, mRNA expression profile (volcano plot) of AlvMs versus PECMs isolated from IL-4c injected mice. Dashed lines represent P <0.01, and about two-fold change. d, Selected pathways from KEGG analysis (Supplementary Fig. 6) of significantly altered mRNA transcripts from c, black lines represent P <0.05. e, Relative transcript expression by AlvMs versus PECMs from IL-4c injected mice that were significantly altered (P <0.01, log2 normalized intensity), as identified from the glycolysis pathway by network analysis (several genes displayed more than one altered transcript variant), n = 2 (PECM PBS, PECM IL-4c, AlvM PBS, IntM PBS, IntM IL-4c), n = 3 (AlvM IL-4c) separate biological replicates, each replicate pooled cells from three to five mice.

Fig. 6 |. Impaired uptake and use of glucose renders AlvMs unresponsive to IL-4.

a, ECAR of AlvMs and PECMs isolated from lung tissue or PEC of naïve mice by flow cytometry, at baseline and after sequential treatment (vertical lines) with glucose, oligomycin (Oligo) or 2-Deoxy-d-glucose (2-DG) to measure glycolysis, glycolytic reserve and glycolytic capacity. Data representative of four independent experiments, n = 6 (AlvM), n = 10 (PECM) glycolytic stress test profile, n = 6 (AlvM) glycolysis, glycolytic capacity and glycolytic reserve, n = 10 (PECM) glycolysis, glycolytic capacity, n = 9 (PECM) glycolytic reserve, wells per group, each group pooled cells from eight mice. b, Flow cytometry plots of 2-NBDG uptake in vitro by BAL AlvMs, or PECMs, from naïve mice cultured separately or at a 50:50 mix for 20 min with fluorescently labeled 2-NBDG. c, 2-NBDG uptake in vivo by donor (CD45.2⁺) and host (CD45.1⁺) macrophage populations identified by flow cytometry in lung tissue or PEC from host mice on day 5 after i.n. PBS or donor PECM transfer on day 0, administration of IL-4c i.p. on day 1 and day 3, and i.p. injection of fluorescently labeled 2-NBDG 20 min before lung tissue and PEC collection. Data representative of two independent experiments, n = 6 mice per group. d, mRNA expression by qPCR of lung tissue AlvMs from naïve mice cultured for 0, 12, 24 or 48 h in media alone. Data representative of six (Eno1) or five (Slc2a6) independent experiments, n = 3 (Eno1), n = 2 (Slc2a6) wells per group, each group pooled cells from six to eight mice. e, mRNA expression by qPCR of lung tissue AlvMs from naïve mice cultured for 48 h in media alone, or with rIL-4 ± 2-DG. Data representative of three independent experiments, n = 2 (media), n = 3 (rIL-4), n = 3 (rIL-4 + 2-DG) wells per group, each group pooled cells from six to eight mice. Data analyzed using unpaired t-test (a) or a one-way ANOVA with Tukey’s post-test for multiple comparisons as indicated (b, c, e) or compared to 0 h (d), displayed as mean ± s.e.m., *P <0.05, **P <0.01, ***P <0.001 and ****P <0.0001.