TREM-2 promotes macrophage survival and lung disease after respiratory viral infection

Abstract

Viral infections and type 2 immune responses are thought to be critical for the development of chronic respiratory disease, but the link between these events needs to be better defined. Here, we study a mouse model in which infection with a mouse parainfluenza virus known as Sendai virus (SeV) leads to long-term activation of innate immune cells that drive IL-13-dependent lung disease. We find that chronic postviral disease (signified by formation of excess airway mucus and accumulation of M2-differentiating lung macrophages) requires macrophage expression of triggering receptor expressed on myeloid cells-2 (TREM-2). Analysis of mechanism shows that viral replication increases lung macrophage levels of intracellular and cell surface TREM-2, and this action prevents macrophage apoptosis that would otherwise occur during the acute illness (5-12 d after inoculation). However, the largest increases in TREM-2 levels are found as the soluble form (sTREM-2) long after clearance of infection (49 d after inoculation). At this time, IL-13 and the adapter protein DAP12 promote TREM-2 cleavage to sTREM-2 that is unexpectedly active in preventing macrophage apoptosis. The results thereby define an unprecedented mechanism for a feed-forward expansion of lung macrophages (with IL-13 production and consequent M2 differentiation) that further explains how acute infection leads to chronic inflammatory disease.

Figure 1.

Effect of macrophage deficiency on postviral lung disease. (A) Lung levels of total cells, tissue monocytes (tissue monos), interstitial macrophages (interstitial macs), and alveolar macrophages (alveolar macs) for wt/wt and wt/opT mice at 5 dpi with SeV or UV-inactivated SeV (SeV-UV) determined by flow cytometry staining with fluorescent bead-based quantitation. (B) For conditions in (A), corresponding values for 49 dpi. (C) Representative photomicrographs of lung sections stained with PAS plus hematoxylin, immunostained for MUC5AC and counterstained with hematoxylin, or immunostained for IL-13 and counterstained with DAPI. Bars, 200 µm. (D) Lung levels of Il13, Muc5ac, Arg1, and Chi3l3 mRNA in wt/wt and wt/opT mice at 49 dpi with SeV or SeV-UV. For A, B, and D, * represents a significant increase from SeV-UV, and ** represents a significant decrease from corresponding wt/wt control value. All experimental data were verified in at least three independent experiments.

Figure 2.

Induction and function of Trem2 gene expression during postviral lung disease. (A) Gene expression microarray analysis of mRNA from lungs of mice at 3 and 49 dpi with SeV (10⁵ pfu) or SeV-UV. Scatter plot depicts log₂ normalized gene expression. Each symbol represents the expression value for an individual gene with the top 20 differentially expressed genes colored red and annotated in black, and others colored gray. Genes encoding Trem family members and Dap12 are colored red if significantly increased, blue if decreased, or white if unchanged, and are annotated in red. (B) Lung levels of Trem1, Trem2, and Il13 mRNA at indicated dpi with SeV. * represents a significant increase from (–) SeV control value. Values at 0 dpi were no different than those for SeV-UV 0–49 dpi. (C) Representative photomicrographs of PAS and Muc5ac staining of lung sections from WT, Trem2^−/−, Dap12^−/−, and Trem1/3^−/− mice at SeV p.i. day 49. Bar, 200 µm. (D) Corresponding lung levels of Il13, Muc5ac, Arg1, and Chi3l3 mRNA for conditions in (A). * represents a significant increase from SeV-UV mice and ** a significant decrease from corresponding WT control mice. All experimental data were verified in at least three independent experiments.

Figure 3.

Similarity of acute illness after viral infection in WT and knockout mice. (A) Body weights in WT, Trem2^−/−, Dap12^−/−, and Trem1/3^−/− mice at the indicated dpi. (B) Viral titers assessed by real-time qPCR assay for SeV N gene RNA in lungs of indicated mouse strains at 5 and 8 dpi. For A and B, values represent mean ± SEM for 4–7 mice. (C) Representative photomicrographs SeV immunostaining in lung sections from WT and Trem2^−/− mice at 0–8 dpi. (D) Representative photomicrographs of hematoxylin and eosin staining of lung sections from indicated mouse strains at 5 dpi. Each section contains an area of bronchiolitis with inflammatory cells surrounding an airway and adjacent blood vessel. Bar, 200 µm. All experimental data were verified in at least three independent experiments.

Figure 4.

Induction and function of TREM-2 in lung macrophages during postviral lung disease. (A) Representative photomicrographs of TREM-2 plus F4/80 or Mac-3 immunostaining of lung sections from WT mice at 49 dpi with SeV or SeV-UV. Bar, 200 µm. (B) Representative photomicrographs of F4/80 plus IL-13 immunostaining of lung sections from WT and Trem2^−/− mice at 49 dpi with SeV-UV or SeV. Bar, 200 µm. (C) Levels of Il13, Arg1, and Chi3l3 mRNA in FACS-purified tissue monos, interstitial macs, and alveolar macs from WT and Trem2^−/− mice at 49 dpi with SeV or SeV-UV. Each value represents mean ± SEM for five mice and is representative of three experiments. * represents a significant increase from corresponding SeV-UV in WT value, and ** represents a significant decrease from corresponding SeV in WT value. All experimental data were verified in at least three independent experiments.

Figure 5.

Regulation of cell surface and intracellular TREM-2 levels during viral replication and postviral lung disease. (A) Representative histograms from FACS analysis of TREM-2 immunostaining for tissue monos, interstitial macs, and alveolar macs from lungs of mice at 5 or 49 dpi with SeV-UV or SeV. (B) Corresponding MFI values for cell surface TREM-2 levels on tissue monos at the indicated dpi based on analysis conditions in A. (C) Representative cytograms from flow cytometry analysis of cell surface TREM-2 immunostaining for BMDMs at 0–3 dpi with GFP-expressing SeV (SeV-GFP). (D) Corresponding MFI values for cell surface TREM-2 and SeV-GFP for conditions in C. (E) Representative histograms for flow cytometry analysis of cell surface and intracellular TREM-2 immunostaining for BMDMs with indicated conditions. (F) Western blotting of TREM-2 in BMDMs at 2 dpi with SeV (MOI 0–10). (G) ELISA of cellular TREM-2 and sTREM-2 in BMDMs at 2 dpi with SeV for indicated MOI or with SeV at MOI 10 for 0–3 dpi. * represents a significant increase from corresponding control at MOI 0 and 0 dpi and Ø represents an undetectable level of sTREM-2. (H) Trem2 mRNA level in BMDMs with SeV at MOI 1 and 10 for 0–1 dpi. All experimental data verified in at least three independent experiments.

Figure 6.

Effect of cellular TREM-2 on macrophage apoptosis during acute viral infection. (A) Flow cytometry analysis of lung levels of total cells, tissue monos, and neutrophils (SSC^highLy6G⁺) for WT and Trem2^−/− mice at 0–5 dpi with SeV. (B) Ccl2 and Cx3cl1 mRNA levels in WT and Trem2^−/− mice at 0–5 dpi. For A and B, values represent mean ± SEM for 5 mice, and * represents a significant increase from 0 dpi and ** a significant decrease from corresponding WT mice. (C) Representative cytograms for flow cytometry analysis of levels of CD45.2⁺ tissue monos in WT and Trem2^−/− mice at 3 dpi after CD45.2 WT and Trem2^−/− BMDMs were introduced into CD45.1 WT mice at 0 dpi. (D) Representative photomicrographs of active caspase-3 and Mac-3 immunostaining of lung sections from WT and Trem2^−/− mice at 8 dpi. Arrows indicate cells with double-positive immunostaining. Bar, 200 µm. (E) Quantification of analysis in (D). Values represent mean ± SEM for 12 randomly selected fields of lung sections from 3 mice, and ** represents a significant increase from corresponding WT mice. (F) Representative photomicrographs of active caspase-3 and SeV immunostaining of lung sections from WT and Trem2^−/− mice at 5 dpi. Arrows indicate cells with active caspase-3⁺ immunostaining. Bar, 200 µm. (G) Lung levels of Csf1 mRNA for WT and Trem2^−/− mice at indicated dpi. (H) Corresponding lung levels of CSF-1 for conditions in (G). For G and H, values represent mean ± SEM for 5 mice, and * represents a significant increase from 0 dpi. (I) Flow cytometry analysis of propidium iodide staining of BMDMs from WT and Trem2^−/− mice cultured with or without CSF-1 for 1 d. (J) Levels of Annexin V immunostaining in PI^– cells for culture conditions in (I). Values represent mean ± SEM for 6 randomly selected fields of replicate wells, and * represents a significant increase from (+) CSF-1, and ** a significant increase from corresponding WT mice. (K) Western blot levels of active caspase-3 for culture conditions in I. All experimental data were verified in at least three independent experiments.

Figure 7.

Effect of IL-13 on production of sTREM-2. (A) Lung levels of TREM-2 determined by ELISA at 5 or 49 dpi with SeV or SeV-UV. Values represent mean ± SEM for 5 mice, and * represents a significant increase from corresponding SeV-UV mice. (B) Representative histograms from flow cytometry analysis of cell surface TREM-2 on BMDMs treated with or without IL-13 (20 ng/ml) or IgG control for 1–3 d. (C) Representative histograms from flow cytometry analysis of cell surface TREM-2 on BMDMs treated with or without IL-13, IL-4, IL-5, or IL-9 (20 ng/ml) or IgG control for 1 d. (D) Levels of cellular and sTREM-2 in BMDMs obtained from WT and Dap12^−/− mice and cultured with IL-13 for 1–3 d post-administration (dpa) as in B. Values represent mean ± SEM for 3 replicate wells, and * represents a significant increase from 0 dpa and ** a significant decrease from corresponding WT cells. (E) Lung levels of TREM-2 in WT and Dap12^−/− mice at 49 dpi. Values represent mean ± SEM for 5 mice, and * represents a significant increase from SeV-UV and ** a significant decrease from corresponding WT mice. All experimental data were verified in at least three independent experiments.

Figure 8.

Effect of sTREM-2 on macrophage apoptosis. (A) Representative photomicrographs of immunostaining for recombinant sTREM-2 after incubation with BMDMs for 30 min with and without cell permeabilization with saponin. Bar, 100 µm. (B) Levels of Arg1 and Chi3l3 mRNA in BMDMs obtained from Trem2^−/− and WT mice and then cultured with sTREM-2 (0–200 ng/ml) or IL-13 (20 ng/ml) for 1 d. Values represent mean ± SEM for 3 replicate wells, and * represents a significant increase from no treatment control (0 ng/ml). (C) Representative photomicrographs of sTREM-2–treated BMDMs cultured in 15% l-cell conditioned medium for 7 d. Bar = 200 µm. (D) XTT assay for BMDMs obtained from WT, Trem2^−/−, and Dap12^−/− mice and then cultured with sTREM-2 (0–200 ng/ml) for 2 d after CSF-1 withdrawal. Values represent mean ± SEM for 3 replicate wells, and * represents a significant increase from corresponding untreated control cells. (E) XTT assay of BMDMs from WT mice cultured with native (unheated) or denatured (heated) sTREM-2 (0–200 ng/ml) with culture conditions in D. * represents a significant increase from corresponding untreated control cells. (F) Flow cytometry analysis of propidium iodide staining of BMDMs obtained from WT, Trem2^−/−, and Dap12^−/− mice and cultured with sTREM-2 (0–200 ng/ml) with or without CSF-1 for 1 d. (G) Western blot levels of active caspase-3 for culture conditions in F. (H) Flow cytometry analysis of BrdU-labeling of BMDMs from Trem2^−/− mice for culture conditions in F. All experimental data were verified in at least three independent experiments.

Figure 9.

Effect of TREM-2 deficiency on apoptosis signaling pathway. (A) Western blot levels of indicated cell survival proteins in BMDMs cultured from WT and Trem22/2 mice after treatment with mouse sTREM-2 (200 ng/ml) for 0–24 h. (B) Representative photomicrographs of phospho-ERK1/2 (p-ERK) and Mac-3 immunostaining with DAPI counterstaining of lung sections from WT and Trem22/2 mice at 5 or 49 dpi with SeV or SeV-UV. Arrows indicate cells with double-positive immunostaining. Bar, 200 µm. (C) Flow cytometry analysis of lung levels of total cells and tissue monos for WT and Trem22/2 mice at 0 and 49 dpi. Values represent mean ± SEM for 5 mice, and * represents a significant increase from 0 dpi and ** a significant decrease from corresponding WT mice. Values at 0 dpi were no different than those for SeV-UV (49 dpi). All experimental data were verified in at least three independent experiments.

Figure 10.

Scheme for regulation and function of TREM-2 in chronic postviral lung disease. Major steps include: (1) early activation of lung macrophages in which viral replication increases TREM-2 at the cell surface; (2) cleavage of TREM-2 from the cell surface to form sTREM-2 in a process that is up-regulated by IL-13 and DAP12; (3) sTREM-2 actions to prevent apoptosis in association with increased ERK1/2 activation and thereby allow for amplified macrophage interaction with iNKT cells and consequent IL-13 production; and (4) IL-13–dependent differentiation of the macrophage population toward an M2 pattern of gene expression (including Arg1 and Chi3l3) and differentiation of an airway progenitor epithelial cell (APEC) niche to airway mucous cells (AMCs) as signatures of chronic lung disease.