Thrombospondin-1 triggers macrophage IL-10 production and promotes resolution of experimental lung injury

Abstract

Mononuclear phagocyte recognition of apoptotic cells triggering suppressive cytokine signaling is a key event in inflammation resolution from injury. Mice deficient in thrombospondin (TSP)-1 (thbs1⁻/⁻), an extracellular matrix glycoprotein that bridges cell-cell interactions, are prone to lipopolysaccharide-induced lung injury and show defective macrophage interleukin (IL)-10 production during the resolution phase of inflammation. Reconstitution of IL-10 rescues thbs1⁻/⁻ mice from persistent neutrophilic lung inflammation and injury and thbs1⁻/⁻ alveolar macrophages show defective IL-10 production following intratracheal instillation of apoptotic neutrophils despite intact efferocytosis. Following co-culture with apoptotic neutrophils, thbs1⁻/⁻ macrophages show a selective defect in IL-10 production, whereas prostaglandin E2 and transforming growth factor beta 1 responses remain intact. Full macrophage IL-10 responses require the engagement of TSP-1 structural repeat 2 domain and the macrophage scavenger receptor CD36 LIMP-II Emp sequence homology (CLESH) domain in vitro. Although TSP-1 is not essential for macrophage engulfment of apoptotic neutrophils in vivo, TSP-1 aids in the curtailment of inflammatory responses during the resolution phase of injury in the lungs by providing a means by which apoptotic cells are recognized and trigger optimal IL-10 production by macrophages.

Figure 1

TSP-1 is required for effective resolution following lung injury. (A–B) Note that the range of the y-axis is separated into two different segments with each lower segment tick interval representing an increase by 2 × 10⁵ cells/mL. Each tick interval within the higher segment represents an increase of 20 × 10⁵ cells/mL. (A) Total cell counts/ml, (B) Total PMN counts/ml; n=8–16 mice/group at day 0, 1, 3, and at day 6. A minimum of 2 independent experiments was performed for each time points in A–B. (C) Total BAL protein concentrations. n= 4–12 mice/group at day 0, 1, 3, and day 6. (D) Evan’s blue dye extravasation, an indicator of lung leak and measured by OD_620-500, is increased in thbs1^−/− mice at 1, 3, and days 6 following injury but not under basal conditions (day 0). n=5–7 mice/group. (E) Representative H&E of lung tissue sections obtained from WT and thbs1^−/− mice at day 0, 6 post-LPS instillation. Scale bar, 100 um. (F) IL-10, (G) IL-6, (H) KC and (I) MCP-1 concentrations in BAL. n=7–8 mice/group at each time point. Data are represented as mean +/− SEM. (J) Bioactive concentrations of soluble TGF-βl as measured by a TGFβl-sensitive PAI-1 promoter luciferase reporter assay, n=3–4 mice/group performed 2 independent times. (K) The number of IL-10 producing cells from CD 11c-selected BAL cells in WT and thbs1^−/− mice at day 6 following LPS instillation determined by ELISPOT. SFC, spot forming cells. n=4–5 mice per group. **p <0.001, *p<0.05.

Figure 2

Impaired resolution in il10^−/− mice and IL-10 administration attenuates neutrophilic inflammation in the airspaces and lung microvascular permeability of thbs1^−/− mice during the resolution phase of LPS-induced lung injury. (A) Total cell counts/ml, (B) Total PMN counts/ml, (C) Total Protein concentrations in the BAL fluid of WT and il10^−/− mice under basal conditions (day 0) and at day 6 following intratracheal LPS instillation. *p < 0.05, n=4 mice per group at day 0; n=7–8 mice per group at day 6. (D) Total cell counts/ml, (E) Total PMN counts/ml, (F) Total protein concentrations in the BAL fluid of WT and thbs1^−/− mice at day 6 following intratracheal LPS instillation. WT and thbs1^−/− mice were administered either recombinant murine IL-10 (1 μg) or vehicle (PBS) intraperitoneally at days 0, 2, and 4 following intratracheal LPS. *p < 0.05, n=7–8 mice per group at day 6. Data are represented as mean +/− SEM.

Figure 3

thbs1^−/− mice show defective IL-10 production but intact efferocytosis in the lungs following intratracheal instillation of apoptotic neutrophils. (A) % of apoptotic neutrophils following 24 h serum starvation, as indicated by 7-AAD^negAnnexinV⁺ events. (B) Confocal image of alveolar macrophages from naïve WT mice immunostained with CD11c (red), white arrow reflects CD11c⁺ alveolar macrophage. (C) CD11c⁺ (red) alveolar macrophages instilled with DDAO-labeled apoptotic neutrophils (green), white arrowhead reflects ingested apoptotic neutrophils. Scale bar=10 microns. (D) % Efferocytosis in WT or thbs1^−/− mice. (E) The number of IL-10 producing cells from CD11c-enriched BAL cells in WT and thbs1^−/− mice with or without intratracheal instillation of apoptotic neutrophils determined by ELISPOT. SFC, spot forming cells. ** p < 0.01, n=4–5 mice per group.

Figure 4

Optimal IL-10 production by macrophages following stimulation with apoptotic neutrophils requires TSP-1. (A) IL-10 production in pg/ml, as measured in the supernatant, following incubation of WT macrophages with fresh or apoptotic neutrophils in the absence of LPS, and (B) following stimulation with LPS. (C) IL-10 production in pg/ml following incubation of WT versus thbs1^−/− macrophages with syngeneic apoptotic neutrophils in the absence of LPS, and (D) following stimulation with LPS. (E) PGE2 production in pg/mL following incubation of WT versus thbs1^−/− macrophages with syngeneic apoptotic neutrophils following stimulation with LPS. (F) Total TGF-βl production in pg/ml following incubation of WT and thbs1^−/− macrophage with syngeneic apoptotic neutrophils following stimulation of macrophages with LPS prior to the addition of neutrophils. Data points represent individual wells performed in duplicate, utilizing macrophages combined from 3–4 mice, and neutrophils harvested from 5–6 mice from respective genotype, mean +/− SEM. *p<0.01, **p<0.001, comparison to respective WT control at the same PMN:Mφ ratio. Experiment was performed twice with similar results, and the results of one representative experiment are shown.

Figure 5

TSR 2 and CD36 engagement is required for macrophage IL-10 production. (A) Macrophages from thbs1^−/− mice were pre-incubated with increasing concentrations of TSR domain 2 peptide prior to LPS stimulation and the addition of syngeneic apoptotic neutrophils to WT and thbs1^−/− macrophages. (B) IL-10 production in pg/ml following incubation of WT macrophages with GST alone, GST-CD36 (81–117), GST-CD36 (90–117), and Thioredoxin-CD36 (81–117) peptides containing the CLESH domain at 1 μg/mL each in the presence of LPS prior to the addition of syngeneic apoptotic neutrophils. (C–D) Macrophages were transfected with siRNA targeted against CD36 and control scramble sequence RNA (scRNA). At 72 hour post-transfection, Macrophages were stimulated with LPS (10 ng/ml) for 1 h prior to the addition of neutrophils and IL-10 in the supernatant was subsequently detected by ELISA. Cells lysates were analysed by SDS-PAGE and immunoblotted with CD36 and β–actin antisera. *p < 0.05. (A–D) are representative of 2 independent experiments. Each condition was performed in duplicates, utilizing macrophages combined from 3–4 mice, and neutrophils harvested from 5–6 mice.