Macrophages are critical to the maintenance of IL-13-dependent lung inflammation and fibrosis

Abstract

The roles of macrophages in type 2-driven inflammation and fibrosis remain unclear. Here, using CD11b-diphtheria toxin receptor (DTR) transgenic mice and three models of interleukin 13 (IL-13)-dependent inflammation, fibrosis, and immunity, we show that CD11b(+) F4/80(+) Ly6C(+) macrophages are required for the maintenance of type 2 immunity within affected tissues but not secondary lymphoid organs. Direct depletion of macrophages during the maintenance or resolution phases of secondary Schistosoma mansoni egg-induced granuloma formation caused a profound decrease in inflammation, fibrosis, and type 2 gene expression. Additional studies with CD11c-DTR and CD11b/CD11c-DTR double-transgenic mice suggested that macrophages but not dendritic cells were critical. Mechanistically, macrophage depletion impaired effector CD4(+) T helper type 2 (Th2) cell homing and activation within the inflamed lung. Depletion of CD11b(+) F4/80(+) Ly6C(+) macrophages similarly reduced house dust mite-induced allergic lung inflammation and suppressed IL-13-dependent immunity to the nematode parasite Nippostrongylus brasiliensis. Consequently, therapeutic strategies targeting macrophages offer a novel approach to ameliorate established type 2 inflammatory diseases.

Figure 1. DTX treatment of primed and challenged CD11b-DTR mice causes direct, partial, and selective depletion of CD11b+ F4/80+ macrophages within the granulomatous lung

CD11b-DTR egg primed and challenged mice were treated with DTX (n=14) or left untreated (n=13) on D3 and harvested on D4. Lung leukocytes were analysed by flow cytometry to identify directly depleted cell types. (a) Representative flow cytometry plots of untreated D4 lung leukocytes (live CD45+) showing the gating strategy used to distinguish eosinophils (Siglec-F+ Ly6G-/low), neutrophils (Siglec-F− Ly6Ghigh), T cells (TCRβ+), macrophages (F4/80+ CD11b+) as well as CD11b− and CD11b+ DCs (CD11c+ MHCII+ F4/80−). DTX treatment (b) directly reduced the number of CD11b+ F4/80+ macrophages but not other leukocytes in the lung. Statistical significance was calculated using unpaired two-tailed Student’s t test. * p<0.05, ** p<0.01, *** p<0.001. Results represent three independent experiments.

Figure 2. Macrophages are critical promoters of both the local induction and maintenance of type 2-dependent lung fibrosis

(a) CD11b-DTR mice were primed by IP injection of 5,000 S. mansoni eggs on D-14 and then challenged by a single IV injection of 5,000 live eggs on D0. Mice were harvested at the initiation (D4), peak (D7) or resolution (D14) stages of lung granuloma formation. Half of the mice were treated with DTX (25ng/g) when indicated. (b) Representative images showing inflammation and collagen (blue) in Masson’s Trichrome stained lung tissue from primed and challenged (CD11b-DTR) (Primary lung granuloma D4 n=5; D7 n=15; D10 n=15; Secondary lung granuloma D4 n=8; D7 n=19; D10 n=19), and primed, challenged, and DTX treated (CD11b-DTR + DTX) (Primary lung granuloma D4 n=8; D7 n=18; D10 n=19; Secondary lung granuloma D4 n=7; D7 n=19; D10 n=15) CD11b-DTR mice. Volumes of (c) primary lung granulomas (intravenous challenge without priming) and (d) secondary lung granulomas (intraperitoneal primed, intravenous challenged) were scored by a pathologist blinded to groups and data presented as average granuloma volume/mouse (top panel) and individual granuloma volume (bottom panel). DTX treatment reduced both primary and secondary lung granuloma volumes at all-time points assessed. (e) Change in collagen deposition in egg primed and challenged CD11b-DTR mice with or without DTX treatment was compared by measuring hydroxyproline content of lung tissue. (f) Collagen was visualized by picrosirius red staining of lung tissue exposed to polarized and normal transmitted light. Dotted lines outline granulomas. DTX treatment reduced lung collagen at D7 and 14. Data are presented as median and statistical significance calculated using Mann-Whitney U test. * p<0.05, *** p<0.001. Results represent two independent experiments.

Figure 3. Type 2 immunity in the granulomatous lung is dependent on macrophages

Relative quantitative gene expression in lung tissue of naïve (n=13), egg primed and challenged (D4 n=8; D7 n=9, D10 n=10), and primed, challenged, and DTX treated (D4 n=8; D7 n=10, D10 n=9) CD11b-DTR mice. Results were normalized to RPLP2 and scaled to naïve mice. All Th2-induced genes were more weakly induced by egg challenge following DTX treatment. Data are presented as mean ± s.e.m. Statistical significance was calculated using unpaired two-tailed Student’s t test. * p<0.05, ** p<0.01, *** p<0.001. Results represent two independent experiments.

Figure 4. Direct depletion of macrophages in the granulomatous lung indirectly reduces inflammation and leads to their replacement by cells with a monocyte phenotype

CD11b-DTR egg primed and challenged mice were treated with DTX (n=15) or left untreated (n=13) on D3, 4, 5 and harvested on D7. Lung leukocytes were analysed by flow cytometry as in Figure 1 to examine the cumulative effects of depletion. The direct, partial, and selective depletion of macrophages within 18 hours (Fig. 1) led to an indirect decline in inflammatory leukocytes. (a) Net changes in lung leukocyte populations. DTX treatments reduced macrophage numbers, but also led to an indirect decline in total leukocytes, eosinophils, T cells, CD11b− DCs, and CD11c+ DCs. Neutrophils were not significantly changed. (b–c) DTX treatment shifts the predominant phenotype of macrophages from Ly6C− CD11c+ MHCII+ to Ly6C+ CD11c− MHCII−, resembling conventional monocytes. Statistical significance was calculated using unpaired two-tailed Student’s t test. * p<0.05, ** p<0.01, *** p<0.001. Results represent three independent experiments.

Figure 5. Reduced local but not systemic CD4+ Th2 cell responses underlie decreased lung inflammation and fibrosis

(a) Mediastinal lymph node and (b) lung leukocytes isolated from naïve (lymph node n=20; lung D7 n=6), egg primed and challenged (lymph node D4 n=8; D7 n=6; D10 n=6; lung D7 n=6), and primed, challenged, and DTX treated (lymph node D4 n=8; D7 n=4; D10 n=7; lung D7 n=6) CD11b-DTR mice were restimulated with PMA plus Ionomycin and stained to compare cytokine producing capabilities of CD4+ T lymphocytes. (c) Total numbers of cytokine-producing inflammatory CD4+ T lymphocytes in the lungs, and (d) magnitude of cytokine production per cell was measured by the mean fluorescence intensity (MFI) of cytokine staining. Little differences in effector CD4+ T lymphocytes were observed in lung-draining lymph nodes. In contrast DTX treatment reduced the frequency, number, and intensity of IL-13, IL-4, and IL-17A-producing CD4+ effector T lymphocytes in the lungs. Data are presented as medians. Statistical significance was calculated using Mann-Whitney U test. * p<0.05, ** p<0.01. Results represent two independent experiments.

Figure 6. CD11c-DTR-sensitive leukocytes are not required after antigen priming to maintain type 2-dependent granulomas and fibrosis in the lung

(a) Wild-type C57Bl/6 mice were irradiated to ablate hematopoiesis and reconstituted with bone marrow cells from wild-type C57Bl/6, CD11b-DTR, CD11c-DTR or mice expressing both CD11b-DTR and CD11c-DTR transgenes (CD11b/c-DTR). Chimeric mice were primed and challenged with S. mansoni eggs, and half were treated with DTX (25ng/g) when indicated. All mice were harvested at D7. (b) Representative images of granulomas and collagen (blue) in lungs stained with Masson’s Trichrome (wild-type control n=8; wild-type DTX n=7; Cd11c-DTR control n=14; Cd11c-DTR DTX n=18; Cd11b-DTR control n=18; Cd11b-DTR DTX n=14; Cd11b/c-DTR control n=19; Cd11c-DTR DTX n=18). (c) Lung granuloma volume was scored by a pathologist blinded to groups and data presented as average granuloma volume/mouse. (d) Lung collagen was measured by hydroxyproline content of all primed and egg challenged groups. DTX treatment of CD11b-DTR mice reduced both secondary lung granuloma volumes and collagen content of the lung. In contrast, DTX treatment of CD11c-DTR or CD11b/c-DTR mice did not change granuloma volumes or collagen content of the lung compared to DTX treated wild-type or CD11b-DTR mice, respectively. Data are presented as median and statistical significance calculated using Mann-Whitney U test. * p<0.05, ** p<0.01, *** p<0.001. Results represent two independent experiments.

Figure 7. Failure to maintain lung inflammation and fibrosis selectively correlates with the depletion of Ly6C+ lung macrophages in CD11b-DTR mice

CD11b-DTR and CD11c-DTR egg primed and challenged transgenic mice were treated with DTX or PBS on D3 and harvested on D4. Lung leukocytes were analysed by flow cytometry to identify directly depleted cell types (CD11b-DTR PBS n=8, CD11b-DTR DTX n=9, CD11c-DTR PBS n=10, CD11c-DTR DTX n=10). (a) Numbers of dendritic cells and total macrophages in the lung, based on gates shown in Fig. 1a. (b) Reciprocal depletion of the Ly6C+ macrophage subset in CD11b-DTR and Ly6C− CD11c+ subset in CD11c-DTR mice, and profiles of CD11c and MHCII co-expression by these subsets. Percentages of gated cells from individual mice are representative or group means. (c) DTX treatment decreased the number of Ly6C+ macrophages in CD11b-DTR but not CD11c-DTR mice, whereas Ly6C− macrophages decreased in CD11c-DTR but not CD11b-DTR mice. Data are presented in (a) and (c) as median. Statistical significance was calculated using Mann-Whitney U test. * p<0.05, ** p<0.01, *** p<0.001. Results represent two independent experiments.

Figure 8. Macrophages regulate chemokine production and recruit effector T cells to the lungs

(a) In vitro activated CFSE labelled OT-II transgenic CD4+ T lymphocytes (4×10⁶ cells) were injected IV into egg primed and challenged CD11b-DTR mice with or without prior DTX treatment on day 6. The homing of donor T lymphocytes was compared 24 hours after transfer by detecting CFSE+ CD4+ T cells in different tissues by flow cytometry. Fewer activated CD4+ T lymphocytes were recruited to and remained in the lung and mediastinal lymph nodes in DTX treated mice. In contrast, similar small numbers of donor cells were present in blood or inguinal lymph nodes. Relative quantitative gene (b) and protein expression of chemokines in lung tissue from naïve (n=13), egg primed and challenged (D4 n=8; D7 n=9, D10 n=10), and primed, challenged and DTX treated (D4 n=8; D7 n=10, D10 n=9) CD11b-DTR mice. Results were normalized to RPLP2 and scaled to naïve mice. Induction of CCL1 and CCL22 were blunted by DTX treatment while CCL2 was further increased at all time points and CCL11 remained mostly unaltered. The data in (a) are presented as median. The data in (b) and (c) are presented as mean ± s.e.m. Statistical significance was calculated using unpaired two-tailed Student’s t test or Mann-Whitney U test as appropriate. * p<0.05, ** p<0.01, *** p<0.001. Results represent two independent experiments.

Figure 9. CD11b-DTR-sensitive cells locally drive airway allergen-induced type 2 immunity

(a) CD11b-DTR mice were primed by IP injection of house dust mite (HDM, 200μg) on D0 and 7 prior to intratracheal challenge with HDM (50μg) on D14 and 16. Half of the mice were treated with DTX (25ng/g) at D13 and 15. Lungs and lung-draining mediastinal lymph nodes were harvested on D17. (b) Representative images of airway epithelial mucus by Alcian blue-periodic acid-Schiff (AB-PAS) staining of saline treated (naïve n=11), HDM primed and challenged (HDM n=12), and primed, challenged, and DTX treated (HDM DTX n=10) CD11b-DTR mice. (c) Relative quantitative gene expression in lung tissue was normalized to RPLP2 and scaled to naïve mice. DTX treatment weakened the HDM-stimulated increase in mucus and Th2-induced genes. (d) AB-PAS staining (scored 0–4 by a pathologist blinded to groups) was less intense and widespread in the airways of DTX treated mice. (e) Total number of leukocytes in perfused lung tissue and bronchoalveolar lavage (BAL). Total number of eosinophils in BAL was calculated from cytospin analysis. DTX treatment decreased the total number of leukocytes in lung tissue and BAL, as well as airway eosinophils. Data are presented as mean ± s.e.m. Statistical significance was calculated using unpaired two-tailed Student’s t test. * p<0.05, ** p<0.01, *** p<0.001. Results represent two independent experiments.

Figure 10. Macrophage depletion suppresses Th2 gene expression and impairs defense against Nippostrongylus brasiliensis hookworm infection

(a) CD11b-DTR mice were injected subcutaneously with N. brasiliensis (500 L3) at D0. Mice were harvested at D4 (lung), or 7 or 10 (gut). Half of the mice were treated with diphtheria toxin (DTX, 25ng/g) when indicated. (b) Intestinal worm burden was equal in control mice and DTX treated mice on D7 (n=13/group) but significantly higher in DTX mice at D10 (n=18/group). (c) DTX treatment increased fecundity of N. brasiliensis adults (eggs/worm) at both D7 and 10. Daily longitudinal fecal egg counts showed (d) a higher egg burden (eggs/mg feces) from D7 (n=8/group) to 10 (control n=10; DTX n=14) and (e) an increased percentage of infected mice above a threshold (dotted line in e) of >10 eggs/mg feces following DTX treatment (control n=22; DTX n=14). Relative quantitative gene expression in (f) D7 and 10 gut (grossly inflamed proximal duodenum) and (g) D4 lung tissue from untreated (naïve n=10), N. brasiliensis infected (D4 n=8; D7 n=13; D10 n=18), and infected DTX treated (DTX) (D4 n=9; D7 n=18; D10 n=28) CD11b-DTR mice. Results are normalized to RPLP2 and scaled to naïve mice. The majority of Th2-dependent genes exhibited weaker induction following DTX treatment in infected mice. The data in (b) and (c) are presented as median. The data in (d), (f) and (g) are presented as mean ± s.e.m. Statistical significance was calculated using unpaired two-tailed Student’s t test or Mann-Whitney U test as appropriate. * p<0.05, ** p<0.01, *** p<0.001. Results represent two independent experiments.