Human monocytes and macrophages regulate immune tolerance via integrin αvβ8-mediated TGFβ activation

Abstract

Monocytes are crucial immune cells involved in regulation of inflammation either directly or via differentiation into macrophages in tissues. However, many aspects of how their function is controlled in health and disease are not understood. Here we show that human blood monocytes activate high levels of the cytokine TGFβ, a pathway that is not evident in mouse monocytes. Human CD14⁺, but not CD16⁺, monocytes activate TGFβ via expression of the integrin αvβ8 and matrix metalloproteinase 14, which dampens their production of TNFα in response to LPS. Additionally, when monocytes differentiate into macrophages, integrin expression and TGFβ-activating ability are maintained in anti-inflammatory macrophages but down-regulated in pro-inflammatory macrophages. In the healthy human intestine, integrin αvβ8 is highly expressed on mature tissue macrophages, with these cells and their integrin expression being significantly reduced in active inflammatory bowel disease. Thus, our data suggest that integrin αvβ8-mediated TGFβ activation plays a key role in regulation of monocyte inflammatory responses and intestinal macrophage homeostasis.

Graphical abstract

Figure 1.

Human peripheral blood monocytes activate TGFβ in an integrin αvβ8–dependent manner. (A) Illustration of TGFβ activation assay whereby cells of interest are co-cultured with active TGFβ reporter cells (Abe et al., 1994) expressing a TGFβ-inducible promoter fused to luciferase. (B) Human CD14⁺ blood monocytes (n = 8) and murine splenic Ly6c⁺ monocytes (n = 3, each data point representing two pooled mice) were isolated and co-cultured with active TGFβ reporter cells overnight, before measuring luciferase activity (active TGFβ concentration calculated from standard curve). Reporter cells alone were used as a negative control (n = 8), and U251 cells (n = 8; Fenton et al., 2017) were used as a positive control. (C) Healthy human PBMCs were analyzed by flow cytometry. Following gating on single, live cells, integrin β8 expression was identified on lymphocyte populations (CD3 for T cells, CD19 for B cells, and CD56 for NK cells), DCs (lineage-negative [CD3, CD14, CD15, CD16, CD19, CD20, and CD56] HLA-DR⁺ CD11c⁺ with subsets identified by CD1c and CD141 staining), and monocytes (HLA-DR⁺, CD14⁺, and CD16⁺) following gating on forward and side scatter (n = 4–19). (D) RNA from murine spleen Ly6c^hi monocytes (lineage-negative [TCRβ, NK1.1, B220, CD19, Ter119, Ly6G, and Siglec F], CD11b⁺, Ly6c⁺) was analyzed for Itgb8 expression by real-time PCR, normalizing to the housekeeping gene Hprt (n = 6, each from two pooled mice). Bone marrow–derived DCs were used as a positive control. (E) TGFβ activation of human CD14⁺ monocytes (n = 6) was measured in the presence of an isotype control antibody or an integrin β8 blocking antibody. Bars show mean and SD. n.s., not significant; n.d., not detected; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; P values were determined by Kruskal–Wallis with Dunn’s multiple comparisons test (B and C) and Wilcoxon matched-pairs signed rank test (E).

Figure 2.

The CD14⁺ classical monocyte subset activates high levels of TGFβ via integrin αvβ8 and MMP14. (A and B) PBMCs were gated on single, live HLA-DR⁺ cells, then as CD14⁺CD16⁻ classical monocytes (green gate), CD14⁺CD16⁺ intermediate monocytes (red gate), and CD14⁻CD16⁺ nonclassical monocytes (blue gate). Levels of integrin β8 were measured on each subset (colored histogram, integrin β8 antibody; gray histogram, isotype control). Representative flow cytometry plots (A) and quantification of integrin β8 levels (B); n = 19 healthy donors. (C and D) Monocyte subsets were isolated by flow cytometry sorting and co-cultured overnight with a TGFβ reporter cell line (C; n = 10–13 donors) in the presence of an isotype or anti–integrin β8 antibody (D; n = 3–6 donors). Bars show mean and SD. (E and F) MMP14 expression was measured by real-time PCR, normalizing to the housekeeping gene B2M (E; n ≥ 5 for each subset) and flow cytometry (F; n = 8 for each subset) in monocyte subsets. Bars show mean and SD. (G) Active TGFβ production by CD14⁺ monocytes in the presence of an isotype control, integrin β8 blocking, or MMP14 blocking antibody (n = 6 donors). Bars show mean and SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant; P values were determined by Friedman test with Dunn’s multiple comparisons post-test (B, F, and G), Wilcoxon matched-pairs signed rank test (D), and Kruskal–Wallis with Dunn’s multiple comparisons post-test (C and E).

Figure 3.

Integrin αvβ8–mediated TGFβ activation reduces monocyte TNFα production in response to LPS and is down-regulated upon differentiation to pro-inflammatory macrophages. (A) TGFβ receptor chain II (TGFβRII, red histogram) levels were assessed by flow cytometry on CD14⁺ monocytes (isotype control, gray histogram; graph shows cumulative data of n = 7 donors). (B) Levels of the TGFβ signaling molecule pSmad2/3 were measured in CD14⁺ classical monocytes (n = 3) after treatment of PBMCs with 5 ng/ml TGFβ for 30 min (isotype control shown in gray). Representative flow cytometry plots are displayed. (C and D) TNFα production by CD14⁺ monocytes was measured by intracellular flow cytometry, following PBMC culture overnight in the presence of an isotype control, anti-TGFβ, or anti–integrin β8 blocking antibody followed by activation with 10 ng/ml LPS for 4 h in the presence of a protein transport inhibitor; C shows representative flow cytometry plots; D shows cumulative data (n = 18). (E) TNFα production by CD14⁺ monocytes as in C and D, with addition of 5 ng/ml active TGFβ in the presence of anti–integrin β8 antibody (n = 9). (F and G) CD14⁺ monocytes were cultured for 6 d with GM-CSF or M-CSF to differentiate to macrophages and integrin β8 expression measured by flow cytometry; F displays representative flow cytometry plots for macrophage expression of integrin β8; G shows pooled data for monocyte and macrophage expression of integrin β8 from 10–19 donors. (H) Monocytes and MDMs were co-cultured overnight with active TGFβ reporter cells (n = 6–8 donors). *, P < 0.05; **, P < 0.01; ****, P < 0.0001, n.s., not significant; P values were determined by Friedman test with Dunn’s multiple comparisons post-test (D and E) or Kruskal–Wallis with Dunn’s multiple comparisons post-test (G and H).

Figure 4.

Integrin αvβ8 is highly expressed on intestinal macrophages and is decreased in patients with IBD. (A) Integrin β8 levels were analyzed by flow cytometry on human colonic monocyte/macrophage populations, gated as single, live, CD45⁺, lineage-negative (CD3, CD15, CD19, CD20, and CD56), HLA-DR⁺CD14⁺CD64⁺ cells. Expression of integrin αvβ8 was compared with equivalent CD14⁻CD64⁻ cells (n = 9). (B) Integrin β8⁺ monocytes/macrophages were analyzed for expression of the tissue macrophage markers CD163 and CD206 by flow cytometry (representative image from n = 9 donors). (C and D) Human monocyte/macrophage populations, identified as described in A, were gated for expression of the monocyte marker CCR2 and expression of integrin β8 on monocytes (CD14⁺CCR2⁺) and macrophages (CD14⁺CCR2⁻) analyzed. Representative flow cytometry plots (C) and cumulative data (D) from 11 donors are depicted. SSC-H, side scatter height. (E) Monocyte and macrophage populations in the intestinal tissue of healthy versus inflamed IBD patients were analyzed by flow cytometry (gated as described in C and D) with histograms showing levels of HLA-DR expression on monocytes (purple) and macrophages (blue) in healthy tissue compared with IBD. Representative plots shown, with F showing cumulative data for CCR2⁺ monocyte and CCR2^– macrophage levels from 11–12 healthy or IBD donors. (G and H) Expression levels of integrin αvβ8 were measured on monocyte/macrophage populations from healthy patients and patients with IBD. Representative histograms (G) and cumulative data (H) from 11 healthy and IBD donors are depicted. Bars show mean and SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; P values were determined by Wilcoxon matched-pairs signed rank test (A and D) or Mann–Whitney (F and H).