The mannose receptor (CD206) identifies a population of colonic macrophages in health and inflammatory bowel disease

Abstract

To understand the contribution of mononuclear phagocytes (MNP), which include monocyte-derived intestinal macrophages, to the pathogenesis of inflammatory bowel disease (IBD), it is necessary to identify functionally-different MNP populations. We aimed to characterise intestinal macrophage populations in patients with IBD. We developed 12-parameter flow cytometry protocols to identify and human intestinal MNPs. We used these protocols to purify and characterize colonic macrophages from colonic tissue from patients with Crohn's disease (CD), ulcerative colitis (UC), or non-inflamed controls, in a cross-sectional study. We identify macrophage populations (CD45⁺CD64⁺ HLA-DR⁺) and describe two distinct subsets, differentiated by their expression of the mannose receptor, CD206. CD206⁺ macrophages expressed markers consistent with a mature phenotype: high levels of CD68 and CD163, higher transcription of IL-10 and lower expression of TREM1. CD206^- macrophages appear to be less mature, with features more similar to their monocytic precursors. We identified and purified macrophage populations from human colon. These appear to be derived from a monocytic precursor with high CCR2 and low CD206 expression. As these cells mature, they acquire expression of IL-10, CD206, CD63, and CD168. Targeting the newly recruited monocyte-derived cells may represent a fruitful avenue to ameliorate chronic inflammation in IBD.

Figure 1

Identification of colonic human macrophage populations from healthy colon resections. (A) Gating strategy to identify human colonic macrophage populations by flow cytometry. Cells belonging to the monocyte: macrophage lineage reside within the live (7AAD-) single CD45 + CD64 + HLA-DR + leukocyte population. (B) Differential CD206 expression within CD64 + HLA-DR + population. (C) CD14 expression on CD64 + CD206 + and CD64 + CD206⁻ myeloid populations as compared to isotype control (histogram counts normalised to mode). (D) Morphological assessment of FACS-purified CD64 + HLA-DR + CD206⁻ and CD64 + HLA-DR + CD206 + cells. Data are representative of results from 10 samples.

Figure 2

Phenotypic characterisation of CD64 + CD206⁻ and CD64 + CD206 + healthy colonic macrophages, and healthy blood monocytes (BM) by flow cytometry. (A) Expression of macrophage associated cell surface markers. (B) Protein expression of monocyte associated cell surface markers; n = 8, 10 and 10 for BM, CD206⁻ and CD206 + , respectively.

Figure 3

Expression of TNFα and IL-10 of FACS-purified healthy intestinal myeloid populations. (A) TNFα and (B) IL-10 expression were determined by qRT-PCR. CD64 + HLA-DR + CD206 + cells were subdivided into CD14LO and CD14HI populations. Expression of both cytokines was compared to blood monocytes (BM). Intestinal macrophages derived from normal colon resections; n = 6, 4, 8 and 10 for BM, CD206⁻, CD206 + CD14LO and CD206 + CD14HI, respectively. Error bars show + SD.

Figure 4

RNAseq analysis of FACS-purified CD206⁻ and CD206 + colonic human macrophage populations from healthy individuals. (A) Expression of differentially expressed genes between macrophage populations (CD206⁻ and CD206 +). Intestinal macrophages derived from normal colon resections; n = 8 and 7 for CD206⁻ and CD206 + , respectively.

Figure 5

Intestinal macrophages in health and disease. (A) Proportion of total macrophages (CD64 + HLA-DR +) inthe lamina propria of healthy colonic tissue, and tissue isolated from CD and UC patients. (B) Ratio of CD206⁻:CD206 + macrophages. UCr UC tissue in remission, UCa active (inflamed) UC tissue, CDn CD tissue in remission and CDd active (inflamed) CD tissue. Sample numbers are n = 18, 8, 8, 9 and 10 for Healthy, UCr, UCa, CDr and CDa, respectively.

Figure 6

Proliferative capacity of human colonic macrophages. (A) Identification of human colonic Ki67 + macrophages isolated from lamina propria tissue of healthy colon biopsies. (B) Quantification of Ki67 + CD206 + and Ki67 + CD206⁻ macrophage populations in healthy and CD tissue. Error bars show + SD. n = 9 for both CD206⁻ and CD206 + . (C) Immunohistochemical staining of representative healthy and CD colonic cells cells for CD68 (left column) and Ki67 (right column). (D) Proliferation comparison between healthy and CD tissue. Example of gating, and frequencies of CD206⁻ and CD206 + macrophages in G2 in healthy and CD tissue.