DHRS9 Is a Stable Marker of Human Regulatory Macrophages

Abstract

Background: The human regulatory macrophage (Mreg) has emerged as a promising cell type for use as a cell-based adjunct immunosuppressive therapy in solid organ transplant recipients. In this brief report, dehydrogenase/reductase 9 (DHRS9) is identified as a robust marker of human Mregs.

Methods: The cognate antigen of a mouse monoclonal antibody raised against human Mregs was identified as DHRS9 by immunoprecipitation and MALDI-MS sequencing. Expression of DHRS9 within a panel of monocyte-derived macrophages was investigated by quantitative PCR, immunoblotting and flow cytometry.

Results: DHRS9 expression discriminated human Mregs from a panel of in vitro derived macrophages in other polarisation states. Likewise, DHRS9 expression distinguished Mregs from a variety of human monocyte-derived tolerogenic antigen-presenting cells in current development as cell-based immunotherapies, including Tol-DC, Rapa-DC, DC-10, and PGE2-induced myeloid-derived suppressor cells. A subpopulation of DHRS9-expressing human splenic macrophages was identified by immunohistochemistry. Expression of DHRS9 was acquired gradually during in vitro development of human Mregs from CD14 monocytes and was further enhanced by IFN-γ treatment on day 6 of culture. Stimulating Mregs with 100 ng/mL lipopolysaccharide for 24 hours did not extinguish DHRS9 expression. Dhrs9 was not an informative marker of mouse Mregs.

Conclusion: DHRS9 is a specific and stable marker of human Mregs.

FIGURE 2

DHRS9 expression distinguishes human Mregs from monocyte-derived macrophages and DCs. A, In immunocytochemistry, the ASOT1 mAb recognized an antigen expressed by Mregs, but not comparator macrophages. B, An antigen of ~35 kDa was specifically immunoprecipitated by ASOT1 and was subsequently identified by MALDI-MS as DHRS9. C, Strong DHRS9 mRNA expression was detected in Mregs, but not comparator macrophage types (n = 6; mean ± SD). D, ASOT1 precipitated an antigen which was also recognized by an anti-DHRS9 rabbit pAb (generated in-house) and a mouse mAb (clone 3C6, Abnova), confirming that ASOT1 recognizes DHRS9. E, Immunoblotting with our custom-made rabbit anti-DHRS9 pAb demonstrated that DHRS9 protein expression distinguishes Mregs from comparator macrophages. F, DHRS9 mRNA expression distinguished human Mregs from a panel of tolerogenic monocyte-derived therapeutic cell products. G, DHRS9 protein was detected in human Mregs but not other tolerogenic monocyte-derived therapeutic cell products using a commercial rabbit anti-DHRS9 pAb (ab98155, Abcam).

FIGURE 1

Comparative phenotyping of Mregs and other human macrophages. Low or absent expression of CD14, CD16, TLR2 and CD163 discriminated human Mregs from a panel of differently stimulated human monocyte-derived macrophages, including resting Mφ, LPS + IFNγ-stimulated Mφ, IL-4-stimulated Mφ, immunoglobulin (Ig)-stimulated Mφ and GC-stimulated Mφ. Values represent mean ± SD of n = 6 donors.

FIGURE 3

Factors affecting DHRS9 expression in human macrophages. A, DHRS9 mRNA expression increased steadily over 7 days of culture as human monocytes transitioned to Mregs and was further induced by IFN-γ stimulation on day 6 (n = 6; mean ± SD). B, Pattern of DHRS9 expression in a publicly available microarray data set encompassing 29 differently stimulated human monocyte-derived macrophages (Xue, J. et al, Immunity; GEO accession: GSE47189). More than twofold upregulation of DHRS9 was elicited by treatment with either 1 μM dexamethasone or 200 IU/mL IFN-γ. HDL, high-density lipoprotein; IC, immune complex; P3C, Pam3CSK4; PGE2, Prostaglandin E2; LA, lauric acid; OA, oleic acid; LiA, linoleic acid; SA, steric acid; PA, palmitic acid; TPP, TNF + PGE2 + P3C. C, Optimization of an intracellular staining method for detection of DHRS9 expression in human macrophages using PE-conjugated anti-DHRS9 mAb clone 3C6. D, Quantification of DHRS9 expression in human Mregs, IFN-γ Mφ and resting Mφ by flow cytometry.

FIGURE 4

Human Mregs express enzymes involved in retinoid metabolism. A, A schematic overview of retinoid metabolism. DHRS9 is a member of the SDR family of NAD(P)(H)-dependent oxidoreductases that catalyse interconversion of retinol and retinal. B, Mregs expressed ALDH1A1, ALDH1A2 and BCO2 mRNA, as well as the RA-responsive gene, CD1D (n = 3).

FIGURE 5

Identification of DHRS9⁺ Mφ in the subcapsular red pulp of human spleen. Immunohistochemical staining of 3 μm, H&E-counterstained sections of fixed, paraffin-embedded human spleen revealed a population of DHRS9⁺ macrophages. A, DHRS9⁺ Mφ detected with mAb 3C6 appeared to be most prevalent in the subcapsular red pulp. Original magnification, 20×. B, Negative control. Original magnification, 20×. C, A high density of DHRS9⁺ macrophages were detected in the subcapsular splenic red pulp using mAb 3C6. Original magnification, 40×. D, DHRS9 staining with mAb 3C6 was restricted to cytoplasm of cells with typical macrophage morphology. Original magnification, 40×.

FIGURE 6

DHRS9 is not a marker of mouse Mregs. Microarray analysis of SDR-family gene expression in monocyte-derived macrophages and DCs from C57BL/6 mice (Riquelme, P. et al, Molecular Therapy; GEO accession: GSE32690). No differential expression of DHRS9 or other SDR family members was observed between mouse Mregs and comparator cell types.