In this study, we define transcriptional profiles of human CD16+ and CD16- monocytes (Mo) and provide new insights into their developmental relationship and biological functions. Despite remarkable transcriptional similarity (approximately 83%), a significant number of transcripts were differentially expressed (n = 2,759), with 228 and 250 >2-fold upregulated and downregulated, respectively, in CD16+ compared to CD16- Mo. Differentially expressed genes related to cell-to-cell adhesion and trafficking, immune responses and inflammation, metabolism and stress response, signaling and signal transduction, cell cycle, proliferation, and differentiation, cytoskeleton, and regulation of transcription. Gene set enrichment analysis (GSEA) demonstrated that CD16+ Mo are enriched in genes related to NK-mediated cytotoxicity, inositol phosphate metabolism, actin binding, and oxidative stress, while CD16- Mo are enriched in genes related to hematopoietic cell lineage, receptor-mediated endocytosis, arginine and proline metabolism, NTHi pathway, and lipid binding. The transcriptional profiles suggest that CD16+ and CD16- Mo subsets originate from a common myeloid precursor, with CD16+ Mo being at a more advanced stage of myeloid differentiation and having distinct biological functions in vivo.
Previous studies in mice provide evidence for a developmental relationship between Ly6ChighCCR2highGr1+CX3CR1low and Ly6ClowCCR2lowGr1-CX3CR1high Mo (homologs of human CD16- and CD16+ Mo, respectively), with Ly6ClowCCR2lowGr1-CX3CR1high Mo being more mature and derived from Ly6ChighCCR2highGr1+CX3CR1low Mo [5, 41, 85]. Likewise, studies on human Mo demonstrated the ability of CD16-CX3CR1low Mo to differentiate into CD16+CX3CR1high Mo upon stimulation with TGF-β, IL-10, M-CSF, or CCL2 [17, 43–45]. Our comparative transcriptome analysis provides further evidence for the idea that CD16- Mo originate from a common granulocyte-macrophage (GM) precursor and give rise to CD16+ Mo, which are more closely related to macrophages (MΦ) and dendritic cells (DC). CD16- Mo preferentially expressed granulocyte-associated transcripts (i.e., CSF3R, formyl peptide receptor 1 (FPR1), the calgranulins S100A8, S100A9, and S100A12), and myeloid markers (i.e., CD14, MNDA, TREM-1, CD1d, and C1qR1/CD93), together with transcripts suggesting an increased potential for receptor-mediated endocytosis via molecules such as CD14 and FCGR1A/CD64 [85, 86]. In contrast, CD16+ Mo preferentially expressed MΦ (i.e., CSF1R/CD115, MafB, EGF module-containing mucin-like hormone receptor (EMR)1-3, CD97, and C3aR)  and DC markers (i.e., SIGLEC10, CD43, CXCL16, and RARA) [56, 74, 87, 88]. CD16+ Mo expressed higher levels of transcripts encoding the cysteine protease cathepsin L (CTSL), which contributes to phagocytic-endocytic proteolysis in DC for subsequent antigen presentation . Upregulation of transcripts encoding dipeptidyl-peptidase I, CTSC  may further enhance antigen processing by CD16+ Mo or DC derived from these cells. Although some studies classified CD16+ Mo as DC based on their increased antigen presenting ability [14–16] and transcriptional profile similarities , a recent compendium analysis of transcriptional profiles demonstrated that CD16+HLA-DR+ cells are more closely linked to myeloid CD14+ cells than to DC subsets in peripheral blood . Our results demonstrate that CD16+ Mo share approximately 83% of their transcripts with CD16- Mo, supporting the idea that these two Mo subsets are developmentally related.
Recruitment of CD16+ and CD16- Mo into tissues is mediated via distinct molecular mechanisms [7, 8]. Our gene expression analysis confirms differential expression of adhesion molecules and chemokine receptors previously reported to be preferentially expressed on CD16+ Mo (i.e., LFA-1, PECAM/CD31, CX3CR1) and CD16- Mo (i.e., CCR1, CCR2, and L-selectin/CD62L) [18–20]. We also identified new cell surface markers and other molecules that are differentially expressed in these Mo subsets and may influence their trafficking and migration into tissues. The tetraspanins MS4A4A and MS4A7, adhesion molecules SIGLEC10 and ICAM-2, and membrane-bound chemokine CXCL16  were preferentially expressed by CD16+ Mo, whereas the tetraspanin MS4A6A, adhesion molecules CD99 and junctional adhesion molecule like (JAML or AMICA) , and chemokine receptor FPR1 were preferentially expressed by CD16- Mo. CD31 and CD99 are involved in distinct steps of Mo transendothelial migration [91, 92]. Expression of CXCL16, a chemokine expressed by DC , on the surface of CD16+ Mo may facilitate interaction with CXCR6+ cells (i.e., NKT and activated CD4+ and CD8+ T-cells ) and retention of CXCR6+ cells in tissues. Similar to mouse and rat CCR2lowCX3CR1high Mo [37, 41], CD16+ Mo expressed higher levels of SPN/CD43 (sialophorin, leukosialin, large sialoglycoprotein or gp115), a ligand for ICAM-1 [93, 94], and the macrophage adhesion receptor sialoadhesin (Siglec-1) . CD43 has both adhesive and anti-adhesive properties , mediates DC maturation , and contributes to regulation of immunological synapse formation . CD47, a receptor for thrombospondin-1 (TSP-1), is preferentially expressed by CD16+ Mo. CD47 ligation selectively inhibits the development of human naive T cells into Th1 effectors by decreasing IL-12 and TNF-α production by Mo-derived DC [98, 99]. Consistent with these findings, CD16- and CD16+ Mo may induce Th1 and Th2-like differentiation, respectively . However, CD16+ Mo express IL-12RB1, which favors Th1 polarization , whereas CD16- Mo express receptors for the Th2 cytokines IL-6 and IL-13  and the anti-Th1 cytokine, IL-27 [60, 61]. Accordingly, the influence of CD16+ and CD16- Mo on Th1 versus Th2 polarization of immune responses is likely to be highly dependent on the local microenvironment within tissues.
CD16+ Mo expressed high levels of transcripts for RARA, which controls transcription of genes involved in cell trafficking and mucosal homing. RA imprints lymphocytes with non-skin mucosal homing properties by decreasing cutaneous lymphocyte-associated antigen (CLA, an epitope on PSGL-1) expression  and increasing expression of CCR9 and integrin beta 7, two mucosal addressins . RA also controls reciprocal differentiation of Th17 and regulatory T cells , modulates myeloid gene expression and differentiation , and regulates survival and antigen presentation by DC . Consistent with our hypothesis that the RARA pathway is activated in CD16+ Mo, we demonstrated CLA downregulation on these cells, together with upregulation of two RA-induced targets: SLP-76  and CXCL16 . RA induces mucosal-type DC, which produce TGF-β and thereby imprints T-cells for gut homing by inducing CCR9 and integrin beta 7 . CD16+ Mo-derived MΦ and DC constitutively produce TGF-β [23, 100], but whether they also instruct T-cells for gut homing remains to be determined.
KLF2 mRNA is expressed at very high levels and significantly upregulated in CD16+ compared to CD16- Mo. KLF2 belongs to a family of zinc-finger transcription factors that is induced by PI3K signaling  and controls expression of several genes including those coding for CD62L, CCR7, integrin beta7, sphingosine-1-phosphate receptor (S1PR1) , and lymphotoxin beta . CCR7 and CD62L are essential for migration into lymph nodes, S1P1 regulates T-cell thymic egress and recirculation , and integrin beta 7 mediates cell recruitment into Peyer's patches and mesenteric lymph nodes . Together, these findings raise the possibility that preferential expression of KLF2 in CD16+ Mo may confer an increased potential for trafficking.
CD16+ compared to CD16- Mo express very high levels of transcripts for cyclin-dependent kinase inhibitor 1C (CDKN1C or p57/KIP2) (18.4-fold increase) and metastasis suppressor 1 MTSS1 (5.7-fold increase). CDKN1C is a potent inhibitor of several G1 cyclin-dependent kinase (cdk) complexes, and negative regulator of G1/S cell cycle transition and cell proliferation . CDKN1C  and MTSS1  are candidate tumor suppressor genes, and their high expression is consistent with the inability of Mo to proliferate . CDKN1C is induced by TGF-β , a cytokine known to induce CD16+ Mo differentiation [17, 43]. Thus, our results are consistent with a potential link between TGF-β pathway activation and CD16+ Mo differentiation in vivo.
Several transcripts related to cell activation were upregulated in CD16+ Mo including LTB, TNFRSF8, leukocyte specific transcript 1 (LST1), IFITM1-3, HMOX1, superoxide dismutase-1 (SOD-1), tryptophanyl tRNA synthetase (WARS), and monoglyceride lipase (MGLL), indicating increased activation of CD16+ compared with CD16- Mo. LST1 , HMOX1 , SOD-1, and WARS are induced by stimulation with lipopolysaccharide . The role of LST1 in immune regulation remains elusive. HMOX1 modulates Mo inflammatory responsiveness by decreasing LPS-induced TNF and IL-1β expression . WARS and indoleamine 2,3-dioxygenase (IDO) are responsible for tryptophan use in protein synthesis and degradation, respectively . WARS was identified as a molecular marker for Mo differentiation into MΦ  and DC . These results suggest increased activation of CD16+ compared to CD16- Mo in vivo.
The CD16+ Mo subset includes two subsets with distinct levels of CD14 expression: CD14highCD16+ and CD14lowCD16+ [11, 112]. CD14highCD16+ Mo exhibit a phenotype intermediate between that of CD14highCD16neg and CD14lowCD16+ Mo in terms of adhesion molecule (e.g., CL62L) and chemokine receptor expression (e.g., CCR2, CXCR2, and CX3CR1) . Both CD14highCD16+ and CD14lowCD16+ Mo contributed to the transcriptional profile of CD16+ Mo in this study. The expression of some genes we identified as markers for CD16+ Mo may be distinct on CD14highCD16+ and CD14lowCD16+ Mo. Consistent with this prediction, we demonstrated intermediate expression of CD115 and CD114 on CD14highCD16+ Mo compared to CD14highCD16neg and CD14lowCD16+ Mo, and high expression of CD93 and C3aR1, similar to that on CD14highCD16- and CD14lowCD16+ Mo, respectively (Additional file 3). These findings suggest a developmental relationship between these Mo subsets in which CD14highCD16neg Mo, CD14highCD16+ Mo, and CD14lowCD16+ Mo represent sequential stages of monocyte differentiation .