Inflammation Response Cytokines IFN-γ and IL-10 Regulate Monocyte Subset Differentiation

Circulating monocytes comprise of a heterogeneous and functionally-diverse cell population which based on surface markers can be divided into three subsets: classical (CMo), intermediate (IMo), and non-classical monocytes/patrolling monocytes (PMo). The frequency/number, gene expression profile and activity of IMo and PMo significantly change in a variety of inflammatory diseases with the changes associated with disease risk and severity as well as response to treatment. While it is believed that CMo differentiate into IMo and that IMo further differentiate into PMo, there is paucity of data on the mechanisms that alter CMo to IMo/PMo differentiation profiles in these conditions. In addition, factors which induce human and mouse IMo and PMo differentiation have yet to be identified. To screen cytokine/chemokine candidates affecting IMo/PMo differentiation, human monocytes were isolated from healthy donors (HD) and cultured with candidates (22 cytokines/chemokines) for 3 days. On day 3, IMo/PMo marker expression was examined by flow cytometry focusing on candidate molecules that led to increased expression of markers (CD16, CX3CR1, CD11c, HO-1, HLA-DR) whose levels are normally found to be higher in IMo/PMo and to decrease in expression of markers (CD14, CD36, CCR2) which are expressed at higher level in CMo as measured by mean fluorescence intension (MFI). We found that of all molecules tested, only two , IFN-γ and IL-10, had significant effects: IFN-γ (10ng/ml) increased expression of CX3CR1 (20 fold), CD16 (60%), HLADR (15%) and inhibited CD36 (42% inhibition) and CD14 expression (45% inhibition) while IL-10 (10ng/ml) increased CD16 (3.3 fold), CD11c (45%), HO-1 (59%) and CX3CR1 (6 fold) expression and inhibited CCR2 (60% inhibition) expression. These data suggest that IFN-γ and IL-10, two key cytokines involved in sterile and infectious inflammation, induce IMo and PMo differentiation. To test whether the effect of IFN-γ and IL-10 in human in vitro cultures can be replicated in vivo, wildtype B6 mice were I.V. injected with IFN-γ or IL-10 for 3 days: IFN-γ, IL-10, or the same volume of PBS. The frequencies of monocyte subsets in blood (gated on CD45+Ly6G-CD11b^highCD115+ for total monocyte population and CMo/IMo/PMo based on Ly6C expression level) on day 4 were analyzed by flow cytometry. We found that IFN-γ (2.5μg/injection/mice twice/day) significantly increased IMo frequencies (from 12% to 35%) but decreased PMo frequencies (from 38% to 26%) while IL-10 (0.25μg/injection/mice twice/day) significantly induced PMo differentiation (from 38% to 63%) without effect on IMo frequencies. The data suggest that IFN-γ increases IMo frequency by simultaneously inducing CMo differentiation into IMo and inhibiting IMo differentiation into PMo. We have previously reported lower PMo frequency in patients with sickle cell disease (SCD), considered an inflammatory disease with altered immune profiles. To test whether altered differentiation programming of IMo/PMo may contribute to reduced PMo frequency in SCD, we analyzed the frequency of CMo/IMo/PMo at baseline and after IFN-γ or IL-10 injection to mimic an inflammatory response in AA mice (expressing normal human hemoglobin) and SS mice (expressing human SCD hemoglobin). We found significantly lower IMo frequency before treatment (AA vs SS:15.0% vs 9.2%) but also lower induction of IMo following IFN-γ treatment in SS mice (18%) relative to AA mice (35%), suggesting that IFN-γ inhibition of IMo differentiation into PMo in SCD is impaired. Furthermore, IL-10 was less effective in inducing PMo in SS as compared to AA mice (SS vs AA: 40% vs 60%). These data suggest that IFN-γ or IL-10-mediated monocyte differentiation in SCD is altered. Altogether, these data have unraveled a novel role for IFN-γ or IL-10, two key cytokines known to be induced during an inflammatory response, in monocyte differentiation, and suggest that IMo/PMo differentiation in a chronic inflammatory disease such as SCD may be defective due to altered response to IFN-γ and IL-10, opening up the potential for identification of novel therapeutic targets for IMo/PMo associated diseases including SCD.