Fig 2: Pro-lymphatic differentiation coincides with IL-10 dependent shift to M2 phenotype.(A) Representative histograms demonstrating expression of M2-specific myeloid markers CD163, CD209, and PD-L1 in freshly-isolated BM cells (Ex-vivo, blue line) and differentiated cells (CSF-1 + LPS, red line). Numbers in black font indicate the percentage of cells expressing an M2-specific marker. (B) The mean percentage of positive cells and (C) MFI (x103) of M2 marker expression in ex-vivo and differentiated cells. (D) Histograms demonstrating the expression of CD204 in CSF-1/LPS differentiated cells derived from wild-type C57BL/6 mice in the presence of rat isotype-matched control (left panel) or anti-IL-10R antibody (center panel). The right panel shows CD204 expression in identically differentiated cells from IL-10R knockout (KO) mice. Secondary controls are shown by the grey area outlined by black line. Transcriptional analysis determined by qPCR on day 6 of differentiation of anti-IL-10R antibody-treated BM cells compared with rat control antibody, and BM cells from IL-10R-/- compared with those from WT mice. Analyzed markers included those for (E) M1 phenotype, (F) immunosuppressive regulators, (G) Th2 regulators, and (H) M2 phenotype. Expression in WT cells in the absence or presence of control rat IgG was taken as 1. Stars represent statistical significance determined by Student’s t-test with P-values indicated by *<0.05, **<0.01, and ***<0.001. All experiments were performed in duplicate and reproduced at least twice.

Fig 3: ACh reduces cytokine release from cultured resident macrophages. (A) GFP-expressing macrophages were isolated by flow cytometry from the colonic muscularis propria of Cx3cr1GFP;CCR2RFP mice. (B) Cx3cr1GFP macrophages were cultured in vitro and formed colonies in the presence of CSF-1 (dashed box enlarged in inset). (C) Transcript levels of Tnf in the presence of LPS (100 ng/mL) were reduced significantly by the addition of ACh in a dose-dependent manner (n = 3 per group). Channelrhodopsin-expressing cholinergic neurons (ChATChR2-tdT) and muscularis macrophages (Cx3cr1tdT) were co-cultured in vitro. (D) After addition of LPS, IL1β can be seen expressed by tdT+/CD11b+ macrophages (white arrows), and not by tdT+/CD11b-negative cholinergic neurons (yellow arrows). (E) Quantification of IL1β immunofluorescence (MFI, mean fluorescence intensity) is shown in control conditions, in the presence of LPS, and with LPS after BLS to activate ChAT-expressing neurons (n = 8 per group). Scale bars: 200 μm (B), 50 μm (D). Data are shown as means ± SEM. ∗∗P < .01, ∗P < .05.

Fig 4: MSU-42011 polarizes bone marrow-derived macrophages (BMDMs) towards an anti-tumor phenotype. BMDMs were isolated from C57BL/6 mice and differentiated with MCSF. On day 5, BMDMs were treated with conditioned media (CM) from E18-14C-27 cells, either alone or with 300 nM RXR agonist. (A) BMDMs were harvested for flow cytometry to detect percentage of live single cells that were F4/80 + CD206+. All data normalized to conditioned media-treated cells and presented as mean of three experimental runs. Error bars represent standard error. (B–E) RNA was extracted, and gene expression was detected by qPCR. Error bars represent standard error of three biological replicates. * p < 0.05.

Fig 5: IL-10 pathway regulates transcription of specific markers of multiple lineages in BM myeloid precursors.Marker expression levels in CSF-1/LPS differentiated BM cells from WT mice in the presence of anti-IL-10R antibodies and from IL-10R KO mice were compared with the levels in WT cells treated with a control antibody. Cells harvested on the sixth day of differentiation were analyzed by RT-qPCR for mRNA expression of markers of (A) lymphatic-specific, (B) endothelial, (C) myeloid, (D) erythroid, (E) T-cell, and (F) B-cell lineages. All analyses were performed in triplicate for each target, normalized to β-actin, and independently reproduced twice. Data for each target are reported as the mean fold-change in treated cells compared with the WT cells treated with control rat IgG ± S.D. Statistical significance of expression changes compared with control group was determined by Student’s t-test with P-values indicated by *<0.05, **<0.01, and ***<0.001.