Fig 2: Treg cell ablation results in an immune microenvironment associated with tumor progression. (A) Multiplex analysis of shown cytokines. Values are expressed as mean ± SEM, n = 3 mice per group. *p = 0.0278 and **p = 0.0082 were calculated by unpaired t-test. (B) Percentage (left) and absolute number (right) of tumor associated macrophages (CD11b+ LY6C- LY6G- F4/80+). ****p < 0.0001 was calculated by Mann–Whitney test. (C) Representative flow cytometric plot (left) and quantification (right) of TAM (Ly6G- Ly6C- F4/80+) cell polarization as relative amounts of MHCII- CD206+ and MHCII+ CD206- cells. ***p < 0.0007 was calculated by two-way ANOVA, followed by Bonferroni's post-hoc test. (D) Mammary gland relative expression of iNOS and Arg-1 (M1 and M2 markers, respectively) quantified by qPCR. Values are expressed as mean ± SEM and were normalized by F4/80 and beta-actin levels. n = 5–6, *p = 0.0479 was calculated by unpaired t-test. Data were pooled from two independent experiments.

Fig 3: sDCs are inhibited during L. donovani infection. (A) Spleens were isolated from uninfected (Uninf) mice or mice infected (Inf) with L. donovani for 45 days. Splenocytes were then prepared and cultured in the presence (+) or absence (-) of LPS for 24 h. Expression of costimulatory molecules on sDCs (i.e., CD11c+ F4/80- gated cells; see Fig. S3A for gating strategy) was assessed via flow cytometry. Results are representative of three analyses. The relative mean fluorescence intensities of costimulatory molecule expression from three different analyses are presented in Fig. S3B. (B and C) Splenocytes of uninfected and 45-day-infected mice were cultured with LPS as in panel A. The frequency of sDCs producing IL-12 (B) and TNF-a (C) was determined via flow cytometry. Numbers in outlined areas represent the percentage of sDCs expressing IL-12 or TNF-a. Gating strategies for flow cytometry analysis are illustrated in Fig. S3C and E. Results are representative of three independent analyses. Compiled data for three separate experiments are presented in Fig. S3D and F.

Fig 4: An HSD activates peripheral nerve macrophages. a Representative flow cytometry dot plots showed an increase in the frequency of peripheral nerve macrophages (CD45+CD11b+F4/80+) at 1 and 3 months of HSD feeding. b Histograms depicted the absolute macrophage (CD45+CD11b+F4/80+) number, as well as those that were CD86+ macrophages. A quantitative analysis indicated a significant increase at 1 and 3 months of HSD feeding, and the number remained elevated after shifting HSD-fed mice to a ND, n = 6–9/group. c Histograms depicted the absolute macrophage (CD45+CD11b+F4/80+) number as well as those that were CD206+ macrophages. An HSD favored the differentiation of CD86+ macrophages in the nerve more than those of CD206+ macrophages, n = 6–9/group. d A representative flow cytometry histogram showed the percentage of Ki67+macrophages (CD45+CD11b+F4/80+) in the sciatic nerves of HSD and ND fed mice. A quantitative analysis showed that HSD did not trigger significant macrophage proliferation in nerves, n = 6–8/group. e Longitudinal sections of mouse sciatic nerves were stained with CD16/32 (Fc?II/III receptor), F4/80, and DAPI, showing an increased F4/80+ macrophage density in HSD nerves primarily colocalized with CD16/32. f Real-time quantitative PCR showed an elevated expression of IL-1ß and IL-6 in the peripheral nerves at three months of HSD feeding, n = 6–10/group. Data were combined with from male and female mice. All data was presented as mean ± SEM and analyzed with an unpaired t test, **p < 0.01, ***p < 0.001; red and green ## represents statistics for CD86+ or CD206+ macrophages, respectively

Fig 5: The effect of CCR2 and CX3CR1 deletion on macrophage and CD8+ T cell number, activation status and cytokine release in the nerve of L31 mice. (A) Quantitative analysis from flow cytometry showed a reduced number of macrophages (CD45+CD11b+F4/80+) in the nerve of L31/CX3CR1KO mice compared to L31 and L31/CCR2KO mice. Of note, two distinct subsets of macrophages were detected in L31 mice, which were distinguished by varying expression level of F4/80 and CD11b. Only one subset (F4/80highCD11blow) was seen in L31/CX3CR1KO and L31/CCR2KO mice, although the latter had significantly higher number. (B) The number of activated macrophages (CD86+) were significantly reduced in L31/CX3CR1KO mice. (C) Frequency and absolute number of infiltrating CD8+ T cells were significantly diminished in nerve of L31/CX3CR1KO mice. Real-time quantitative PCR showed a significantly reduced/undetectable expression of pro-inflammatory molecules (D) IFN-?, (E) TNF-a, (F) IL-6, and (G) IL-1ß, in sciatic nerve of L31/CX3CR1KO mice. (H) The mRNA level of the anti-inflammatory molecule, IL-10 was significantly enhanced in L31/CX3CR1KO mice. Disease was induced by PSNL and experiments done 30 days post PSNL. Quantification in a-c is shown as number of cells per a segment of 2 cm long sciatic nerve. n = 5-8/group; student’s t test; t*p < 0.05; **p < 0.01; ***p < 0.001.