Fig 2: Effect of in vivo CD200R1 and miR-129-5p inhibition on the level of microglial TNF-a and IL-6. Animals were exposed to arsenic (0.38 mg/kg bd. wt.) for 2 months, and in the last week of exposure, animals were treated with CD200R1 siRNA and anti-miR-129 intracerebrally by stereotaxic method and sacrificed on 60th day. A, experimental scheme, (B) exposure to arsenic increased the level of miR-129-5p, which was brought down by the treatment of anti-miR-129 (n = 3 mice/group). CD200R1 siRNA did not show any significant effect on the level of miR-129-5p. C, arsenic exposure did not alter the mRNA level of CD200R1 like in vitro compared with control, whereas siRNA significantly inhibited it. D, Western blot analysis showed that arsenic exposure inhibited CD200R1 protein, which was rescued by anti-miR (n = 5 mice/group). E, immunofluorescence staining of CD200R1 in brain sections (n = 3 mice/group). Scale bar: 50 µm for uncropped images and 17 µm for the cropped images. The effects of CD200R1 siRNA and anti-miR-129 on the level of (F) TNF-a (n = 3–4 mice/group) and (G) IL-6 (n = 5 mice/group) were measured in the ex vivo microglial culture supernatant. Bar graphs represent mean ± SEM. “p” denotes the level of significance in comparison to control; *p < 0.05, **p < 0.01, ***p < 0.001; ns, nonsignificant.

Fig 3: Effect of arsenic stress on the expression of microglial CD200R1. BALB/c mice were exposed to sodium arsenite (arsenic) for 2 months and checked the expression of CD200R1 in the whole brain (n = 4 mice/group). A, Western blots, (B) immunostaining of CD200R1 in brain section. Scale bar: 50 µm for uncropped images and 11 µm for the cropped images. Mouse primary neonatal microglia were treated with arsenic as well as LPS in vitro for 72 h, and CD200R1 expression was checked (n = 4). C, Western blot, (D) immunostaining of CD200R1 in mouse primary neonatal microglia. Scale bar: 10 µm. The levels of mRNA of (E) CD200R1 were measured 72 h following treatment with arsenic and LPS in primary microglia and, (F) CEBPß was measured (n = 3) in primary microglia 2 h following treatment with arsenic and LPS. Another set of cells was treated in vitro with arsenic, and Taqman Low-Density Array (TLDA) was run to detect the changes in the global miRNA profile (n = 2). G, fold change in the miRNA level was presented as a heatmap, (H) miRNA upregulated more than 1.5-fold and downregulated more than 0.8-fold was presented in the table; (I) Network generated by IPA with the up- and downregulated genes and miRNA in arsenic-treated microglia indicated a possible relationship between CD200R1 and miR-129-5p. The symbols used in the network map are shown in the legend. “n” denotes the number of independent study for in vitro experiments. Bar graphs represent mean ± SEM. “p” denotes the level of significance in comparison to control; *p < 0.05, **p < 0.01, ***p < 0.001; ns, nonsignificant.

Fig 4: Proposed model for Foxp3 modulation of microglial polarization.Under normal physiological conditions, microglia exist in resting state, expressing CD200R, while nearby neurons express CD200. In the absence of neuronal expression of CD200, or when CD200R or Foxp3 expression in microglia is low, microglia undergo pro-inflammatory polarization, promoting neuroinflammation. When expression of CD200R or Foxp3 is elevated, anti-inflammatory polarization is enhanced, contributing to tissue repair and resolution of neuroinflammation.

Fig 5: Foxp3 expression is dependent upon CD200-CD200R-mediated signalling in microglia.(A) One day after KA treatment, mice were sacrificed and the hippocampus was double-labelled for Foxp3 and CD200R. Foxp3 IR (red, a) co-localized with that of CD200R (green, b; yellow, merge, c). Scale bar, 20 µm. (B) Primary microglia were collected from neonatal rats, plated, and treated with recombinant CD200 protein for the indicated length of time (left). Foxp3 expression (middle), but not CD200R (right), increased 3 h after CD200 treatment. (C) Treatment of rat microglia with CD200 up-regulated expression of the beneficial microglia activation marker, Arg1. (D) BV2 cells were transfected with control (scr) siRNA or siRNA targeting CD200R (si_CD200R) for 24 h, followed by treatment with CD200 for 3 h. Resultant immunoblots (left) and corresponding quantification (middle, CD200R; right, Foxp3) are shown. CD200R siRNA treatment reduced Foxp3 expression, which recovered slightly with CD200 treatment. ***P < 0.001 vs. control.