Fig 1: rTMS treatment improved motor functions and increased the proportion of peripheral Tregs in PD patients. A Study flow chart. Sixty patients with PD were screened for eligibility in this clinical study between Sep 2021 and Jan 2022. A total of 6 PD patients were ultimately excluded. Finally, the 54 PD patients were randomized (1: 1) to divided into sham and rTMS group. Demographic data, basic examinations including electroencephalography, medication dosage before and after rTMS or sham treatment were carried out in both groups of patients. Tregs, aTreg, nTreg, proportion and the levels of IFN-γ, TNF-α, IL-17α, IL4, IL10 and TGF-β1 in the peripheral blood were measured, H&Y and MDS-UPDRS III scores were performed at the same time point before and after rTMS or sham treatment. B Tregs, and aTreg/nTreg proportion in the peripheral blood were assessed by flow cytometry prior to treatment and 1 day after the treatment. Hoehn and Yahr and MDS-UPDRS III were performed prior to treatment and on Days 13, 19, and 40 after the treatment. C No significant difference between the Mean H & Y ± SD on Days -1, 13, 19 and 40 in the sham and rTMS groups. (sham vs. rTMS groups on days -1: n = 27, P = 0.761, t = -0.306, d. f. = 52; sham vs. rTMS groups on days 13: n = 27, P = 0.761, t = -0.306, d. f. = 52; sham vs. rTMS groups on days 19: n = 27, P = 0.761, t = -0.306, d. f. = 52; sham vs. rTMS groups on days 40: n = 27, P = 0.761, t = -0.306, d. f. = 52). D There was a significant difference in the difference between the MDS-UPDRS III scores of the rTMS group and the sham group on day 13, 19, and 40 and their respective MDS-UPDRSIII scores on day -1.(rTMS vs. sham groups between days 13 and -1: n = 27, P < 0.0001, t = 15.64, d. f. = 208; rTMS vs. sham groups on 19 and -1: n = 27, P < 0.0001, t = 15.38, d. f. = 208; rTMS vs. sham groups on on 40 and -1: n = 27, P < 0.0001, t = 15.77, d. f. = 208). E The baseline of the proportion of CD4 + CD25 + CD127- Treg cells in the peripheral blood displays no significant difference between sham and rTMS group. (sham vs. rTMS groups: n = 27, P = 0.796, t = 0.26, d. f. = 52). F The peripheral blood CD4 + CD25 + CD127- Treg cells before and after sham rTMS treatment in the sham treatment group shows no significant difference. (before vs. after: n = 27, P = 0.185, t = 1.363, d. f. = 26). G The peripheral blood CD4 + CD25 + CD127-Treg cells before and after rTMS treatment in the rTMS treatment group shows significant difference. (before vs. after: n = 27, P < 0.000, t = -14.88, d. f. = 26). H Mean change in the proportion of Tregs/CD4 + T cells in the peripheral blood ± SD between days -1 and 13 in the sham and rTMS groups. (n = 27, P < 0.0001, t = 14.98, d. f. = 52). I No correlation between the change in the proportion of Tregs/CD4 + T cells and the change in MDS-UPDRSIII scores (between days -1 and 13) in sham group (n = 27, r2 = 0. 0007, p = 0.895). J Negative correlation between the change in the proportion of Tregs/CD4 + T cells. and the change in MDS-UPDRSIII scores (between days -1 and 13) in rTMS group. (n = 27, r2 = 0.8261, p < 0.0001). rTMS = repetitive transcranial magnetic stimulation; H & Y = Hoehn-Yahr; MDS UPDRS III = Movement Disorder Society Unified Parkinson's Disease Rating Scale part III; SD = Standard Deviation; Data are presented as mean ± SD; two-tailed unpaired t test (C, D, E, H); paired sample t test (F, G); linear regression analysis (I, J). ****P < 0.0001. Each data point represents an individual subject. Comparisons with no asterisk had a P > 0.05 and were considered not significant
Fig 2: Schematic illustration of the mechanism by which MIIP haploinsufficiency promotes CRC progression through the modulation of CIN and M2 macrophage infiltration in the CRC microenvironment. In our previous study, MIIP haploinsufficiency was shown to induce excess APC/CCdc20 ubiquitin ligase activity and the deregulation of Topo II activity, leading to CIN in CRC cells (in the red dotted line). The results of the current study further revealed that MIIP downregulation increases IL10 secretion via the dsDNA–STING–NFκB2–IL10 signaling axis in CRC cells and drives M2 macrophage polarization in the CRC tumor microenvironment. M2 macrophages promote CRC cell migration and invasion in turn in an IL-10-dependent manner (created with Figdraw.com). APC/C, anaphase-promoting complex/cyclosome; MIIP, migration and invasion inhibitory protein; CIN, chromosomal instability; STING, stimulator of interferon genes; CRC, colorectal cancer.
Fig 3: MIIP inhibits tumor growth and metastasis and M2 macrophage infiltration in a syngeneic CRC mouse model through the STING–NFκB2–IL10 axis. (A) Tumors in situ (indicated by green arrows) and liver metastatic nodules (indicated by black arrows) were observed in a syngeneic CRC mouse model. Statistics for the size of tumors in situ (B) and the number of metastatic foci (C). (D) Statistical analysis of IL10 levels in mouse blood. (E) Representative images of HE and IHC staining for SATB2, MIIP, STING, IL10, p52, and CD163 (green arrows) in tumor tissues from the different groups. The bar graphs show the percentages of high/low STING (F), IL-10 (G), and p52 (H) expression in the different groups. (I) Comparison of the number of CD163-positive cells among the different groups. All data are presented as the means ± SDs (n = 3). ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; scale bar, 50 μm.
Fig 4: MIIP suppresses M2 macrophage polarization and decreases macrophage-derived IL10 levels in a co-culture system. MIIP knockdown in SW480 cells increased CD163 expression (A) and CD206 levels (B) in PMA-induced THP-1 cells, the CD163 level in the co-culture medium (C), the mRNA expression of IL-10 (D), and the IL10 levels (E) in PMA-induced THP-1 cells. MIIP overexpression in CT26 cells decreased the CD163 level in the co-culture medium of the co-cultured system (F) and macrophage-derived IL10 expression (G) and IL-10 levels in RAW264.7 cells (H). Effects of MIIP knockdown, STING knockdown, and dual MIIP and STING knockdown in CRC cells on CD163 expression in co-cultured PMA-treated THP-1 cells (I), CD163 levels in the co-culture medium (J), CD206 levels in PMA-treated THP-1 cells (K), and macrophage-derived IL10 expression (L) and IL-10 levels (M). All data are presented as the means ± SDs (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; scale bar, 10 μm.
Fig 5: MIIP modulates the STING–NFκB2–IL10 signaling pathway in CRC cells. Immunofluorescence staining showing the effects of MIIP knockdown and MIIP overexpression on dsDNA levels (A) and the distribution and expression of STING (B) in SW480 and SW620 cells. (C) Western blots showing the effects of MIIP knockdown and overexpression on STING, TRAF3, p-p100, and p52 levels. (D) ELISA results showing the effects of MIIP knockdown and overexpression on IL10 levels in the cell culture medium. (E and F) Effects of MIIP knockdown and/or STING knockdown on p52 expression (E) and IL10 levels in the cell culture medium (F). All data are presented as the means ± SDs (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001; scale bar, 50 μm.
Supplier Page from Abcam for Human IL-10 ELISA Kit