Additional file 2: Figure S1. of Increased serum concentrations of IL-1 beta, IL-21 and Th17 cells in overweight patients with rheumatoid arthritis

Frequencies of peripheral immune cells (Th17 cells and Plasmablast (PB)) and BMI. A comparison of the frequencies of Th17 cells and PB between the three BMI groups among healthy donors. A p value <0.05 was defined as a significant difference. ns not significant (TIF 569 kb

Additional file 3: of Decreased severity of experimental autoimmune arthritis in peptidylarginine deiminase type 4 knockout mice

Padi2 and Padi4 are expressed in the spleens of wild-type mice. (a–e) Immunofluorescence staining (×40) shows that Padi2 is expressed ubiquitously in the spleen. Spleens were probed with anti-Padi2 (green fluorescent signal) and cell surface markers (red fluorescence signal; a, CD3; b, B220; c, Gr-1; d, CD56; e, F4/80). The nuclei were stained with DAPI (blue fluorescence signal). The arrows indicate the colocalization of Padi2 and each cell surface marker. (f–k) Immunofluorescence staining (×40) shows that Padi4 was expressed in splenocytes. The spleens were probed with anti-Padi4 (green fluorescence signal), and cell surface markers (red fluorescence signal; f, CD3; g, B220; h, Gr-1; i, CD56; j, F4/80). Nuclei were stained with DAPI (blue fluorescence signal). (K) Immunofluorescence staining (left, ×40; right, ×280) shows that Padi4 was localized in both the nuclei and cytoplasm of macrophages, which expressed F4/80. (PDF 286 KB

Gene expression of Prox1 and FOXC2 by qRT-PCR.

<p>The mRNA expression levels of Prox1 (P<0.01) and FOXC2 (P<0.01) in OSCCs were higher than normal oral mucosa. Prox1 expression was upregulated in nodal metastasis positive OSCCs than in those with negative OSCCs (P<0.01). GAPDH was used for internal control. Error bar, standard deviation (S.D.).</p

bmjopen-2017-015952R2data

bmjopen-2017-015952R2dat

Univariate and multivariste analysis of disease free survival.

<p>Univariate analysis was performed by log lank test. Multivariate analysis was calculated by means of Cox proportional hazard model. HR and 95% CI mean hazard ratio and 95% confidence intervals, respectively.</p

Cell growth and migration of endothelial cells affected by OSCC cells.

<p><b>a</b>, Cell growth of endothelial cells treated with conditioned medium from OSCC cells. The growth of HUVECs and HDLMVECs were significantly enhanced by the addition of negative siRNA treated KON cell culture supernatant. Further, HUVECs or HDLMVECs proliferation was inhibited when added to FOXC2 or Prox1 siRNA treated KON cell culture supernatant, respectively. <b>b</b>, Migration of endothelial cells co-cultured with OSCC cells. The migration of HUVECs and HDLMVECs were enhanced by co-culture with negative siRNA treated KON cells. Moreover, co-cultivation with FOXC2 or Prox1 siRNA treated KON cells suppressed HUVECs or HDLMVECs migration, respectively.</p

<i>In vitro</i> analysis of Prox1 and FOXC2 using human OSCC cells.

<p><b>a</b>, Expression of Prox1 and FOXC2 in 6 OSCC cells measured using realtime RT-PCR. Highly metastatic KON cells showed higher expression of both genes. Expression levels of GAPDH was used for internal control. <b>b</b>–<b>d</b>, Effects of Prox1 or FOXC2 siRNA treatment in KON cells on cell growth (b), invasive ability (c),and mRNA expression levels of Prox1, FOXC2, VEGF-A, VEGF-C, VEGF-D (d). Growth and invasive ability were examined by the MTT assay and an in vitro invasion assay. Expression levels of Prox1 and VEGF-C were decreased by Prox1 siRNA treatment and decreases in FOXC2, Prox1, and VEGF-A expression were observed in FOXC2 siRNA treated KON cells (d).</p

Immunohistochemical analysis of Prox1 and FOXC2 in human OSCC cases.

<p>Prox1 (a) and FOXC2 (b) expression were not observed in non-tumor oral mucosa. The lymphoendothelial cells (arrow) showed immunoreactivity to Prox1 (c) and expression of FOXC2 was found in lymphoendothelial cells (arrow) and vascular endothelial cells (arrow head) (d). Expression of Prox1 (e) and FOXC2 (f) were observed in cytoplasm of the cancer cells. LYVE1 positive lymphovessels (g) and CD34 positive blood vessels were counted for MVD and LVD, respectively. Original magnification was 200-fold. Bar, 100 μm. LEC; lymphoendothelial cells, VEC; vascular endothelial cells.</p

DataSheet_1_Upregulated Fcrl5 disrupts B cell anergy causes autoimmune disease.pdf

B cell anergy plays a critical role in maintaining self-tolerance by inhibiting autoreactive B cell activation to prevent autoimmune diseases. Here, we demonstrated that Fc receptor-like 5 (Fcrl5) upregulation contributes to autoimmune disease pathogenesis by disrupting B cell anergy. Fcrl5—a gene whose homologs are associated with human autoimmune diseases—is highly expressed in age/autoimmunity-associated B cells (ABCs), an autoreactive B cell subset. By generating B cell-specific Fcrl5 transgenic mice, we demonstrated that Fcrl5 overexpression in B cells caused systemic autoimmunity with age. Additionally, Fcrl5 upregulation in B cells exacerbated the systemic lupus erythematosus-like disease model. Furthermore, an increase in Fcrl5 expression broke B cell anergy and facilitated toll-like receptor signaling. Thus, Fcrl5 is a potential regulator of B cell-mediated autoimmunity by regulating B cell anergy. This study provides important insights into the role of Fcrl5 in breaking B cell anergy and its effect on the pathogenesis of autoimmune diseases.</p

Relationship between Prox1 or FOXC2 expression and clinicopathological parameters.

<p>Relationship between expression of Prox1 or FOXC2 and parameters excluding MVD and LVD were calculated by chi-square test. Relationship between expression of Prox1 or FOXC2 and MVD or LVD were calculated by one-factor ANOVA test. T classification and clinical stage were classified according to the TNM classification.</p><p>*Histological differentiation: Well, well-differentiated squamous cell carcinoma; Mod, moderately differentiated squamous cell carcinoma; Por, poorly differentiated squamous cell carcinoma.</p><p>**MVD and LVD were Means ± S.D. (standard deviation), each S.D. was less than 10% in all cases.</p