2222dc6e5b2d5afea19385529bf04435 301293.pdf 2222c66f763468c818de25f169ac94c04f7d28e7 301293.pdf 61a30b690c9af87c3c0607355413c690b497e9b4c1d39c39dd2e281556be0be5 301293.pdf Title: Impaired Regulation by IL-35 in Systemic Sclerosis Subject: This study investigated the role of IL-35 in systemic sclerosis (SSc) patients, focusing on CD4+ T cell response and immunomodulatory cytokine production. By comparing the cytokine levels in healthy donors (HD) and SSc patients using ELISAs, we found a significantly lower plasma IL-35 concentration in the SSc patients (52.1 5.6 vs. 143 11.1, p < 0.001). Notably, the IL-35 levels showed a negative correlation with TGF- (p < 0.001) and IL-17 (p = 0.04). Assessing the IL-35R expression across cell types in the SSc patients and HDs via flow cytometry, we found higher levels on monocytes (40.7 + 5.7 vs. 20.3 1.9, p < 0.001) and lower levels on CD8+ T cells (61.8 9.2 vs. 83.4 0.8, p < 0.05) in the SSc patients. The addition of recombinant IL-35 to stimulated peripheral blood mononuclear cells reduced the IL-17+CD4+ T cell percentage (9.0 1.5 vs. 4.8 0.7, p < 0.05) and increased the IL-35+CD4+ T percentage (4.1 2.3 vs. 10.2 0.8, p < 0.001). In a Treg:Tresponder cell Sco-culture assay with HD and SSc samples, rIL35 decreased the cell proliferation and levels of IL-17A (178.2 30.5 pg/mL vs. 37.4 6.4 pg/mL, p < 0.001) and TGF- (4194 777 pg/mL vs. 2413 608 pg/mL, p < 0.01). Furthermore, we observed a positive correlation between the modified Rodnan skin score (mRSS) and TGF- (p < 0.001), while there was a negative correlation between mRSS and IL-35 (p = 0.004). Interestingly, higher levels of plasmatic IL-35 were detected in individuals with limited disease compared to those with diffuse disease (60.1 8.0 vs. 832.3 4.1, p < 0.05). These findings suggest that IL-35 exhibits anti-inflammatory properties in SSc and it may serve as a marker for disease severity and a therapeutic target. Keywords: systemic sclerosis; interleukin-35; CD4+ T lymphocytes; regulatory T cells Author: Rubén Osuna-Gómez, Ivan Castellví, Maria Mulet, Mª Àngels Ortiz, Douglas E. Brough, Helen Sabzevari, Roshanak T. Semnani and Silvia Vidal Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Jun 28 10:50:04 2023 CEST ModDate: Wed Jun 28 10:59:05 2023 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2987064 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CUEFWC+VnURWPalladioL Type 1 Custom yes yes no 10 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 15 0 NPKGUR+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 XJEJGX+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 VSVFUS+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 WTAUCZ+CMSY10 Type 1 Builtin yes yes no 32 0 ADUODH+VnURWPalladioL-Bold Type 1 Custom yes yes no 36 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 54 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 57 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 87 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 91 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 95 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 99 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 111 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 115 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 119 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 123 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 139 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 143 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 147 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 151 0 BCDIEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 158 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 174 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 178 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 182 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 186 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 202 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 206 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 210 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 214 0 BCDIEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 221 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 237 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 241 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 245 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 249 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 271 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 275 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 279 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 283 0 CRJHCU+PazoMath-Italic Type 1 Builtin yes yes no 294 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-04 02:39:33 CEST RepresentationInformation: 301293.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-10-03 14:01:15 CEST Size: 2987064 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 484 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Population Destination: subsection.2.1 Item: Title: Plasma Levels of IL-35 and Association with Circulating Subsets of Leukocytes Destination: subsection.2.2 Item: Title: Expression of IL35R on PBMCs Destination: subsection.2.3 Item: Title: Effect of rIl-35 on Intracellular Cytokine Production by CD4+ T Cells Destination: subsection.2.4 Item: Title: Effect of rIl-35 on Suppression Treg Assay Destination: subsection.2.5 Item: Title: Plasma Levels of IL-35 and Association with Clinical Features and Progression Destination: subsection.2.6 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Patients and Samples Destination: subsection.4.1 Item: Title: Cell Culture and IL-35R Expression on PBMC Subpopulations Destination: subsection.4.2 Item: Title: Intracellular Cytokine Production by Flow Cytometry Destination: subsection.4.3 Item: Title: Suppression Assay Destination: subsection.4.4 Item: Title: Determination of IL-35, TGF-, IL-17A and IL-10 Destination: subsection.4.5 Item: Title: Statistics Destination: subsection.4.6 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.6 Info: Title: Impaired Regulation by IL-35 in Systemic Sclerosis Author: Rubén Osuna-Gómez, Ivan Castellví, Maria Mulet, Mª Àngels Ortiz, Douglas E. Brough, Helen Sabzevari, Roshanak T. Semnani and Silvia Vidal Subject: This study investigated the role of IL-35 in systemic sclerosis (SSc) patients, focusing on CD4+ T cell response and immunomodulatory cytokine production. By comparing the cytokine levels in healthy donors (HD) and SSc patients using ELISAs, we found a significantly lower plasma IL-35 concentration in the SSc patients (52.1 5.6 vs. 143 11.1, p < 0.001). Notably, the IL-35 levels showed a negative correlation with TGF- (p < 0.001) and IL-17 (p = 0.04). Assessing the IL-35R expression across cell types in the SSc patients and HDs via flow cytometry, we found higher levels on monocytes (40.7 + 5.7 vs. 20.3 1.9, p < 0.001) and lower levels on CD8+ T cells (61.8 9.2 vs. 83.4 0.8, p < 0.05) in the SSc patients. The addition of recombinant IL-35 to stimulated peripheral blood mononuclear cells reduced the IL-17+CD4+ T cell percentage (9.0 1.5 vs. 4.8 0.7, p < 0.05) and increased the IL-35+CD4+ T percentage (4.1 2.3 vs. 10.2 0.8, p < 0.001). In a Treg:Tresponder cell Sco-culture assay with HD and SSc samples, rIL35 decreased the cell proliferation and levels of IL-17A (178.2 30.5 pg/mL vs. 37.4 6.4 pg/mL, p < 0.001) and TGF- (4194 777 pg/mL vs. 2413 608 pg/mL, p < 0.01). Furthermore, we observed a positive correlation between the modified Rodnan skin score (mRSS) and TGF- (p < 0.001), while there was a negative correlation between mRSS and IL-35 (p = 0.004). Interestingly, higher levels of plasmatic IL-35 were detected in individuals with limited disease compared to those with diffuse disease (60.1 8.0 vs. 832.3 4.1, p < 0.05). These findings suggest that IL-35 exhibits anti-inflammatory properties in SSc and it may serve as a marker for disease severity and a therapeutic target. 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