Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: (A, E) Pretreatment with IL-35 neutralizing antibodies (anti-P35 or anti-Ebi3) aggravated LPS- and CLP-induced ARDS. Lungs from each experimental group were processed for histological examination after H&E staining. Compared with the LPS + IgG group, thickened alveolar wall, alveolar hemorrhage and collapse, inflammatory cells in filtration were more severe and pretreated with IL-35 neutralizing antibodies prior to LPS challenge. (B, D) Lung injury scores were estimated by the method of Mikawa, which is from the following four indicators of lung injury score: alveolar congestion; bleeding; gap or vascular wall neutrophil infiltration or aggregation; alveolar septal thickening or transparent membrane formation. 0 marks: no or very slight damage, 1 marks: mild injury, 2 marks: moderate injury, 3 marks: severe injury, 4 marks: very severe damage, the cumulative increase in the number of lesions of the total score is the pathological score of the ARDS. Pretreatment with IL-35 neutralizing antibodies lungs injury were more severe than LPS + IgG group. **p < 0.01, ***p < 0.001, by the two-way ANOVA followed by LSD multiple comparisons test, compared with the LPS + IgG group.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Staining, Filtration, Membrane

Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: Interleukin 35 (IL-35) blockade upregulated the production of proinflammatory cytokines and downregulated anti-inflammatory cytokines during ARDS models. Cytokine and chemokine concentrations in BALF and blood specimens from five mice treated with or without anti–Ebi3 or anti–P35 blocking antibodies were determined by mice cytokine/chemokine magnetic bead panel kit assays 24 h after onset of ARDS. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by the one-way ANOVA followed by LSD multiple comparisons test, compared with mice treated with isotypical IgG control. ARDS featured increased CXCL-1, TNF-α, IL-6, and IL-17A and reduced IFN-γ, IL-10, IL-2, and IL-13 after neutralizing antibody treatment (anti-IL-35 Ebi3 or anti-IL-35 P35) in BALF and serum.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Blocking Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Photoreceptor Cells Constitutively Express IL-35 and Promote Ocular Immune Privilege

doi: 10.3390/ijms23158156

Figure Lengend Snippet: Generation and characterization of IL-35 reporter mouse strain. ( A ) Schematic of the DNA and cDNA constructs used to genetically engineer the il-12a-venus yellow fluorescent protein (yfp) knock-in reporter mouse by CRISPR/Cas9-mediated homologous recombination at the C57BL/6J il-12a locus. ATG in exon 1 indicates integration site of the guide RNA (gRNA), which is 5′ proximal to the Venus cDNA and bGHPA (bovine growth hormone polyadenylation signal), as shown. The complete 3814 bp il-12a-yfp knock-in allele cassette was inserted between the left and right homologous arms, and PCR primers used to validate homologous recombination (HR) are shown. ( B ) Tail DNA genotype analysis showing PCR products used to identify mice with successful homologous recombination. Wild-type control mice (WT) express the IL-12p35 protein but not yfp ( il12 +/+ ); mice expressing yfp from both chromosomes ( il12a yfp/yfp ) do not express IL-12p35 ( il12 −/− ) and are henceforth referred to as IL-12p35 knock-KO (p35-KO); mice expressing yfp only from one chromosome ( il12a yfp/− ) express IL-12p35 and yfp proteins ( il12 +/ − ) and are henceforth referred to as IL-12p35 knock-in (p35-KI). Details of the entire construct delineate various components of the construct and expected PCR products derived from analysis of tail DNA of WT, p35-KI or p35-KO mice. ( C ) Western blot analysis of whole cell protein extracts of spleen cells from WT, p35-KI or p35-KO mice. ( D ) B cells isolated from the spleen of WT or p35-KI mice were sorted for CD19 + B cells, and cell surface FACS analysis established that ~6.4% CD19 + cells in the spleen of p35-KI mice express yfp. ( E ) Total RNA isolated from CD19 + B cells of the WT, p35-KI or p35-KO mice were analyzed by qPCR using primers specific to mouse to determine the relative abundance of b-actin, IL-12p35 or ebi3 mRNA transcripts. Data represent at least 3 independent experiments. * p < 0.05, **** p < 0.0001; N.S, no significant differences.

Article Snippet: Blots were probed with antibodies specific to mouse β-actin (sc-47778), and EBI3 (sc-166158) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Construct, Knock-In, CRISPR, Homologous Recombination, Control, Expressing, Derivative Assay, Western Blot, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Photoreceptor Cells Constitutively Express IL-35 and Promote Ocular Immune Privilege

doi: 10.3390/ijms23158156

Figure Lengend Snippet: IL-35 is highly expressed in retinal rod and cone photoreceptors. ( A ) qPCR analysis of p35, ebi3 IL-10 mRNA transcripts in the retina and spleen of WT mice. ( B ) Western blot analysis of whole cell extracts prepared from sorted CD4 + T cells, CD19 + B cells, dendritic cells (CD11c) and monocytes (Ly6C). Antibodies used were specific to mouse p35 or β-actin. ( C ) Immunohistochemistry (IHC) analyses of frozen eye sections were performed with or without primary mouse IL-12p35 antibodies and goat anti-mouse (fab’2) secondary antibody. Slides were then stained with a DAB kit and counter-stained with methyl blue. IL-12p35-expressing cells, dark brown; microglia, white arrows; photoreceptor layers, red arrows. ( D , E ) Detection of IL-12p35-expressing cells by immunohistochemistry and confocal microscopy. Paraffin-fixed whole eye sections were subjected to antigen-retrieval, blocked in mouse blocking solution, incubated without or with primary rabbit IL-12p35 antibody and then goat anti-rabbit conjugated with AF647 antibody. Far-red magenta color indicates IL-12p35 expression; red arrow, photoreceptor layers; white arrow, microglial cells. GC, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL outer plexiform layer, ONL, outer nuclear layer; IS, inner section; OS, outer segment; RPE/choroid, retinal pigmented epithelial and choroid. Data represent at least 3 independent experiments. *** p < 0.001.

Article Snippet: Blots were probed with antibodies specific to mouse β-actin (sc-47778), and EBI3 (sc-166158) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Immunohistochemistry, Staining, Expressing, Confocal Microscopy, Blocking Assay, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Photoreceptor Cells Constitutively Express IL-35 and Promote Ocular Immune Privilege

doi: 10.3390/ijms23158156

Figure Lengend Snippet: Decrease of IL-35 at EAU onset correlates with increase of inflammatory cytokines. WT C57BL/6J mice were immunized with IRBP 651-670 in CFA, and mice were sacrificed at several time points (day 0, day 7, day 14 and day 21) after EAU induction. After perfusing with PBS, retina was isolated, RNA was immediately isolated, and cDNA was subjected to qPCR analysis. ( A , B ) Histograms show relative abundance of IL-35 subunit mRNA transcripts IL-12p35 and ebi3 ( A ) and mRNA transcripts for the proinflammatory cytokines IL-6, IL-1β and TNF-α ( B ). Results represent 3 independent studies. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Blots were probed with antibodies specific to mouse β-actin (sc-47778), and EBI3 (sc-166158) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Isolation