Journal: Science Advances

Article Title: Pregnancy enhances antiviral immunity independent of type I IFN but dependent on IL-17–producing γδ + T cells in the nasal mucosa

doi: 10.1126/sciadv.ado7087

Figure Lengend Snippet: ( A ) Model of murine intratracheal IAV infection (50 PFU in 50 μl of PBS). Created with BioRender.com . ( B ) Kinetics of pulmonary viral titers by MDCK plaque assay following intratracheal IAV infection ( n = 7 to 9). ( C ) Graphical representation of upper airway infection (50 PFU IAV in 10 μl of PBS). Created with BioRender.com . ( D ) Kinetics of the viral load measured by MDCK plaque assay on nasal tissues from WT nonpregnant and pregnant mice ( n = 7 to 10). ( E ) Graphical representation of nasal tissue regions and fluorescence microscopy of infected olfactory epithelial cells (zone III) with quantification 3 days postinfection with Ruby-NS1 PR8 (500 PFU in 10 μl of PBS) ( n = 3 to 4). Scale bars, 200 μm. ( F ) Kinetics of viral load measured by MDCK plaque assay on nasal tissues of nonpregnant and pregnant Ifnar1 −/− mice (50 PFU; n = 4 to 7). ( G ) Kinetics of nasal il-17A expression by RT-qPCR (50 PFU; n = 4 to 5). ( H ) Representative FACS plots and frequency of γδ + T cells, ( I ) absolute number of γδ + T cells, ( J ) proportion of IL-17A–producing cells and ( K ) total number of IL-17A + γδ + T cells in nasal tissues of nonpregnant and pregnant mice at 0 and 1 day post-IAV infection following 4 hours of stimulation with rIL-23 (10 ng/ml) and rIL-1β (10 ng/ml) (50 PFU; n = 7 to 10). ( L ) Ns1 and ( M ) M1 expression in nasal tissues of WT and TCR δ −/− pregnant mice by RT-qPCR 3 days post-IAV infection (50 PFU; n = 5). ( N ) Ns1 and ( O ) M1 expression in nasal cavities of TCR δ −/− nonpregnant and pregnant mice 3 days post-IAV infection ( n = 5 to 8). Data produced from . Data are presented as mean ± SEM. with * P < 0.05, ** P < 0.01, and *** P < 0.001. Data were analyzed using two-way ANOVA followed by Sidak’s multiple comparisons [(B), (D), and (F) to (K)], unpaired Student’s t test [(E), (L), (N), and (O)], or Mann-Whitney U test (M).

Article Snippet: Nonpregnant mice were administered rIL-17A (1 μg per mouse; R&D Systems, #7956-ML-025/CF) intranasally in 10 μl of PBS (5 μl per naris) 2 hours before IAV infection (50 PFU intranasally).

Techniques: Infection, Plaque Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Produced, MANN-WHITNEY

Journal: Science Advances

Article Title: Pregnancy enhances antiviral immunity independent of type I IFN but dependent on IL-17–producing γδ + T cells in the nasal mucosa

doi: 10.1126/sciadv.ado7087

Figure Lengend Snippet: ( A to E ) Muc5ac , Cramp , Mbd-3 , Mbd-4 , and Mbd-14 , expression in the nasal cavities of nonpregnant and pregnant IAV-infected mice measured by RT-qPCR at various time points postinfection (50 PFU; n = 4 to 5). ( F ) Loading plot and correlation coefficients of Ns1 and AMP expression by RT-qPCR 3 days post-IAV infection ( n = 4 to 5). ( G ) Intraperitoneal (i.p.) administration of anti-IgG1 (αIgG1) or anti–IL-17A (αIL-17A) (200 μg per mouse in 200 μl of PBS) to nonpregnant and pregnant mice 2 days before and the day of IAV infection (50 PFU in 10 μl). Created with BioRender.com . ( H to K ) Quantification of nasal Muc5ac , Cramp , Mbd-3 , and Mbd-4 expression by RT-qPCR ( n = 10 to 11) and ( L ) nasal viral titers quantified by MDCK plaque assay 1 day post-IAV infection following intraperitoneal administration of αIL-17A ( n = 7 to 8). ( M to O ) Neutrophil, monocyte, and macrophage absolute numbers in the nasal cavities of nonpregnant and pregnant mice at days 0 and 1 post-IAV infection measured by flow cytometry (50 PFU; n = 7 to 11). Viral ( P ) Ns1 and ( Q ) M1 gene expression measured by RT-qPCR in murine epithelial cells 24 hours post-IAV infection (MOI of 1) with/without recombinant mBD-3 (rmBD-3; 100 μg/ml) ( n = 3). ( R ) Intranasal administration of rmBD-3 (1 μg per mouse; in 10 μl PBS) 1 hour post-IAV infection (50 PFU in 10 μl of PBS) to nonpregnant mice. Created with BioRender.com . Viral ( S ) Ns1 and ( T ) M1 gene expression measured by RT-qPCR in nasal tissues of nonpregnant mice 1 day post-IAV infection with/without rmBD-3 ( n = 7 to 8). Data are presented as mean ± SEM. with * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Data were analyzed using two-way ANOVA followed by Sidak’s multiple comparisons [(A) to (E) and (M) to (O)], Pearson’s correlation (F), unpaired Student’s t test [(H), (K), (L), (P), and (Q)], or Mann-Whitney U test [(I), (J), (S), and (T)].

Article Snippet: Nonpregnant mice were administered rIL-17A (1 μg per mouse; R&D Systems, #7956-ML-025/CF) intranasally in 10 μl of PBS (5 μl per naris) 2 hours before IAV infection (50 PFU intranasally).

Techniques: Expressing, Infection, Quantitative RT-PCR, Plaque Assay, Flow Cytometry, Recombinant, MANN-WHITNEY

Journal: European Journal of Immunology

Article Title: Innate T‐cell‐derived IL‐17A/F protects from bleomycin‐induced acute lung injury but not bleomycin or adenoviral TGF‐β1‐induced lung fibrosis in mice

doi: 10.1002/eji.202451323

Figure Lengend Snippet: IL‐17A and IL‐17F gene and protein expression in lungs of BLM challenged mice. WT mice were treated with saline (CL, white bars) or BLM (grey bars) for 6 h, 24 h, 72 h, 7 days, 14 days, or 21 days, as indicated. (A, B) Gene expression levels of IL‐17A and IL‐17F in sorted TCRαβ pos (A) and TCRγδ pos T cells collected from lungs of BLM‐exposed WT mice. (C, D) Intracellular IL‐17A and IL‐17F protein expression of CD4 pos T cells (C) and TCRγδ pos T cells (D) of WT and IL17af −/− mice was measured 72 h post‐BLM by flow cytometry. (E, F) IL‐17A protein levels in BALF (E) and lung (F) of saline vs. BLM‐treated mice quantified by ELISA. Data are shown as scatter plots with mean values indicated as horizontal lines (A, B) or as mean ± SD of n = 9–12 (A, B), or n = 5–11 (C, D), or n = 4–8 (E, F) mice per group and time point. Note that in A and B, one data point represents a pool of T cells sorted from n = 2–3 mice per experimental group and time point. Data in (C–F) are representative of two independent experiments. * p ≤ 0.05, ** p ≤ 0.01 compared with mice from the control group (Mann–Whitney U ‐test).

Article Snippet: Recombinant mouse IL‐17A/F protein (rIL‐17A/F) was purchased from R&D Systems.

Techniques: Expressing, Saline, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY

Journal: European Journal of Immunology

Article Title: Innate T‐cell‐derived IL‐17A/F protects from bleomycin‐induced acute lung injury but not bleomycin or adenoviral TGF‐β1‐induced lung fibrosis in mice

doi: 10.1002/eji.202451323

Figure Lengend Snippet: Therapy of BLM‐treated IL17af −/− mice with recombinant IL‐17A/F protein. (A) Experimental design. BLM‐exposed IL17af −/− mice were treated i.v. with rIL‐17A/F protein or vehicle, as indicated. (B) Histopathology of hematoxylin/eosin‐stained lung tissue section of IL17af −/− mice with or without rIL‐17A/F protein therapy at day 6 post‐BLM treatment. Closed arrows in (B, left histology) indicate angiocentric neutrophilic infiltrations, while open arrows in (B, left histology) denote intravascular coagulation in IL17af −/− mice treated with BLM for 6 days. Closed arrows in (B, right histology) indicate lymphoplasmacellular infiltrates in BLM‐treated IL17af −/− mice with rIL‐17A/F protein therapy. Representative histology images from a total of n = 4 mice per experimental group are shown at original magnifications of ×40 (Scale bar 20 µm). (C–E) Immunophenotypic analysis of neutrophils (C), CD4 pos T cells (D), and CD8 pos T cells (E) in BALF of BLM‐treated IL17af −/− mice in the absence or presence of rIL‐17A/F protein therapy. Data are shown as mean±SD of n = 5–6 mice per group and time point and are representative of two independent experiments. * p ≤ 0.05, ** p ≤ 0.01 compared with mice from the control group (CL). ++ p ≤ 0.01 rIL‐17A/F application relative to vehicle (Mann–Whitney U ‐test).

Article Snippet: Recombinant mouse IL‐17A/F protein (rIL‐17A/F) was purchased from R&D Systems.

Techniques: Recombinant, Histopathology, Staining, Coagulation, Control, MANN-WHITNEY

Journal: European Journal of Immunology

Article Title: Innate T‐cell‐derived IL‐17A/F protects from bleomycin‐induced acute lung injury but not bleomycin or adenoviral TGF‐β1‐induced lung fibrosis in mice

doi: 10.1002/eji.202451323

Figure Lengend Snippet: AdTGF‐β1 induced lung fibrosis in WT and IL17af −/− mice. WT and IL17af −/− mice were left untreated (grey bars) or were treated with either AdCL (white bars) or AdTGF‐β1 (black bars) for up to 21 days. (A, B) IL‐17A protein levels in BALF (A) and lung (B) of AdCL vs. AdTGF‐β1‐treated mice. (C) Hydroxyproline levels in lung tissue of AdCL‐ vs. AdTGF‐β1‐treated WT and IL17af −/− mice. (D) Histopathology of hematoxylin/eosin‐stained lung tissue sections of WT and IL17af −/− mice at day 14 post‐AdCL vs. AdTGF‐β1 application. Data are shown as mean ± SD of at least n = 5–8 mice per experimental group and time point and are representative of two independent experiments. * p ≤ 0.05, ** p ≤ 0.01 compared with AdCL.

Article Snippet: Recombinant mouse IL‐17A/F protein (rIL‐17A/F) was purchased from R&D Systems.

Techniques: Histopathology, Staining

Journal: bioRxiv

Article Title: An IL-17-DUOX2 axis controls gastrointestinal colonization by Candida albicans

doi: 10.1101/2024.08.16.608271

Figure Lengend Snippet: A,B. Colonoids were prepared from control mice ( Duoxa1/a2 fl/fl ) or those lacking functional intestinal DUOX2 ( Duoxa1/a2 ΔIEC mice) to evaluate H 2 O 2 production and gene expression in response to recombinant murine IL-17 (rmIL-17). Colonoids were stimulated for 24 h with 5 ng/mL of rmIL-17A or the equivalent amount of carrier BSA protein (n=6 cultures). Lipopolysaccharide (LPS) was used as a positive control. A. H 2 O 2 production rates were normalized to MTT viability values. Data were analyzed by two-way ANOVA followed by Tukey’s post-hoc test. B. Transcript expression levels for Duox2 and Duoxa2 were determined in colonoids stimulated for 24 h with BSA, rmIL-17A, or LPS (n=6 cultures). Data were analyzed by means of Kruskal-Wallis test for each individual gene. *- p≤0.05, **- p≤0.01, ns- not significant. C . WT C57BL/6J and Il17ra -/- mice were treated with fluconazole (Flz) and the antibiotics penicillin, streptomycin, and vancomycin (Ab) followed by colonization with C. albicans SC5314 for 7 days (with Ab treatment continued throughout the experiment). D. Fungal colonization levels were determined from fecal pellets and GI organs at 7 dpi. Duo-Duodenum, Jej-Jejunum, Ile-Ileum, Col-Colon. E. The proportion of yeast and hyphal cells was determined from the ileum and colon of C. albicans- colonized WT (n=6 per group) and Il17ra -/- (n=5 per group) mice. Paraffin embedded tissue sections were deparaffinized and stained with an anti- Candida antibody, epithelial nuclei were stained with DAPI and mucus was stained with rhodamine-conjugated UEA-1 and WGA-1. 500-1000 cells were counted from each tissue section. Data is presented as standard error of mean (SEM). Statistical significance was determined using unpaired t-test and **- p≤0.01, ***- p≤0.001. F,G . Duox2 / Duoxa2 expression was determined by qRT-PCR in ileum ( F ) and colon ( G ) tissues of C. albicans WT SC5314-colonized mice (n=6 per group) and Il17ra -/- mice (n=5 per group). Data is presented as relative expression with SEM. Unpaired t-test was used to determine statistical significance; ns-not significant and *- p≤0.05, **- p≤0.01, ***- p≤0.001.

Article Snippet: On day 4, colonoids were challenged with sonicates of C. albicans SC5314 in yeast and hyphal forms (10 7 cells/mL); β-1,3-curdlan from Alcaligenes faecalis (100 µg/mL in DMSO; Invivogen); mannan (250 µg/mL in 1:1 PBS/DMSO solution; Millipore-Sigma), zymosan A (250 µg/mL in 1:1 PBS/DMSO solution; Millipore-Sigma), β-glucan from Saccharomyces cerevisiae (100 µg/mL in 1:1 PBS/DMSO solution; Millipore-Sigma); recombinant mouse IL-17A (5 ng/mL; R&D Systems); or the appropriate vehicles and carrier proteins for 24 h. All ligands and cytokines were preincubated with polymyxin B (PMB; 25 µg/mL; Millipore-Sigma) for 30 min at 37°C to prevent activation by lipopolysaccharide (LPS) contamination.

Techniques: Control, Functional Assay, Expressing, Recombinant, Positive Control, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Investigating Polyreactivity of CD4+ T Cells to the Intestinal Microbiota

doi: 10.1101/2024.08.15.607895

Figure Lengend Snippet: IL-17A cytokine production quantification after ex-vivo restimulation of CD4+ T cells from mice immunized with ( a ) BT and ( b ) AKK peptides in CFA, respectively. Controls are mice immunized with CFA only and naïve nonimmunized mice. CD4+T cells from spleen and lymph nodes are sorted and restimulated with bone marrow derived dendritic cells (BMDCs) pulsed with an array of BT, AKK, mammalian, viral, and literature peptides, each individually. IL-17A ELISA is performed on co-culture supernatant after 3 days. Heatmap (left panel) depicts averaged IL-17A quantity in nanogram/ml (4 mice per peptide immunization). Boxplot (right panel) shows individual quantitative values. Data are representative of two independent experiments.

Article Snippet: For standards, 50 ng/ml of the following proteins were used: mouse IFN-γ recombinant protein (catalog # 14-8311-63) and mouse IL-17A recombinant protein (catalog # 14-8171-62, eBioscience™).

Techniques: Ex Vivo, Derivative Assay, Enzyme-linked Immunosorbent Assay, Co-Culture Assay