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Bar graph shows statistical analysis of absolute numbers of neutrophils and their frequencies among total CD11b+ cells

Bar graph shows statistical analysis of absolute numbers of neutrophils and their frequencies among total CD11b+ cells. outnumber all the other type 17 cells such as Th17, ILC3, NKT17, and MAIT17 cells. Upon PM exposure, IL-1-secreting neutrophils and IL-17-generating T17 cells entice each other round the airways. Accordingly, Prostratin PM-induced neutrophilia was significantly relieved in T- or IL-17-deficient and germ-free mice. Collectively, these findings show the commensal microbiome promotes PM-induced neutrophilia in the lung T17 cells. (B6.129P2-(B6) mice were received from Dr. Charles D. Surh (POSTECH, Korea). CD45.1/2 B6 mice were received from Dr. Sin-Hyeog Im (POSTECH, Korea). All mice were used at the age of 6-12 weeks unless indicated, and age- and sex-matched animals were used as settings. Germ-free (GF) mice were bred and used as previously explained (24). All mouse experiments were performed using protocols authorized by the Institutional Animal Care and Use Committees (IACUC) of the POSTECH. PM-Induced Pulmonary Swelling and Treatments Mice were intranasally given with 250 g of particulate matter (PM) in saline or saline only as settings. PM was from Sigma (PM10-like ERMCZ100-1VL and ERMCZ120-1VL) and used like a 1:1 combination. All intranasal administration (20 l/nostril) was performed under anesthesia (i.p.) with ketamine (Yuhan)/xylazine (Rompun, BAYER) answer, as Prostratin explained (21). Mouse Models of Chronic HDM/PM-Induced Allergic Asthma We used a previously explained house dust mite (HDM)-induced mouse asthma model with small changes (25). HDM (the intranasal route with 250 g of PM. Parabiosis Five-week-old B6 CD45.1/2 and CD45.2/2 female mice were joined together by Prostratin parabiosis for 2 or 7 weeks, as previously described (11). Weight-matched mice were anesthetized and shaved. An incision was made along the side of each mouse and the skin was connected using medical clips. Circulation Cytometry and Antibodies Single-cell suspensions were isolated and stained with fluorescein-conjugated antibodies. For cytokine detection experiments, Rabbit Polyclonal to p47 phox (phospho-Ser359) lymphocytes were stimulated with Cell Activation Cocktail and protein transport inhibitors (eBioscience) for 4 hours. Cells were washed twice in FACS buffer and stained with surface markers for 30?min at 4C. For intracellular staining, single-cell suspensions were surface-stained, fixed, and permeabilized with the eBioscience Foxp3 staining buffer collection. Following antibodies were used; anti-CD4-BUV395 (BD, GK1.5), anti-CD8-BV650 (BD, 53-6.7), anti-SiglecF-PE (BD, E50-2440), anti-CD11b-PerCP-Cy5.5 (BD, M1/70), anti-CD11c-PE-Cyanine7 (eBioscience, N418), anti-TCR-PE-CF594 (BD, H57-597), anti-Ly6G-APCCy7 (BD, IA8), anti-CD45.2-BV605 (BD, 104), anti-B220-BV711 (BD, Prostratin RA3-6B2), anti-CD8-BV510 (BD, 53-6.7), anti-CD44-redFluor 710 (TONBO, IM7), anti-TCR-APCCy7 (BD, H57-597), anti-CD11c-BV650 (BD, HL3), anti-Thy1.2-BV786 (BD, 53-2.1), anti-GL3-BV421 (BD, GL3), anti-CD45.2-BV650 (Biolegend, 104), anti-CD11c-BV711 (BD, HL3), anti-GATA3-PE (ebioscience, TWAJ), anti-Tbet-PE-Cyanine7 (eBioscience, 4B10), anti-ROR-PE-CF594 (BD, Q31-378), anti-PLZF- Alexa Fluor 647 (BD, R17-809), Zombie-Aqua (Biolegend), anti-RORt- PerCP-Cy5.5 (BD, Q31-378), anti-IL-17A-BV650 (BD, TC11-18H10), anti-IFN-BV786 (BD, XMG1.2), anti-CD11b-BV711 (BD, M1/70), anti- IFN-PE (Invitrogen, XMG1.2), anti-proIL-1- PE-Cyanine7 (Invitrogen, NJTEN3), anti-CD11c-APC (Invitrogen, N418), anti- CD121a (IL-1R, Type I/p80)-PE (Biolegend, JAMA-147), anti-IFN-BV421 (BD, XMG1.2), anti-GL3- PE-CF594 (BD, GL3), anti-CD24-BV605 (Biolegend, M1/69), anti-?V2 TCR-BV786 (BD, UC3-10A6), anti-CD23p19-Alexa Fluor 488 (Invitrogen, fc23cpg), anti-Ki-67- PerCP-eFluor 710 (Invitrogen, SolA15), anti-GL3-PE (BD, GL3), anti-IL-17F- Alexa Fluor 488 (Biolegend, 9D3.1C8), anti-V1.1 TCR-BV421 (BD, 2.11), anti- V1.1+ V1.2 TCR-PE (Biolegend, 4B2.9), anti-V3 TCR-BV510 (BD, 536), anti-CD45.1- Pacific Blue (Biolegend, A20), anti-CD3-APCCy7 (BD, 145-2C11), anti-V6.3/2 TCR (BD, 8F4H7B7). 17D1 hybridoma (anti-V6 antibody) and biotinylated anti-V7 antibody was used as previously explained (21). Cells were analyzed on an LSR (Foretessa BD Biosciences) and data were processed using FlowJo software (Tree Celebrity). Tetramers and Cell Enrichment Biotinylated PBS57 loaded CD1d monomers and 5-OP-RU loaded MR1 monomers were from the tetramer facility of the US National Institutes of Health (NIH). Biotinylated monomers were tetramerized using streptavidin-phycoerythrin (PE) (Prozyme), streptavidin-allophycocyanin (APC) (Prozyme), and streptavidin-PE-Cy7 (BD). For simultaneous enrichment of NKT, MAIT, and T cells, solitary cell suspensions of lung had been stained with PBS57-Compact disc1d PE-Cy7, 5-OP-RU-MR1 PE, and anti-TCR (GL3) PE-TR and enriched with anti-PE microbeads (Miltenyi) based on the producers instructions. Cell Planning Mice were sacrificed on the indicated period BAL and factors liquids were collected in 1 mL PBS. To eliminate circulating cells, 15?ml of PBS was injected in to the center after incision from the abdominal aorta. Gathered lung tissues had been minced by.