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Isolated kidney CD45⁺ cells were used to analyze immune subtypes. The cells were surface-stained with fluorophore-conjugated antibodies against the following mouse antigens: CD45 (FITC, APC), CD11b (PE), CD11c (FITC), F4/80 (PE/CY7), CX3CR1 (BV510), CD24 (APC), GR-1 (APC), I-A/I-E (APC/CY7), and CD103 (BV421) (BioLegend, San Diego, CA, USA). A FACSAria SORP flow cytometer (BD Biosciences, CA, USA) and FlowJo software were used to acquire and analyze the flow cytometric data, respectively.

Mice were sacrificed, and tumor tissues were harvested. Single-cell suspensions were prepared using Collagenase B (1 mg/ml, 11088807001, Roche) and Hyaluronidase (1 mg/ml, abs47014926, Absin). After digestion, the suspensions were filtered by 40 μm Nylon cell strainers (352340, Corning). Before staining, cells were suspended in PBS and dyed by Fixable Viability Stain 700. Fluorescent staining was performed according to the manufacturer’s recommendations. Fluorescent antibodies recognizing murine CD45 (557659, BD Biosciences), CD3 (100306, BioLegend), CD8a (100722, BioLegend), CD107a (121629, BioLegend), granzyme B (372204, BioLegend), CD11b (101206, BioLegend), I-A/I-E (107608, BioLegend), CD11c (566504, BD Biosciences), CD206 (141708, BioLegend), F4/80 (565411, BD Biosciences) were used in this assay. Flow cytometry buffers utilized in the experiment included Brilliant Stain Buffer (563794, BD Biosciences) and eBioscience™ FOXP3/Transcription Factor Staining Buffer Set (00-5523-00, Invitrogen). Flow cytometry was performed using Beckman CytoFLEX S or Beckman CytoFLEX LX. Flow cytometry data were analyzed by Flowjo v10 (Ashland, OR).

Replicates were conducted with five mice per group (1–2 mock-infected with PBS and 3–4 E. coli UTI89-infected). At 12 days post-infection, mice were humanely euthanized without perfusion and the vagina, cervix and uterine horns were collected and kept on ice in 1 ml RPMI buffer containing 1% fetal bovine serum, 1% penicillin/streptomycin, and 50 μM 2-mercaptoethanol. Single cell suspensions were generated and flow cytometry was performed as previously described [24 (link)]. The following antibodies were used for this study: TruStain fcX (BioLegend, CA, USA, clone 93), Siglec-F (BD Pharmingen, NJ, USA, clone E50-2440), LyG6 (BioLegend, clone 1A8), CD11b (BioLegend, clone M1/70), CD11c (BioLegend, clone N418), Ly6C (BioLegend, clone HK1.4), F4/80 (BioLegend, clone BM8), and I-A/I-E (BioLegend, clone M5/114.15.2). CountBright beads (Invitrogen, CA, USA) were added to samples prior to acquisition to determine cell counts.

Mice were humanely euthanized, and mouse tumors were harvested. Whole tumors were cut and minced into small pieces, followed by digestion in Liberase TL Research Grade 10 (2 µg/mL, Roche, 05401020001) and DNase I (Sigma, 260913-10 MU) in DMEM at 37 °C for 30 min. The digested tissue was filtered through a 70-mm cell strainer (Thermo Fisher Scientific). Cell suspensions were stimulated with PMA (100 µg/mL) plus the protein transport inhibitors Golgi stop and Golgi plug (1:1000, BD Bioscience, 51-2301 KZ) at 37 °C. After 4 h, live cells were identified by vivid yellow staining (Invitrogen, cat# L34959). Cells were stained for cell surface markers including CD45.2 (1:100, BioLegend, 109814), CD8 (1:100, BioLegend, cat# 100708), CD11c (1:100, BioLegend, 117311), and IA/IE (1:100, BioLegend, 107608) at 4 °C for 30 min. For intracellular staining, cells were fixed and permeabilized with a fixation/permeabilization kit (BD Bioscience, 554714) for 30 min at 4 °C and then stained with anti-IFN-γ (1:100, BioLegend, 505813) and anti-granzyme B (1:100, BioLegend, 515406) antibodies. Cells were imaged on a BD LSRFortessa (BD Biosciences) and analyzed using FlowJo software (TreeStar).

C57Bl/6J, Tlr2^-/-, Mavs^-/-, Nod2^-/- and Trif^-/- and mice were from Jackson Laboratories, and Tlr7^-/- mice were from Regeneron. Mice were intranasally infected with 2–5×10⁷ cfu of S. aureus for pneumonia studies, or 7×10⁸ cfu for mortality for 20 h as previously described [10] (link). Protein content in bronchoalveolar lavage fluid (BALF) was measured using Bradford protein assay. Staining cells for flow cytometry has been described elsewhere [45] (link). Cells were labeled with combinations of fluorescein isothiocyanate-labelled anti-Ly-6G (Gr-1; RB6-8C5; Biolegend), PerCP-Cy5.5-labelled anti-CD11c (N418; Biolegend), allophycocyanin-labelled anti-MHC II (I-A/I-E; Biolegend) and phycoerythrin-labelled anti-NK 1.1 (NKR-P1C, Ly-55; Biolegend). Neutrophils were defined as Ly6G⁺/MHCII⁻, macrophages as CD11C⁺/MHCII^low-mid and dendritic cells as CD11c⁺/MHCII^high. Data were analyzed using WinMDI (version 2.8; Joseph Trotter).