Mice
C57BL/6 (B6) mice homozygous for loxP-flanked caspase-8 allele (Casp8
fl/fl) [11] were crossed with mice expressing Cre under control of murine lysozyme M gene promoter (Cre
LysM; The Jackson Laboratory, Bar Harbor, ME, USA), generating Cre
LysM
Casp8
fl/fl mice. Cre
LysM
Casp8
fl/fl mice were crossed with RIPK3
−/− (Genentech, South San Francisco, CA, USA) to generate RIPK3
−/−
Cre
LysM
Casp8
fl/fl mice. OT-II/RAG
−/− and B6.CD45.1 were purchased from The Jackson Laboratory. B6.CD45.1/2 mice were generated from a cross of B6 (The Jackson Laboratory) and B6.CD45.1 mice. Female mice were used in all studies. Proteinuria was assessed using Uristix reagent strips (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). Transnetyx (Cordova, TN, USA) performed all genotyping of mice. All animal experiments were approved by the Northwestern University Institutional Animal Care and Use Committee.
Histopathologic studies
Paraffin-embedded kidney sections (5 μm) were treated with periodic acid–Schiff stain, and a pathologist blinded to the study scored kidney sections using an Olympus BS40 microscope (Olympus Life Science, Center Valley, PA, USA) as previously described [12]. Frozen kidney sections (10 μm) were stained with anti-IgG-fluorescein isothiocyanate [12]. All images were photographed at × 40, ×200, ×400, or × 600 magnification on an Olympus BX41 microscope equipped with an Olympus DP20 camera.
Flow cytometry
Surface staining of cell suspensions and gating strategies were carried out as previously described [9, 10, 13]. At least 100,000 events were captured on a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Dead cells were excluded using the Molecular Probes LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, Carlsbad, CA, USA). For cell-sorting studies, splenocytes preincubated with Fc block antibody were stained with fluorescent antibodies (information available upon request). Splenocyte populations sorted on a BD FACSAria II instrument (BD Biosciences) at the University of Chicago and Northwestern University Cancer Center Flow Core had an average purity of 97 %.
Mixed bone marrow chimeras
Bone marrow was aseptically harvested from tibias, femurs, and humeri from 9-week-old mice and erythrocytes were lysed (BD Pharm Lyse buffer; BD Biosciences). Cells were incubated with Fc block antibody followed by incubation with fluorochrome-conjugated antibodies against B220, CD4, CD8, CD11b, Ly6G, NK1.1, Siglec F, Ter119, c-Kit, and Sca-1 (BD Biosciences; and eBioscience and BioLegend, both located in San Diego, CA, USA). Cell suspensions were subjected to fluorescence-activated cell sorting (FACS) analysis to obtain the Lin−Sca-1+c-kit+ (LSK) cell population. Three-month-old B6.CD45.1/2 received a single 1000-cGy γ-irradiation dose using a Cs-137-based Gammacell 40 irradiator (Best Theratronics, Ottawa, ON, Canada). After 12 h, 5 × 105 LSK cells were intravenously injected from Casp8
fl/fl, Cre
LysM
Casp8
fl/fl, a mixture of Casp8
fl/fl plus B6.CD45.1 or Cre
LysM
Casp8
fl/fl plus B6.CD45.1 (1:1 ratio). Chimeric mice were maintained on trimethoprim/sulfamethoxazole (40 mg/5 mg, respectively; Hi-Tech Pharmacal/Akorn, Amityville, NY, USA) diluted in autoclaved water (2 ml antibiotics/500 ml water) and phenotyped 8 months posttransfer.
In vitro assays
For mixed leukocyte reactions, splenocytes were incubated with anti-CD19 beads and negative fractions were incubated with anti-CD11b magnetic-activated cell sorting beads (Miltenyi Biotec, Bergisch Gladbach, Germany) to purify antigen-presenting cells (APCs). Purified APCs were pulsed with 10 μg/ml ovalbumin (OVA) peptide (amino acids 323–339) for 60 minutes at 37 °C. OVA-specific splenic CD4+ T cells were isolated from B6.CD45.1/OT-II/RAG
−/− mice using CD4+ T-cell isolation kits (Miltenyi Biotec) according to the manufacturer’s instructions. Purity of APCs and T cells was 90 %. T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (500 nM for 12 minutes at 37 °C; Invitrogen, Carlsbad, CA, USA). Pulsed APCs at various ratios were incubated with 2 × 105 CFSE-labeled T cells with or without 5 μg/ml class B CpG (ODN 1668; InvivoGen, San Diego, CA, USA) in triplicate in 96-well flat-bottomed plates at 37 °C for 3 days. Cell clusters were dissociated with 7.5 mM ethylenediaminetetraacetic acid for 15 minutes, and stained with anti-CD4 (BD Biosciences). 7-Aminoactinomycin D (0.25 mg/test; BD Biosciences) was used to exclude dead cells. A constant number of CaliBRITE beads (BD Biosciences) were added for acquisition of equal parts in each culture. Live T cells were gated, and the number of divided cells showing less than maximal CFSE fluorescence intensity was determined.
BMDMs were generated by culturing bone marrow in media (Dulbecco’s modified Eagle’s medium with 10 % fetal calf serum, 2 mM glutamine, 100 U penicillin/0.1 mg streptomycin/1 ml and 1 mM sodium pyruvate) plus macrophage colony-stimulating factor (M-CSF; 100 ng/ml). On day 6, cells were replated at 1.75 × 106 cells/well in a 6-well plate, and on day 7 cells were stimulated with the indicated treatments. For cytokine levels, BMDMs were stimulated for 3, 6, and 12 h with LPS (10 ng/ml), imiquimod (5 μg/ml), or CpG (5 μg/ml) with or without necrostatin-1 (Nec-1; 30 μM), and cells were harvested for cytokine transcript levels while supernatants were evaluated for cytokine levels (see below). Following TLR stimulation for the indicated time, ATP (5 mM; Sigma-Aldrich, St. Louis, MO, USA) was added to BMDMs for 45 minutes for evaluation of IL-1β levels in supernatants. For BMDM death assays, BMDMs at a concentration of 2 × 106 cells/ml were stimulated for 48 h with superFasL (100 ng/ml; Enzo Life Sciences, Farmingdale, NY, USA), etoposide (10 μM; Alexis Biochemicals, Lausen, Switzerland) with or without carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK, 20 μM; Promega, Madison, WI, USA), and supernatants were evaluated for lactate dehydrogenase (LDH) activity according to the manufacturer’s instructions (Sigma-Aldrich). For splenocyte death assays, 3 × 106 total splenocytes were treated for 10 h with superFasL (100 ng/ml; Enzo Life Sciences) or etoposide (10 μM; Alexis Biochemicals) and stained with annexin-V and Molecular Probes LIVE/DEAD Fixable Aqua Dead Cell Stain. For macrophage polarization assays, BMDMs were cultured with M1-polarizing conditions (primed overnight with interferon [IFN]-γ 100 ng/ml and stimulated for 3 h with LPS [10 ng/ml]) and M2-polarizing conditions (stimulated for 24 h with IL-4 [40 ng/mL]) with or without Nec-1 (30 μM) and/or Z-Ile-Glu(O-ME)-Thr-Asp(O-Me)-FMK (Z-IETD-FMK, 20 μM; BD Biosciences). Polarized BMDMs were lysed directly in QuantiGene Lysis Mixture, and gene expression profiles were determined using a custom QuantiGene 2.0 assay (Affymetrix, Santa Clara, CA, USA) and a Luminex 200 instrument (Luminex Corporation, Austin, TX, USA) (see Additional file 1: Table S1 for panel description).
In vivo assays
For TLR ligand injection studies, 3-month-old mice were intraperitoneally injected with LPS, imiquimod, or CpG (200 μg/20 g body weight; InvivoGen) and analyzed after 4 h by FACS analysis. For oral antibiotic treatment, 3-week-old mice were given autoclaved water plus ampicillin (1 g/L), vancomycin (0.5 g/L), neomycin sulfate (1 g/L), metronidazole (1 g/L), and sucrose (10 g/L) twice weekly for 8 weeks with no observable weight loss.
Western blot analysis, antibody/cytokine measurements, and gene expression quantification
Sorted splenocytes and BMDMs were lysed in sample buffer, and equal amounts of proteins were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and analyzed by immunoblotting with the appropriate primary antibodies (caspase-8, Enzo Life Sciences; glyceraldehyde 3-phosphate dehydrogenase [GAPDH], US Biological, Salem, MA, USA) and horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rat immunogloblulin G [IgG]-HRP; Santa Cruz Biotechnology, Santa Cruz, CA, USA), electrochemiluminescence detection (Pierce/Thermo Scientific, Waltham, MA, USA), and image acquisition (Ultra-Lum, Claremont, CA, USA). Single-stranded DNA (ssDNA)-, double-stranded DNA (dsDNA)-, histone-, or chromatin-reactive IgG antibodies were measured as previously described [12]. Total IgM and IgG isotypes and cytokine/chemokine expression were quantified using Luminex bead-based assays (Affymetrix) and enzyme-linked immunosorbent assays (ELISAs). Total RNA was isolated from BMDMs using the RNeasy Plus Mini Kit according to the manufacturer’s instructions (Qiagen, Valencia, CA, USA). RNA was reverse-transcribed with the High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed on an ABI 7300 real-time PCR machine (Applied Biosystems) using the TaqMan gene expression system (Applied Biosystems) and predesigned fluorescein-labeled primer/probes (β-actin, IL-6: Mm00446190_m1; IL-10: Mm00439614_m1; TNF-α: Mm00443258_m1; IL-1β: Mm00434228_m1; IL-12b: Mm00434174_m1; Life Technologies). The results are presented as relative expression compared with β-actin.
Data analysis
For analysis of macrophage polarization, data were normalized to expression of housekeeping genes and imported into Partek Genomics Suite V6.6 software (Partek, St. Louis, MO, USA). Differentially expressed genes between the different groups of stimulated macrophages, as well as transcripts with variable expression within the data set, were calculated using one-way analysis of variance (ANOVA). Differentially expressed genes between two analyzed macrophage populations were defined by a Bonferroni-corrected p value <0.05 unless stated otherwise. Principal component analysis (PCA) using all transcripts was performed for visualization of sample relationships. Hierarchical clustering of the differentially expressed genes was performed based on a Euclidean algorithm for dissimilarity and average linkage method to determine distance between clusters. All other data are shown as mean ± SD and were compared by Mann–Whitney U test using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA).