IRGM1 supports host defense against intracellular bacteria through suppression of type I interferon in mice

IFN-I


Listeria infection
Listeria monocytogenes 10403S strain (LM) were grown in brain-heart infusion (BHI) broth overnight at 37°C, resuspended in sterile PBS and diluted appropriately to inject ~1 x 10 4 CFU in 100 μl PBS intra-peritoneally.Mice were monitored for survival with a 20% weight loss end point.GFP-expressing L. monocytogenes 10403S strain was a kind gift from Dr. Darren E. Higgins (3).
For in vitro studies, bacteria were diluted in DMEM/10% FBS without antibiotics and infected at MOI of 7-12 for one hour.Cells were then washed twice gently with warm PBS and incubated in media containing 50 μg/ml gentamicin (Sigma) for required time.
Bacteria killing assay F4/80 Hi peritoneal macrophages (0.3 x 10 6 ) seeded in 24-well plates were infected with LM in 200 μl media, as described above.After 1h and 24h, cells were washed once with PBS and permeabilized with sterile 0.5% Triton X-100 for 2 min, mixed by pipetting, serially diluted, and plated on BHI agar plates.

Peritoneal lavage measurements
Peritoneum was lavaged using 10 ml sterile PBS.Lavages were spun down, peritoneal cells were used for flow cytometry or macrophage isolation and supernatants were stored at -80°C.For analysis of peritoneal fluid, supernatants were thawed and concentrated 7.5 to 8 times using Amicon Ultra 0.5 ml centrifugal unit (Millipore UFC501024).For cytokine analysis, concentrated lavages were diluted twice, and cytokines were quantified by multiplex assay (Bio-Plex; Bio-Rad Laboratories).Final cytokine concentrations were adjusted by respective concentration or dilution factor.For citrullinated histone H3 (Cayman #501620), similar concentrated lavages were used.To determine LDH activity (Sigma), neat (without concentration) peritoneal lavages were used.
Lysosome staining 1 x 10 6 peritoneal cells were stained with LysoTracker Deep Red (Invitrogen no.L12492) at 50 nM in 250 ul complete DMEM media for 30 min at 37°C, washed and surface stained as mentioned above for Flow cytometry.

TUNEL assay
Tissue sections were deparaffinized and treated with Proteinase K for 30 min at 37°C, washed with PBS, incubated with 100 μl TUNEL labeling enzyme (Roche #11684795910) for 1h at 37°C, washed with PBS again and mounted for microscopy.

Bacteria count in tissues
Spleen and liver were homogenized in 0.1% Triton X-100 PBS using OMNI TH hand-held homogenizer.Fresh peritoneal lavage in PBS was treated with Triton X-100 to a concentration of 0.1%.Bacteria were plated on BHI agar after appropriate serial dilution.
For F4/80 Hi macrophages, 8 slices were taken in the z plane.Surfaces of Listeria and lysosomes were constructed using Imaris software.To determine the voxels of Listeria that colocalized with lysosomes, lysosomal channel was masked by setting the inside voxel to 1.The intensity sum of the new masked channel was then divided by the total voxels of Listeria to determine percentage of Listeria localizing with the lysosome.For peritoneal lavage cells, the total intensity of phospho-MLKL only in F4/80-positive cells was determined using Imaris.For TUNEL-stained tissue sections, ImageJ was used to determine numbers of TUNEL-positive foci.

Histopathology and immunohistochemical analysis
Harvested tissues were fixed in 10% neutral buffered formalin, trimmed, processed for paraffin embedding, sectioned (5 μm), and stained with hematoxylin and eosin.The slides were scanned using an Aperio slide scanner (Leica Biosystems) and images were captured using Aperio's ImageScope.Tissues were evaluated for pathology by a board-certified veterinary pathologist.Immunohistochemical staining for Listeria using the Rb DAB Xtra Wash IHC protocol on the Leica Bond stainer, NR, anti-Listeria (BD, Cat# 223021, Lot# 2069070, assumed 1.0 mg/ml) or normal rabbit IgG at 1:5000, Bond Refine HRP detection and DAB visualization was done.

Enzyme activity in serum
ALT, AST, LDH, and CK reagents were purchased from Beckman Coulter, Inc.All enzymes were assayed using the AU480 clinical analyzer (Beckman Coulter, Inc).

Statistical analysis
Analysis was performed using GraphPad Prism software.Data are represented as mean ± SEM.One-way ANOVA and two-tailed Student's t test were used.For all tests, P<0.05 was considered significant.In Figure 1A, a single datapoint in Irgm1 -/-Ifnar -/-group (S. typhimurium) was found to be an outlier by ROUT (Q=1%) test and was excluded from analysis.

Study approval
Animals were used in accordance with the Animal Welfare Act and the US Public Health Service Policy on Humane Care and Use of Laboratory Animals.Experiments were reviewed by the Animal Care and Use Committee of the National Institute of Environmental Health Sciences (NIEHS) and the National Institute of Allergy and Infectious Diseases (NIAID).