Gasdermin-D-dependent IL-1α release from microglia promotes protective immunity during chronic Toxoplasma gondii infection

Microglia, resident immune cells of the CNS, are thought to defend against infections. Toxoplasma gondii is an opportunistic infection that can cause severe neurological disease. Here we report that during T. gondii infection a strong NF-κB and inflammatory cytokine transcriptional signature is overrepresented in blood-derived macrophages versus microglia. Interestingly, IL-1α is enriched in microglia and IL-1β in macrophages. We find that mice lacking IL-1R1 or IL-1α, but not IL-1β, have impaired parasite control and immune cell infiltration within the brain. Further, we show that microglia, not peripheral myeloid cells, release IL-1α ex vivo. Finally, we show that ex vivo IL-1α release is gasdermin-D dependent, and that gasdermin-D and caspase-1/11 deficient mice show deficits in brain inflammation and parasite control. These results demonstrate that microglia and macrophages are differently equipped to propagate inflammation, and that in chronic T. gondii infection, microglia can release the alarmin IL-1α, promoting neuroinflammation and parasite control.


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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Pilot studies and power analysis determined that quantification of immune cell populations by flow cytometry requires 6 animals to achieve a power of 0.8. Where possible, 6 animals per group was the goal, though controlled groups often were not able to reach 6. Thus, we combined experiments statistically using a Randomized Block ANOVA to correct for any effect of experiment date.
No data were excluded from the analysis.
All major phenotypes were replicated at least 2-3 times (different animals and different infections). Trends were consistent across experiments.
For experiments using naive and infected wild-type mice, different cages of mice were randomly assigned to groups. For experiments involving treatment with chemical inhibitors on ex vivo samples, wells were randomly assigned to treatment and samples deriving from different animals were equally represented in each treatment group. For experiments involving genetic knockout mice, mice groups were defined by genotype.
The investigator was blinded to sample identity during counting of parasite cysts, which was determined to be the measure most likely to be affected by unintended bias. The investigator was not blinded during tissue harvest or processing, but all samples were handled in the same way. The investigator was not blinded during acquisition or analysis of flow cytometry data, but the same gates were applied to all samples in a given experiment. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
The study did not involve field-collected samples.
Animal protocol was approved by the Institutional Animal Care and Use Committee at the University of Virginia.
Tissue processing: Immediately after sacrifice mice were perfused with 30 mL of cold 1X PBS. Brains and spleens were harvested and put into cold complete RPMI media (cRPMI) (10% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% non-essential amino acids, and 0.1% 2-ME). If peritoneal lavage fluid was collected, prior to perfusion, 5 mL of cold 1X PBS was injected through the intact peritoneal membrane with a 26-gauge needle, and removed with a 22-gauge needle. If serum was collected, blood from the heart was collected and allowed to clot at 4C overnight to separate serum. After harvest, brains were minced with a razor blade, passed through an 18-gauge needle, and then enzymatically digested with 0.227 mg/mL collagenase/dispase and 50 U/mL DNase (Roche) at 37C for 45 minutes. After digestion, brains homogenate was passed through a 70m filter (Corning) and washed with cRPMI. To remove myelin from samples, filtered brain homogenate was then resuspended with 20 mL of 40% Percoll and spun at 650 x g for 25 minutes. Myelin was aspirated, samples were washed with cRPMI, and then resuspended in cRPMI. Spleens were mechanically homogenized and passed through a 40 um filter (Corning). Samples were washed with cRPMI and then resuspended in 2 mL of RBC lysis buffer (0.16 M NH4Cl) for 2 minutes. Cells were then washed with cRPMI and then resuspended. Peritoneal lavage fluid was washed with cRPMI, pelleted and resuspended. Flow Cytometry and cell sorting: Single cell suspensions from tissue samples were plated in a 96-well U-bottom plate. Cells were initially incubated with 50 uL Fc block (1 ug/mL 2.4G2 Ab (BioXCell), 0.1% rat gamma globulin (Jackson ImmunoResearch)) for 10 minutes at room temperature. Cells were then surface stained with antibodies and a Live/Dead stain for 30 minutes at 4 degrees C. After surface staining, cells were washed with FACS buffer (0.2% BSA and 2 mM EDTA in