Supplementary Figure 1

IRF1 is essential for assembly of the AIM2 inflammasome by infection with F. novicida

Nature Immunology: doi:10.1038/ni.3118

(a,b) IL-1β release in unprimed bone marrow-derived macrophages (BMDMs) infected with F. novicida (MOI 100) for 20 h. (c) Induction of IRF1 expression in unprimed BMDMs stimulated with 25 U/ml of recombinant mouse IFN-β. (d,e) Confocal microscopy analysis of ASC and caspase-1 in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h or transfected with poly(dA:dT) for 5 h. Quantification of the prevalence of ASC inflammasome speck. F. novicida-infected cells: WT n=228; Irf1–/– n=550; Aim2–/– n=599; Poly(dA:dT)-transfected cells: WT n=470; Irf1–/– n=583; Aim2–/– n=838. Arrowheads indicate inflammasome specks. Scale bar indicates 10 μm. Graphs show mean and s.e.m. of two (b,c-e) or three (a) independent experiments. *P  <  0.01; **P  <  0.0001. NS, not significant. Two-tailed t-test (b) and One-way ANOVA with a Dunnett’s multiple comparisons test (a,e).

Nature Immunology: doi:10.1038/ni.3118

Supplementary Figure 2

The role of IRF1 in the induction of pro-inflammatory cytokines and IFN-β in response to infection with F. novicida.

(a) Relative gene expression of Aim2 and Il1β in BMDMs infected with F. novicida (MOI 100). (b) The levels of TNF-α, IL-6, and KC released by BMDMs infected with F. novicida (MOI 100) for 20 h. (c) Relative gene

Nature Immunology: doi:10.1038/ni.3118

expression of Il12p40 in BMDMs infected with F. novicida (MOI 100). (d) Relative gene expression of Ifnβ in BMDMs infected with F. novicida (MOI 100) for 8 h. Graphs show mean of one experiment representative of three independent experiments (a,c,d) or mean and s.e.m. of three independent experiments (b). NS, not significant. One-way ANOVA with a Dunnett’s multiple comparisons test (b).

Nature Immunology: doi:10.1038/ni.3118

Supplementary Figure 3

GBP2 and GBP5 are needed to activate the AIM2 inflammasome by infection with F. novicida.

(a,b) WT BMDMs were transfected with siRNA for 48 h and infected with F. novicida (MOI 100) for 20 h. Levels of caspase-1 activation and IL-1β release were determined. (c) The knockdown efficiency of Gbp3 and Gbp7 by real time qRT-PCR in WT BMDMs. Graphs show mean and s.e.m of three independent experiments (b). *P  <  0.05; **P  <  0.001; NS, not significant (two-tailed t-test).

Nature Immunology: doi:10.1038/ni.3118

Supplementary Figure 4

IRF1 controls bacterial replication during infection with F. novicida.

Nature Immunology: doi:10.1038/ni.3118

Immunofluorescence staining of GBP5 in unprimed BMDMs infected with GFP-expressing F. novicida for 4, 8 and 16 h. Arrowheads indicate colocalization of GBP5 and GFP-expressing bacteria. Scale bar indicates 20 μm. Data are representative of two independent experiments.

Nature Immunology: doi:10.1038/ni.3118

Supplementary Figure 5

Escape from the vacuole is essential for IRF1-dependent activation of the AIM2 inflammasome by F. novicida.

Nature Immunology: doi:10.1038/ni.3118

(a) Confocal microscopy images of ASC, caspase-1 and DNA in unprimed WT BMDMs infected with F. novicida for 20 h. Arrowheads indicate inflammasome specks. (b) Induction of IRF1, GBP2 and GBP5 expression in WT BMDMs infected with WT F. novicida or F. novicida ΔmglA (MOI 50). (c) Caspase-1 activation, IL-1β and IL-18 release and (d) cell death in unprimed BMDMs infected with WT F. novicida or F. novicida ΔmglA (MOI 100) for 20 h. (e) The levels of TNF-α, KC and IL-6 released by WT BMDMs infected with WT F. novicida or F. novicida ΔmglA (MOI 100) for 20 h. Graphs show mean and s.e.m of two (a) or three (b-e) independent experiments. *P  <  0.01; **P  <  0.0001; NS, not significant (two-tailed t-test).

Nature Immunology: doi:10.1038/ni.3118

Supplementary Figure 6

Cytosolic recognition of infection with F. novicida is mediated by cGAS and STING.

Nature Immunology: doi:10.1038/ni.3118

(a) Caspase-1 activation by F. novicida-mediated delivery of Salmonella LPS. Caspase-1 activation in BMDMs infected with F. novicida (MOI 100) for 20 h in the presence of 500 ng/ml ultrapure LPS from Salmonella minnesota R595. (b) The levels of IL-18 and cell death in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h. (c) Caspase-1 activation and the levels of IL-1β and IL-18 release in unprimed BMDMs infected with F. novicida (MOI 100) for 20 h. (d) Induction of IRF1 expression in unprimed BMDMs infected with F. novicida (MOI 50). (e) A model for the activation of the AIM2 inflammasome by F. novicida, transfected DNA and mCMV. Graphs show mean and s.e.m of two (b,d) or three (a,c) independent experiments. *P  <  0.05; **P  <  0.01; ***P  <  0.001; NS, not significant. Two-tailed t-test (b) and One-way ANOVA with a Dunnett’s multiple comparisons test (c).

Nature Immunology: doi:10.1038/ni.3118

Supplementary Table 1. siRNA sequences Gene Primer Sequences Gbp2 5’-UGA AGA AGC UGA CUG AGA A-3’ 5’-GAA GUG CAC UAU ACU CUG A-3’ 5’-GGA GCU GUG UGG UGA AUU U-3’ 5’-GGA GCU AAC UGA UCU UAU C-3’ Gbp3 5’-GAA CGA AGC AGC AUC UAU U-3’ 5’-GGA CUU UGG UGC AGA CCU A-3’ 5’-GGC CAU UGU UGG UUU AUA U-3’ 5’-CAC AAG CGA CAA ACG UUU A-3’ Gbp5 5’-CGC AGG AGU UCU AUC AUA A-3’ 5’-UGA CUG UGU UAU AAG CUA A-3’ 5’-ACU CAG AUC UUU GCA CUA G-3’ 5’-GCG AGA GGC CAU AGA AAU C-3’ Gbp7 5’-AAG AAU AGC UCA UUG GGU G-3’ 5’-CGA CCU ACG UGG AUG CUA U-3’ 5’-GGA CUA UAC CGU ACG GGA A-3’ 5’-GAC GUG CCG UGU UUA GAG A-3’  

Nature Immunology: doi:10.1038/ni.3118

Supplementary Table 2. Real time qPCR primer sequences Gene Primer Sequences Gapdh Forward: 5’-CGT CCC GTA GAC AAA ATG GT-3’ Reverse: 5’-TTG ATG GCA ACA ATC TCC AC-3’ β-actin Forward: 5’-GGC TGT ATT CCC CTC CAT CG-3’ Reverse: 5’-CCA GTT GGT AAC AAT GCC ATG T-3’ Aim2 Forward: 5’-GAT TCA AAG TGC AGG TGC GG-3’ Reverse: 5’-TCT GAG GCT TAG CTT GAG GAC-3’ Il1β Forward: 5’-GAT CCA CAC TCT CCA GCT GCA-3’ Reverse: 5’-CAA CCA ACA AGT GAT ATT CTC CAT G-3’ Il12p40 Forward: 5’-GGA ATT GTA ATA GCG ATC CTG AGC-3’ Reverse: 5’-ACA TCT ACC GAA GTC CAA TGC A-3’ Ifnβ Forward: 5’-GCC TTT GCC ATC CAA GAG ATG C-3’ Reverse: 5’-ACA CTG TCT GCT GGT GGA GTT C-3’ mGbp2 Forward: 5’-CTG CAC TAT GTG ACG GAG CTA-3’ Reverse: 5’-CGG AAT CGT CTA CCC CAC TC-3’ mGbp3 Forward: 5’-CTG ACA GTA AAT CTG GAA GCC AT-3’ Reverse: 5’-CCG TCC TGC AAG ACG ATT CA-3’ mGbp4 Forward: 5’-GGA GAA GCT AAC GAA GGA ACA A-3’ Reverse: 5’-TTC CAC AAG GGA ATC ACC ATT TT-3’ mGbp5 Forward: 5’-CTG AAC TCA GAT TTT GTG CAG GA-3’ Reverse: 5’-CAT CGA CAT AAG TCA GCA CCA G-3’ mGbp7 Forward: 5’-TCC TGT GTG CCT AGT GGA AAA-3’ Reverse: 5’-CAA GCG GTT CAT CAA GTA GGA T-3’ mGbp8 Forward: 5’-ACA TCT GTC CAT GAA CCA TGA AG-3’ Reverse: 5’-AAA CCG TGA TTC TGT CCT GCC-3’ mGbp9 Forward: 5’-ACC GGG AAT AGA CTG GGT ACT-3’ Reverse: 5’-CCG GGC CAC ACT TGT CAT A-3’    

Nature Immunology: doi:10.1038/ni.3118