SUPPLEMENTARY INFORMATION

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Reginald Bell
5 years ago
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doi:10.1038/nature11419 Supplementary Figure 1 Schematic representation of innate immune signaling pathways induced by intracellular Salmonella in cultured macrophages.

1 doi: /nature11419 Supplementary Figure 1 Schematic representation of innate immune signaling pathways induced by intracellular Salmonella in cultured macrophages. a, During the infection Salmonella LPS activates TLR4, thus inducing pro-il-1" expression via MyD88 and contributing to pro-caspase-11 expression via both signaling adaptors. In addition, a non-tlr dependent pathway contributes to pro-caspase-11 induction (grey arrows). b, Trif also activates IRF3 and initiates the production of type-i-interferon. Paracrine and/or autocrine signaling by IFN-" through the type-i-ifn receptor (IFN#R) results in the expression of a interferon-inducible activator of caspase-11, which is required for the assembly or activity of non-canonical inflammasomes. c, Salmonella establishes itself in its intracellular niche, a vacuolar compartment called Salmonella Containing Vacuole (SCV), requiring the expression and activity of the T3SS encoded by the S. Typhimurium Pathogenicity Island 2 (SPI-2). Intracellular Salmonella subsequently activate a multitude of inflammasomes: Flagellin secreted by the SPI-2 T3SS activates NLRC4 and induces caspase-1-dependent pyroptosis and cytokine maturation. Intracellular Salmonella also induce non-canonical cell death via caspase-11, which requires the expression of an interferon-inducible activator (b). Caspase-11 also triggers the activation of a canonical NLRP3-dependent signaling pathway leading to cytokine maturation. 1

2 RESEARCH Supplementary Figure 2 Intracellular S. Typhimurium induces cell death through caspase-11 and the NLRC4-caspase-1 axis. a, b, IL-1" secretion, LDH release and immunoblots for processed caspase-1, caspase-11 and IL-1 " released from unprimed BMDMs infected with wild-type (wt) S. Typhimurium, SPI-2-deficient (#SPI-2) or flagellin-deficient (#flag) strains grown to stationary phase for 17 h. c, d, LDH release at 17 h or during timecourse (0-16 h) from unprimed BMDMs infected with the indicated strains. Graphs show the mean ± s.d. of quadruplicate wells and are representative of three (a, b) and two (c, d) independent experiments. $ P < 0.01 as compared to WT macrophages. 2

3 RESEARCH Supplementary Figure 3 Cell death and cytokine release require live S. Typhimurium. Unprimed primary WT bone-marrow derived macrophages were infected with untreated or heat-killed (HK), UV-killed (UVK), Paraformaldehyde-treated (PFAK) or 70% Ethanol treated (EK) S. Typhimurium (grown to stationary phase) for 17 hours. IL-1" release (± s.d.) was measured by ELISA. Cell death (± s.d.) was determined by LDH release. Data shown are representative of three independent experiments. 3

RESEARCH Supplementary Figure 4 Activity of the NLRP3 inflammasome triggered by S. Typhimurium infection requires both caspase-1 and caspase-11.

4 RESEARCH Supplementary Figure 4 Activity of the NLRP3 inflammasome triggered by S. Typhimurium infection requires both caspase-1 and caspase-11. Unprimed primary bone-marrow derived macrophages were infected with the indicated S. Typhimurium strains for 17 h. IL-1" release (± s.d.) was measured by ELISA. Immunoblotting was used to measure pro- and cleaved forms of caspase-1, caspase-11, IL-1" and IL-18 in the culture supernatant and cell lysates. Loading was controlled by blotting for "-actin. Data shown are representative of at least two independent experiments. 4

5 RESEARCH Supplementary Figure 5 Assembly of ASC foci by NLRP3 requires preceding caspase-11 activation. a, Fluorescence microscopy of primary murine macrophages from the indicated genotypes, infected with S. Typhimurium "SPI-2 for 17 h. Cells were stained for ASC, Caspase-1 and DNA (with DAPI). Arrowhead point to ASC foci. b, Percentage of ASC foci in (a). Images (original magnification, 63) and quantification are a representative of three independent experiments with a mean of 100 ASC foci counted in each experiment. Error bars represent the mean SD of triplicate experiments. Scale bars, 10 µm. 5

RESEARCH Supplementary Figure 6 Pro-caspase-11 protein levels in different knockout macrophages at selected timepoints or during timecourse.

6 RESEARCH Supplementary Figure 6 Pro-caspase-11 protein levels in different knockout macrophages at selected timepoints or during timecourse. a, c, Unprimed primary bone-marrow derived macrophages were infected with S. Typhimurium "flag mutant for 8, 12 and/or 16 hours. b, Induction of pro-caspase-11 and pro-il-1# expression by immunoblot in unprimed BMDMs infected with "flag S. Typhimurium. Immunoblotting was used to measure pro-forms of caspase-11 and IL-1# in cell lysates. Loading was controlled by blotting for pro-caspase-1 and #-actin. Graphs show the mean ± s.d. of quadruplicate wells and are representative of two independent experiments. 6

7 RESEARCH Supplementary Figure 7 Exogenous IFN- restores caspase-11 activation in MyD88 -/- /Trif -/- macrophages. Caspase-11 and caspase-1 processing were examined in unprimed macrophages infected with S. Typhimurium "flag for 17 hours. Cells were left untreated or treated with the indicated amount of recombinant murine IFN-# at 2 hours post-infection for the remainder of the experiment. Immunoblotting was used to measure pro- and cleaved forms of caspase-1, caspase-11 in the culture supernatant and cell lysates. Supplementary Figure 8 Bacterial burden in Nlrp3 -/- /Nlrc4 -/- mice phenocopies Casp-1 -/- mice. WT, Casp-1 -/- /Casp-11 -/-, Nlrp3 -/- /Nlrc4 -/- mice were infected orally with 2.5x10 7 wild-type S. Typhimurium for 5 days. Bacterial counts were determined by plating serial dilutions. Graphs show ± s.e.m of 6-10 mice per genotype and are representative of three independent experiments. ", P < Dashed line: detection limit. 7

RESEARCH Supplementary Figure 9 Immunofluorescence analysis of tissue sections from mice of different genotypes infected with S. Typhimurium.

8 RESEARCH Supplementary Figure 9 Immunofluorescence analysis of tissue sections from mice of different genotypes infected with S. Typhimurium. WT, Casp-11 -/-, Casp-1 -/- /Casp-11 -/- and Casp-1 -/- (Casp-1 -/- /Casp-11 tg ) mice were infected orally with 1x10 8 wild-type S. Typhimurium for 4 days. a, Immunostaining of liver tissue sections, stained with a Salmonella-specific antibody (green) and for Phalloidin (actin, red) to visualize cell bodies. Images were taken at 20x original magnification. Scale bars are 30 µm. Stars indicate extracellular Salmonella filling sinusoids. b, Close-up views of boxed areas in (a) taken at 63x original magnification. 8

RESEARCH Supplementary Figure 10 Extracellular Salmonella in Casp-1-/- mice. a, Histopathology of livers from Casp-1-/- (Casp-1-/-/Casp-11tg) mice infected with 1x108 wild-type S.

9 RESEARCH Supplementary Figure 10 Extracellular Salmonella in Casp-1-/- mice. a, Histopathology of livers from Casp-1-/- (Casp-1-/-/Casp-11tg) mice infected with 1x108 wild-type S. Typhimurium for 4 days. Tissue sections stained with H&E or Gram stain. Original magnification 10x (overview) or 100x (close-up of boxed areas). Stars indicate mats of extracellular bacteria, arrowheads point out single S. Typhimurium bacteria, arrows indicate the border of large necrotic areas surrounding typhoid nodules. b, In vivo gentamicin protection assay. Comparison of bacterial burden in the spleen of WT, Casp1-/-/Casp-11-/-, Casp-1-/- (Casp-1-/-/Casp-11tg) and Casp-11-/- mice treated with gentamicin or a vehicle control. Animals were infected orally with 1x108 wild-type S. Typhimurium for 4 days and injected intra-peritoneally with 1 mg gentamicin at 48 h, 24 h and 12 h before euthanizing. Bacterial numbers in the spleen were determined by plating serial dilutions. Circles indicate control animals, boxes gentamicin treated animals. Statistical significance was determined using the unpaired Mann-Whitney U test. ", P < Dashed lines represent the detection limit of the CFU counts. W W W. N A T U R E. C O M / N A T U R E 9

Supplementary Figure 1. AnnexinV FITC and Sytox orange staining in wild type, Nlrp3 /, ASC / and casp1/11 / TEC treated with TNF /CHX.

Supplementary Figure 1. AnnexinV FITC and Sytox orange staining in wild type, Nlrp3 /, ASC / and casp1/11 / TEC treated with TNF /CHX. Phase contrast and widefield fluorescence microscopy (20x magnification).