Caspase-1 promotes the control of Salmonella replication within human macrophages.

WT and two independent clones of CASP1-/- THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. Cells were then infected with PBS (Mock), WT S. Typhimurium (A, B, C), or WT S. Typhimurium constitutively expressing GFP (D,E) at an MOI = 20. (A) Release of IL-1β into the supernatant was measured by ELISA at 6 hpi. (B) Cell death (percentage cytotoxicity) was measured by lactate dehydrogenase release assay and normalized to mock-infected cells at 6 hpi. (C) Cells were lysed at 1 hpi and 6 hpi, and bacteria were subsequently plated to calculate CFU. Fold-change in CFU/well was calculated. (D, E) Cells were fixed at 6 hpi and stained for DAPI to label DNA (blue). (D) The number of bacteria per cell at 6 hpi was scored by fluorescence microscopy. Each small dot represents one infected cell. 150 infected cells were scored for each genotype. (E) Representative images are shown. Scale bar represents 10 µm. Bars represent the mean for each genotype, and error bars represent the standard deviation of triplicate wells from one experiment (A, B, C, D). ***p < 0.001, ****p < 0.0001 by Dunnett’s multiple comparisons test (A, B, C, D). Data shown are representative of at least three independent experiments.

Caspase-4 contributes to the control of Salmonella replication within human macrophages later during infection.

WT and two independent clones of CASP4-/- THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. Cells were then infected with PBS (Mock), WT S. Typhimurium (A, B, C), or WT S. Typhimurium constitutively expressing GFP (D, E) at an MOI = 20. (A) Release of IL-1β into the supernatant was measured by ELISA at 24 hpi. (B) Cell death (percentage cytotoxicity) was measured by lactate dehydrogenase release assay and normalized to mock-infected cells at 24 hpi. (C) Cells were lysed at 1 hpi and 24 hpi, and bacteria were subsequently plated to calculate CFU. Fold-change in CFU/well was calculated. (D, E) Cells were fixed at 24 hpi and stained for DAPI to label DNA (blue). (D) The number of bacteria per cell at 24 hpi was scored by fluorescence microscopy. Each small dot represents one infected cell. 150 infected cells were scored for each genotype. (E) Representative images are shown. Scale bar represents 10 µm. Bars represent the mean for each genotype, and error bars represent the standard deviation of triplicate wells from one experiment (A, B, C, D). ns – not significant, *p < 0.05 by Dunnett’s multiple comparisons test (A, B, C, D). Data shown are representative of at least three independent experiments.

GSDMD promotes the control of Salmonella replication within human macrophages.

WT and GSDMD-/- THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. Cells were then infected with PBS (Mock), WT S. Typhimurium (A, B, C), or WT S. Typhimurium constitutively expressing GFP (D,E) at an MOI = 20. (A) Release of IL-1β into the supernatant was measured by ELISA at 6 hpi. (B) Cell death (percentage cytotoxicity) was measured by lactate dehydrogenase release assay and normalized to mock-infected cells at 6 hpi. (C) Cells were lysed at 1 hpi and 6 hpi, and bacteria were subsequently plated to calculate CFU. Fold-change in CFU/well was calculated. (D, E) Cells were fixed at 6 hpi and stained for DAPI to label DNA (blue). (D) The number of bacteria per cell at 6 hpi was scored by fluorescence microscopy. Each small dot represents one infected cell. 150 infected cells were scored for each genotype. (E) Representative images are shown. Scale bar represents 10 µm. Bars represent the mean for each genotype, and error bars represent the standard deviation of triplicate wells from one experiment (A, B, C, D). **p< 0.01, ***p < 0.001, ****p < 0.0001 by Šídák’s multiple comparisons test (A) or by unpaired t-test (B, C, D). Data shown are representative of at least three independent experiments.

NINJ1 contributes to the control of Salmonella replication within human macrophages.

WT THP-1 monocyte-derived macrophages were treated with siRNA targeting a control scrambled siRNA or siRNA targeting NINJ1 for 72 hours prior to infection. Cells were primed with 100 ng/mL Pam3CSK4 for 16 hours. Cells were then infected with PBS (Mock) or WT S. Typhimurium at an MOI = 20. (A) Release of IL-1β into the supernatant was measured by ELISA at 6 hpi. (B) Cell death (percentage cytotoxicity) was measured by lactate dehydrogenase release assay and normalized to mock-infected cells at 6 hpi. (C, D, E) Cells were lysed at 1 hpi and 6 hpi, and bacteria were subsequently plated to calculate CFU. (C) CFU/well at 1 hpi. (D) CFU/well at 6 hpi. (E) Fold-change in CFU/well was calculated. Bars represent the mean for each condition. Error bars represent the standard deviation of triplicate wells from one experiment. ns – not significant, *p< 0.05, **p< 0.01 by unpaired t-test. Data shown are representative of at least three independent experiments.

Inflammasome activation primarily controls cytosolic Salmonella replication in human macrophages.

WT, NAIP-/- (A), and CASP1-/- (A, B) THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. Cells were then infected with PBS (Mock) or WT S. Typhimurium (A) or ΔsipB S. Typhimurium (B) at an MOI = 20. At 5 hpi, cells were left untreated or treated with 500 µM CHQ for 1 hour. Then, at 6 hpi, cells were lysed, and bacteria were subsequently plated to calculate CFU. CFU/well of vacuolar (CHQ-sensitive) and cytosolic (CHQ-resistant) bacteria were calculated from the total bacteria. (C,D) WT, NAIP-/-, and CASP1-/- THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. Cells were then infected with PBS (Mock) or WT S. Typhimurium constitutively expressing mCherry and harboring the GFP cytosolic reporter plasmid, pNF101, at an MOI = 20. Cells were fixed at 8 hpi and stained for DAPI to label DNA (blue). (C) The number of GFP-positive, mCherry-positive bacteria (cytosolic) per cell and the number of GFP-negative, mCherry-positive bacteria (vacuolar) per cell were scored by fluorescence microscopy. Each small dot represents one infected cell. 150 total infected cells were scored for each genotype. (D) Representative images from 8 hpi are shown. Scale bar represents 10 µm. White arrows indicate cytosolic bacteria (GFP-positive, mCherry-positive). Bars represent the mean for each genotype (A, B). Error bars represent the standard deviation of triplicate wells from one experiment (A, B). ns – not significant, *p< 0.05, ****p < 0.0001 by Dunnett’s multiple comparisons test (A) or by Tukey’s multiple comparisons test (B). Data shown are representative of at least three independent experiments (A, B, C).

Inflammasome activation modulates the cytosolic exposure of Salmonella in human macrophages.

(A, B) WT and CASP1-/- THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. Cells were then infected with WT S. Typhimurium at an MOI = 20. At 8 hpi, cells were fixed and collected to be processed for transmission electron microscopy. Representative transmission electron micrographs are shown. Salmonella (green) in an SCV (maroon) with cytosolic or vacuolar clearance (yellow). Scale bar represents 1 µm [A(i), B(i)] or 400 nm [A(ii), B(ii)]. (A) WT THP-1s, (ii) is an inset from (i). White arrows indicate vacuolar bacteria. (B) CASP1-/- THP-1s, (ii) is an inset from (i). White arrows indicate vacuolar bacteria, cyan arrows indicate partially cytosolic bacteria, and black asterisk indicates a fully cytosolic bacterium. (C) CASP1-/- THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. Cells were then infected with WT S. Typhimurium at an MOI = 20. At 8 hpi, cells were fixed and collected to be processed for transmission electron microscopy. Representative tomogram slices are shown, depicting Salmonella (green) in an SCV with a discontinuous vacuolar membrane (maroon).