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 (% cytotoxicity) was measured by LDH 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 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 (% cytotoxicity) was measured by LDH 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 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 (% cytotoxicity) was measured by LDH 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 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 (% cytotoxicity) was measured by LDH 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, B), and CASP1-/- (A, B, C) THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. (A) Cells were then infected WT S. Typhimurium (A) at an MOI = 20. At 1 hpi, cells were left untreated or treated with 500 µM CHQ for 1 hour. Then, at 2 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. (B, C) Cells were then infected WT S. Typhimurium (B) or ΔsipB S. Typhimurium (C) 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. (D,E) 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). (D) 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 dot represents one infected cell. 150 total infected cells were scored for each genotype. (E) 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).

Caspase activity promotes the control of Salmonella replication within human macrophages.

WT THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. One hour prior to infection, cells were treated with 20 μM of the pan-caspase inhibitor Z-VAD(OMe)-FMK, 25 μM of the caspase-1 inhibitor Ac-YVAD-cmk, or DMSO as a vehicle control. Then, cells were infected with PBS (Mock), WT S. Typhimurium (A, B, C), or WT S. Typhimurium constitutively expressing GFP (D) 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) Cells were fixed at 6 hpi and stained for DAPI to label DNA (blue). 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. Bars represent the mean for each condition, and error bars represent the standard deviation of triplicate wells from one experiment. ns – not significant, ***p < 0.001, ****p < 0.0001 by Dunnett’s multiple comparisons test. Data shown are representative of at least three independent experiments.

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 at an MOI = 20. (A, B, C) Release of IL-18, IL-1α, and TNF-α into the supernatant were measured by ELISA at 6 hpi. (D, E) Cells were lysed at 1 hpi and 6 hpi, and bacteria were subsequently plated to calculate CFU. (D) CFU/well at 1 hpi. (E) CFU/well at 6 hpi. Bars represent the mean for each genotype, and error bars represent the standard deviation of triplicate wells from one experiment. ns – not significant, ***p < 0.001, ****p < 0.0001 by Dunnett’s multiple comparisons test. Data shown are representative of at least three independent experiments.

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

WT and one independent clone of CASP1-/- THP-1 monocyte-derived macrophages were left unprimed for 16 hours. Cells were then infected with WT S. Typhimurium at an MOI = 20. Cells were lysed at 1 hpi and 6 hpi, and bacteria were subsequently plated to calculate CFU. (A) CFU/well at 1 hpi. (B) CFU/well at 6 hpi. (C) Fold-change in CFU/well was calculated. Bars represent the mean for each genotype, and error bars represent the standard deviation of triplicate wells from one experiment. ns – not significant, **p < 0.01 by unpaired t-test. Data shown are representative of three independent experiments.

Stationary phase Salmonella does not replicate effectively in human macrophages.

WT and one independent clone of CASP1-/- THP45 1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. Cells were then infected with stationary phase WT Salmonella at an MOI = 20. Cells were lysed at 1 hpi and 6 hpi, and bacteria were subsequently plated to calculate CFU. (A) CFU/well at 1 hpi. (B) CFU/well at 6 hpi. (C) Fold-change in CFU/well was calculated. ns – not significant by unpaired t-test. Bars represent the mean for each genotype, and error bars represent the standard deviation of triplicate wells from one experiment. Data shown are representative of three independent experiments.

Gentamicin does not contribute to the control of Salmonella replication in human macrophages.

. WT and one independent clone of CASP1-/- THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. Cells were then infected with WT Salmonella at an MOI = 20. . At 30 minutes post-infection, cells were treated with either 25 μg/ml or 100 μg/ml of gentamicin. For the 25 μg/ml gentamicin condition, at 1 hpi, cells were washed extensively with RPMI to remove the gentamicin, and then the media was replaced with fresh media containing no gentamicin. For the 100 μg/ml gentamicin condition, at 1 hpi, cells were washed extensively with RPMI to remove the gentamicin, and then the media was replaced with fresh media containing 10 μg/ml gentamicin. At 1 hpi and at 6 hpi the cells were lysed, and bacteria were subsequently plated to calculate CFU. (A) CFU/well at 1 hpi. (B) CFU/well at 6 hpi. (C) Fold-change in CFU/well was calculated. ns – not significant, ***p < 0.001 by Dunnett’s multiple comparisons test. Bars represent the mean for each genotype, and error bars represent the standard deviation of triplicate wells from one experiment. Data shown are representative of three independent experiments.

Validation of CASP4 70 THP-1 clones generated with CRISPR/Cas9-mediated genome editing.

(A) Schematic representations of the CASP4 gene with exons (filled boxes) and introns (lines) The guide RNA target sequence for CASP4 is highlighted in red. (B, C) Shown are the mutations of the two alleles for CASP4 clone #2 or CASP4 clone #6 THP-1 genomic DNA by electropherogram and sequence alignment with WT THP-1 genomic DNA. The CASP4 target sequence is underlined. Nucleotide deletions are indicated by the red filled-in boxes. Nucleotide insertion or switch is marked by a red outline. Purple text represents the predicted impact of the mutation on the amino acid sequence. (D, E) qRT-PCR was performed to quantitate CASP4 mRNA levels in WT THP-1s and CASP4-/- 79 THP-1s. For the CASP4-/- 80 THP-1s, CASP4 mRNA levels were normalized to human HPRT mRNA levels and WT THP-1s. (F) Immunoblot analysis was performed on cell lysates for human CASP4 and β-actin as a loading control.

Caspase-4 is dispensable for the control of Salmonella replication within human macrophages early 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 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). 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.

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 at an MOI = 20. (A, B, C) Release of IL-18, IL-1α, and TNF-α into the supernatant were measured by ELISA at 24 hpi. (D Cells were lysed at 1 hpi and 24 hpi, and bacteria were subsequently plated to calculate CFU. CFU/well of bacteria at 24 hpi. Bars represent the mean for each genotype, and error bars represent the standard deviation of triplicate wells from one experiment. ns – not significant, *p < 0.05 by Dunnett’s multiple comparisons test. Data shown are representative of at least three independent experiments.

GSDMD-mediated pore formation promotes the control of Salmonella replication within human macrophages.

(A, B,C) WT THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. (D, E, F) Primary human monocyte-derived macrophages (hMDMs) were primed 500 ng/mL LPS for 3 hours. One hour prior to infection, cells were treated with 40 μM of disulfiram, to inhibit GSDMD pore formation, or DMSO as a vehicle control. Then, cells were infected with PBS (Mock), WT S. Typhimurium at an MOI = 20. (A, D) Release of IL-1β into the supernatant was measured by ELISA at 6 hpi. (B, E) Cell death (percentage cytotoxicity) was measured by lactate dehydrogenase release assay and normalized to mock-infected cells at 6 hpi. (C, F) Cells were lysed at 1 hpi and 6 hpi, and bacteria were subsequently plated to calculate CFU. Fold-change in CFU/well was calculated. (A, B, C) Bars represent the mean for each genotype, and error bars represent the standard deviation of triplicate wells from one experiment. Data shown are representative of at least three independent experiments. (D, E, F) The pooled results of three independent experiments using hMDMs from three different healthy human donors are shown. Each data point represents the mean of triplicate infected wells from an individual donor. ns – not significant, *p<0.05, **p< 0.01, ***p < 0.001, ****p < 0.0001 by unpaired t-test (A, B, C) or by paired t-test (D, E, F).

GSDMD promotes the control of Salmonella replication within human macrophages.

WT and GSDMD-/- 134 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 at an MOI = 20. (A, B, C) Release of IL137 18, IL-1α, and TNF-α into the supernatant were measured by ELISA at 6 hpi. (D, E) Cells were lysed at 1 hpi and 6 hpi, and bacteria were subsequently plated to calculate CFU. (D) CFU/well at 1 hpi. (E) CFU/well at 6 hpi. Bars represent the mean for each genotype, and error bars represent the standard deviation of triplicate wells from one experiment. ns – not significant, **p< 0.01, ****p < 0.0001 by Šídák’s multiple comparisons test (A, B, C) or by unpaired t-test (D, E). Data shown are representative of at least three independent experiments

Cytoprotection by glycine promotes Salmonella replication within human macrophages.

WT and NAIP-/- 146 THP-1 monocyte-derived macrophages were primed with 100 ng/mL Pam3CSK4 for 16 hours. 30 minutes prior to infection, cells were treated with 20 mM glycine, to prevent cell lysis, or distilled water, as a vehicle control. Cells were then infected with PBS (Mock) or WT S. Typhimurium at an MOI = 20. (A) Cell death (percentage cytotoxicity) was measured by lactate dehydrogenase release assay and normalized to mock-infected cells at 6 hpi. (B, C) Release of IL-1β and TNF-α into the supernatant was measured by ELISA at 6 hpi. (D, E, F) Cells were lysed at 1 hpi and 6 hpi, and bacteria were subsequently plated to calculate CFU. (D) CFU/well at 1 hpi. (E) CFU/well at 6 hpi. (F) 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. *p < 0.05, **p< 0.01, ****p < 0.0001 by Tukey’s multiple comparisons test. Data shown are representative of at least three independent experiments.

Inflammasome activation primarily controls the cytosolic population of Salmonella in primary human macrophages.

(A, B) One hour prior to infection, human monocyte-derived macrophages (hMDMs) were treated with 20 μM of the pan-caspase inhibitor Z-VAD(OMe)-FMK or DMSO as a vehicle control. Then, cells were 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). (A) The number of GFP-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. 100 total infected cells were scored for each genotype. (B) Representative images from 8 hpi are shown. Scale bar represents 10 µm. White arrows indicate cytosolic bacteria (GFP-positive). ns – not significant, ****p < 0.0001 by Tukey’s multiple comparisons test (B). Bars represent the mean for each condition, and error bars represent the standard deviation of triplicate wells from one experiment (B). Data shown are representative of two independent experiments using hMDMs from two different healthy human donors (A, B).

Characterization of Salmonella subpopulations in human macrophages.

(A, B) WT and CASP1-/- 178 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. (A) The percentage of cytosol-exposed Salmonella from the total number of bacteria was quantified in 20 infected cells per genotype. (B) Unannotated representative transmission electron micrographs are shown. (C) CASP1-/- 184 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. Unannotated representative tomogram slices are shown.