Fig 1.
S. marcescens causes PFT-dependent cell death in macrophages.
A-C) BALB/c mice were infected with 1.0 x 106 CFU of wildtype S. marcescens (Sma), S. marcescens with an isogenic deletion of shlA (ΔshlA) at the same dose, or mock infected with and equal volume of PBS (Mock). Fluorescent images show macrophages (F4/80+, green) and all nucleated lung cells (DAPI, blue) present in lung tissue taken at 48h post-infection. (D) Mean pixels per frame analysis of images captured corresponding to punctate points of F4/80 (4 images per mouse, n = 4–6 mice/cohort) as determined using ImageJ64. E) Number of F4/80 positive cells in 50 μl BALF as determined using FACs analysis. F) LDH cell death assay of primary mouse AMs, mouse AM cell line MH-S, mouse bone marrow-derived macrophages (BMDM), and the human macrophage-like cell line THP-1, following infection with Sma or ΔshlA; dashed line shows baseline LDH release from uninfected cells. G) Kinetics of Sma cytotoxicity in MH-S cells as determined by LDH. H) LDH release assay from MH-S cells infected with S. marcescens mock or pretreated with different concentrations of dipalmitoylphosphatidylcholine (DPPC). I) MH-S cells pretreated with Cytochalasin D were infected with Sma and cytotoxicity measured using LDH release assay. For multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001. Data from in vitro experiments are representative of ≥3 separate experiments each with 8 biological replicates.
Fig 2.
RIP1 and not caspases are required for S. marcescens induced macrophage cell death.
A) LDH release assay of MH-S macrophages infected with a high and low MOI of S. marcescens (Sma) following mock or pretreatment with the general caspase inhibitor Z-VAD-FMK (GI), caspase-1 inhibitor Z-WEHD-FMK (CI) caspase-3 inhibitor Z-DEVD-FMK (C3), caspase-8 inhibitor Z-YVAD-FMK (C8), and caspase-9 inhibitor Z-LEHD-FMK (C9), all at a 10 μM concentration. Percent positive cells for B) caspase-1 and C) caspase-3/7 activity 2h after Sma infection following FLICA staining and measured by FACs analyses. For controls, cells with nigericin induced pyroptosis (Ni +LPS; lipopolysaccharide at 10 ng/mL for 4h then Ni at 10 μM for 6 h) and cycloheximide (CHX; 2,000 μg/ml) induced apoptosis were measured, respectively. D) LDH release assay of MH-S macrophages infected with Sma at an MOI of 1 following their mock or pretreatment with necrostatin-1s, -1, -5, or -7 at a concentration of 100 μM. E) LDH release assay of MH-S macrophages infected with Sma after pretreatment with necrostatin-5 at increasing concentrations. F) LDH release assay of MH-S macrophages treated with CHX after pretreatment with necrostatin-5 (Nec 5; 100 μM) or ZVAD (10 μM). For multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001. Data are representative of ≥3 separate experiments, each with 8 biological replicates.
Fig 3.
PFT-producing pathogens and pneumolysin induce RIP1 dependent macrophage cell death.
A) LDH release assay of MH-S, BMDM, and THP-1 macrophages infected either with S. pneumoniae (Spn) or an isogenic mutant deficient in pneumolysin (Δply) both at MOI of 100. B) LDH release assay of MH-S macrophages infected with S. aureus (Sau) (MOI 10), L. monocytogenes (Lmo) (MOI 10), S. pneumoniae (MOI 100), UPEC (MOI 100), A. baumannii (MOI 100), and F. tularensis novicida (MOI 100). Black bars show LDH release when cells were pretreated with necrostatin-5 (100 μM), dashed line shows base line LDH release from uninfected cells. Percent positive cells for C) caspase-1 and D) caspase-3/7 activity 2h after UPEC, Spn and Lmo infection, or recombinant pneumolysin (rPly) challenge following FLICA staining and measurement by FACs analyses. For controls pyroptosis (Ni + LPS) and apoptosis (CHX) induced cells were included in the analyses. E) LDH release assay of MH-S macrophages treated with different concentrations of recombinant pneumolysin (rPly) following mock or pretreatment with necrostatin-5 at 100 μM. For multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001. Data are representative of ≥3 separate experiments, each with 8 biological replicates.
Fig 4.
RIP3 is required for PFT induced necroptosis.
A) LDH release assay of THP-1 cells transfected with siRNAs targeting RIP3, caspase-1 (Casp-1), caspase-8 (Casp-8) and a scramble control, infected with (A) Sma or challenged with B) recombinant pneumolysin (rPly). C) LDH release assay of MH-S macrophages infected with Sma at an MOI of 0.1 mock or following pretreatment with RIP3 inhibitor GSK’872. D) LDH release assay of BMDM from WT-C57BL/6, Caspase 1/11 KO, Caspase 3 KO and RIP3 KO mice, infected with Sma at an MOI of 0.5 or 0.1. E) LDH release of BMDM from wildtype mice, Caspase 1/11 (Casp1/11) KO, RIP3 KO, and BMDM from wildtype mice pretreated with necrostatin-5 (100μM) following their infection with S. aureus (MOI 10), L. monocytogenes (MOI 10) and S. pneumoniae (MOI 100). F) LDH release assay of BMDM from wild type C57BL/6 or RIP3 KO mice following their challenge with recombinant pneumolysin (rPly). G) LDH release assay of MH-S macrophages challenged with rPly with and without pretreatment with GSK’872 (10 μM). H) Alveolar macrophage numbers in BALF of RIP3 KO mice 18h after intratracheal infection with S. marcescens. Mann-Whitney tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05. Data are representative of ≥3 separate experiments, each with 8 biological replicates.
Fig 5.
MLKL is required for PFT induced necroptosis.
Western blot for pMLKL in MH-S whole cell lysates following their challenge with A) S. marcescens (Sma), ΔshlA, S. pneumoniae (Spn) and Δply and B) S. aureus (Sau), L. monocytogenes (Lmo) and UPEC. C) Immunofluorescence for MLKL (Green) and lipid membrane (Red) in uninfected and Sma infected MH-S cells. Nuclei were stained with DAPI (blue). D) LDH release assay of infected THP-1 macrophages following mock or pretreatment with the MLKL inhibitor necrosulfonamide (100μM). MOIs were: Sma = 1,Spn = 100, Sau = 10, UPEC = 100. E) LDH release assay of THP-1 macrophages following challenge with recombinant pneumolysin (rPly), with and without pretreatment with necrosulfonamide (NSA, 100μM, black bars). LDH release assay of F) Sma infected or G) rPly challenged THP-1 macrophages transfected with siRNA targeting MLKL. As control scrambled (Scram) siRNA was used. H) Alveolar macrophage numbers in BALF of MLKL KO mice 18h after intratracheal infection with S. marcescens. Student t-tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05. Data are representative of ≥3 separate experiments, during LDH assays each with 8 biological replicates.
Fig 6.
Inflammasome components can be linked to PFT-induced necroptosis in macrophages.
LDH release assay of BMDM from C57BL/6 wildtype (WT), NLRP3 KO, ASC KO and MyD88 KO mice, infected with A) S. marcescens (Sma; MOI = 0.5), B) S. aureus (Sau; MOI = 10), C) UPEC (MOI = 100), D) S. pneumoniae (Spn; MOI = 100) and challenged with E) recombinant pneumolysin (rPly; 100 μg/ml). F) Levels of IL-1β detected by ELISA in supernatants from MH-S macrophages infected with Sma or ΔshlA. Necrostatin-5 (Nec-5) was added at 100μM. G) Levels of IL-1β detected in supernatants from WT, NLRP3 KO, ASC KO BMDMs infected with Sma, Sau, UPEC and Spn and uninfected controls. For multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05. Data are representative of ≥3 separate experiments, each with 8 biological replicates.
Fig 7.
Ion loss is sufficient to induce macrophage necroptosis.
A) LDH release cytotoxicity assay of MH-S macrophages mock or pretreated with glycine (2 mM) then infected with S. marcescens. B) LDH release cytotoxicity assay of MH-S macrophages following their treatment with ionomycin (io; 20μM), nigericin (ni; 20μM) and gramicidin (gr; 20μM) for 6 hours, with and without pretreatment with necrostatin-5 (N5; 100μM). C) LDH release cytotoxicity assay of THP-1 macrophages treated with io, ni, and gr, with and without pretreatment with necrosulfonamide (NSA; 100μM). D) Western blot for pMLKL in whole cell lysates from MH-S macrophages following their treatment with io, ni, gr. Mann-Whitney U tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001. Data are representative of ≥3 separate experiments, each with 8 biological replicates (during LDH assays).
Fig 8.
Mitochondrial damage and ATP depletion are required during PFT-induced necroptosis.
A) Cytochrome C (cyt C) levels in culture supernatants of MH-S cells infected with S. marcescens (Sma), S. pneumoniae (Spn), ΔshlA, and Δply. B) ATP levels in MH-S cell lysates following their infection with Sma, ΔshlA, and recombinant pneumolysin (rPly) or heat-inactivated rPly (HI-rPly) as measured using a luminescent cell viability assay. LDH release assay of MH-S cells infected with Sma following mock or pretreatment with C) soluble ATP (10 μg/mL), D) CoQ10 (100μM), necrostatin-5 (N5; 100μM), N5/C10 (100μM each), or E) resveratrol (Resv; 100μM). In vitro experiments were done at an MOI of 1 and 100 for Sma and Spn, respectively. Mann-Whitney U tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001. Data are representative of ≥3 separate experiments; during LDH assay each with 8 biological replicates.
Fig 9.
Reactive oxygen species mediate PFT induced necroptosis in macrophages.
A) Levels of ROS in MH-S cells infected with S. marcescens (Sma), ΔshlA, S. pneumoniae (Spn), or Δply as measured using the indicator H2-DCF. B) LDH release cytotoxicity assay of MH-S macrophages infected with Sma following pretreatment with the general ROS inhibitor DPI, complex I ROS inhibitor rotenone, complex II ROS inhibitor TTFA, and complex III ROS inhibitor antimycin at the designated concentrations. C) LDH release cytotoxicity assay of MH-S macrophages infected with Sma, S. aureus (Sau), UPEC, Spn, or challenged with recombinant pneumolysin (rPly) following mock or pretreatment with n-acetyl cysteine (NAC; 100 μM), DPI (10 μM), and with catalase (10μM), respectively. (D) LDH release cytotoxicity assay of MH-S macrophages treated with ROS inducer paraquat (pqt) at a concentration of 500uM for 12h following mock or pretreatment with necrostatin-1 (N1, 100μM), CoQ10 (100μM) or DPI (10 μM). Mann-Whitney U tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001. Data are representative of ≥3 separate experiments, each with 8 biological replicates.
Fig 10.
Inhibition of RIP1 or MLKL decreases morbidity and mortality during S. marcescens hemorrhagic pneumonia.
BALB/c mice were infected intratracheally with Sma at high dose (5.0 x 106 CFU) or low dose (1.0 x 106 CFU). A) CFU recovered from the BALF of BALB/c mice pretreated with PBS or clodronate liposomes 24h after infection with low dose Sma. Each symbol represents an individual mouse. B) Survival of mice infected with the high dose of Sma that received intraperitoneal pre-treatment with CoQ10, necrostatin-5 (N5), or N5 along with CoQ10 (N5/C10). Mice received 100 μl of a 100 μM solution of each drug intraperitoneally from time of challenge every 4h for the first 12h post-infection. C) Percent weight change and D) airway bacterial burden of Sma infected mice (n = 4–5/cohort) (low dose) when treated with PBS or the described N5/C10 therapy. The concentration of E) total leukocytes, F) neutrophils, and G) monocytes (y-axis is log scale) in Hema-3-stained cytospins of BALF from Sma infected mice (low dose) that were received mock or N5/C10 therapy. H) Airway bacterial burden, I) number of F4/80 positive cells in 50 μl, J) IL-1β levels, and K) TNFα levels in BALF from Sma infected mice (low dose) that received treatment with PBS, GW806742X (GW80; 100 μl of 100 μM), GSK’872 (100 μl of 10 μM) or ZVAD (100 μl of 10 μM). Mann-Whitney U tests were applied for two-group comparisons, for multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001. Data are representative of ≥3 separate experiments, each with 8 biological replicates.