Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria
- University of Miami, United States
- Cornell University, United States
Figures
Figure 1 with 4 supplements
Perforin-2 deficiency or siRNA knockdown abrogates intracellular killing of pathogenic bacteria.
(A-C) Perforin-2 knockout, heterozygous, and wild-type macrophages and neutrophils were infected with Mycobacterium species (A) PEM infected with Mycobacterium smegmatis, (B) Neutrophils infected …
Figure 1—figure supplement 1
Perforin-2 genotype reflects total amount of Perforin-2 protein produced.
Murine peritoneal macrophages (A, B) or neutrophils (C, D) were isolated from Perforin-2 wild-type (wt), Perforin-2 heterozygous (het), or Perforin-2 homozygous knockout (ko) mice. Western blot …
Figure 1—figure supplement 2
Perforin-2 dependent growth inhibition of Mtb in BMDM requires activation by LPS and IFN-γ.
BMDM were collected from Perforin-2 wild-type animals (
), Perforin-2 heterozygous animals (
), or Perforin-2 knockout animals (•). After differentiation to BMDM, macrophages were infected with …
Figure 1—figure supplement 3
Perforin-2 is present in human MDM and PMN and can be knocked down in RA treated HL60.
(A) Western blot analysis of human Perforin-2 expression from two donors for primary monocyte derived macrophages and neutrophils (PMN). In addition, the efficiency of human Perforin-2 knockdown is …
Figure 1—figure supplement 4
Human HL-60-differentiated neutrophils require Perforin-2 to eliminate pathogens.
Human HL-60-neutrophils were generated by differentiating HL-60 with RA. One day prior to the experiment, HL-60/PMN cells were transfected with either a pool of scramble (□) or human Perforin-2 …
Figure 2 with 2 supplements
Antimicrobial compounds (ROS and NO) enhance Perforin-2 mediated killing of S. typhimurium by PEM but have limited activity in the absence of Perforin-2.
(A–D) Wild-type S. typhimurium infection of PEMs isolated from either MPEG1 (Perforin-2) +/+ (A, C), or MPEG1 (Perforin-2) −/− mice (B, D). Non-filled symbols indicated MPEG1 (Perforin-2) +/+ PEMs; …
Figure 2—figure supplement 1
Nitrite and reactive oxygen production in PEMs following addition of inhibitors.
(A) PEM nitrite production following stimulation with LPS and IFN-γ and incubation with ROS (NAC) or NO (NAME) inhibitors as indicated. (B) Reactive oxygen production of PEMs after LPS or PMA …
Figure 2—figure supplement 2
Antimicrobial compounds (ROS and NO) enhance Perforin-2 mediated killing of M. smegmatis by PEM.
PEMs were isolated from either (A, C) wild-type, or (B, D) Perforin-2 knockout mice. □= MPEG1 (Perforin-2) +/+ PEMs, ■= MPEG1 (Perforin-2) −/− PEMs. Cells were incubated with NAC (blue line), NAME …
Figure 3 with 2 supplements
Perforin-2 significantly contributes to intracellular killing in non-hematopoietically derived cells.
One day prior to the infection, cells were transfected with either a pool of scramble (□) or Perforin-2 specific (■) siRNA and 14 hr prior to the infection induced with IFN-γ. (A) HUVEC cells …
Figure 3—figure supplement 1
Perforin-2 knockdown is complementable and is able to replicate endogenous Perforin-2 bactericidal function.
Murine cells were transfected with either a pool of murine Perforin-2 specific siRNA and a RFP vector control plasmid (■); a pool of scramble siRNA and a RFP vector control plasmid (□); or a pool of …
Figure 3—figure supplement 2
Perforin-2 protein expression in human primary keratinocytes after knockdown compared to human monocyte derived macrophages (MDM).
Human primary keratinocytes were transfected with a pool of scramble siRNA or human Perforin-2 specific siRNA 24 hr prior to cell collection for western blot. (A) Western blot analysis demonstrating …
Figure 4 with 3 supplements
Endogenous Perforin-2 is located in intracellular sites allowing for rapid translocation to bacteria.
(A) Schematic demonstrating proposed orientation of Perforin-2 in vesicles. (B) Fractionation results of endogenous Perforin-2 from human macrophages. (Lane L) is a post-nuclear lysate control, …
Figure 4—figure supplement 1
Perforin-2-RFP colocalizes with ER, Golgi, and early endosomes.
(A-L) We used RAW264.7 macrophages which constitutively express Perforin-2 and studied the subcellular localization of fluorescent Perforin-2. RAW264.7 macrophages were transfected with …
Figure 4—figure supplement 2
S. typhimurium infection of macrophages with Perforin-2 and DAPI localization through the cell (multiple Z-sections).
(A-F) BV2 cell line overexpressing Perforin-2-GFP infected with S. typhimurium. Images are collected 5 min post-infection. Arrows indicate extracellular S. typhimurium that has maintained the normal …
Figure 4—figure supplement 3
Perforin-2-RFP colocalizes with E. coli-GFP within minutes of infection.
BV2 microglia were transfected with Perforin-2-RFP and stimulated overnight with IFN-γ. Transfected cells were infected with E. coli-GFP for several minutes upon which the cells were fixed and …
Figure 5 with 1 supplement
Perforin-2 forms pores in bacterial surfaces after infection and in Perforin-2 overexpressed eukaryotic membranes that are visible by negative stain transmission electron microscopy (TEM).
(A, B) Electron micrograph of polymerized Perforin-2 membrane lesions from Perforin-2-GFP transfected HEK-293 cells, with Perforin-2 activated to form pores by trypsin digestion to the enriched …
Figure 5—figure supplement 1
Quantification of pores from negative stain transmission electron microscopy.
10 blinded fields were counted from the above eight conditions. Pores are defined as regions containing stain (corresponding to a ‘hole” in the surface) that is approximately 10 nm in diameter. …
Figure 6 with 1 supplement
Cleaved Perforin-2 recovered after cellular infection with bacteria.
Perforin-2 −/− MEFs were transfected with human Perforin-2-GFP or GFP induced with murine IFN-γ and infected with the extracellular bacteria MRSA or EPEC. Following infection, bacteria were …
Figure 6—figure supplement 1
Human Perforin-2 peptide antibody validation.
(A) Six peptide antibodies were generated against different regions of human Perforin-2. Two antibodies detect in the MACPF domain of Perforin-2: C252 detects in the N-terminal portion; C186 detects …
Figure 7 with 3 supplements
Perforin-2 is required for in vivo survival after MRSA epicutaneous challenge.
(A) Aggregated survival curves of 60 C57BL/6 × 129 × 1/SJV mice challenged epicutaneously with 109 MRSA. (B) Aggregated survival curves of 75,129X1/SVJ mice challenged epicutaneously with 109 MRSA. …
Figure 7—figure supplement 1
Characterization of lymphocytes in Perforin-2 knockout mice.
Frequency of CD4, CD8, and NK cells in the peripheral blood of Perforin-2 knockout and wild-type littermate mice. The phenotype of peripheral blood lymphocytes was determined by 7-color flow …
Figure 7—figure supplement 2
Weight loss curves after MRSA epicutaneous infection.
Mice were shaved, tape-stripped, weighed for preinfection baseline, and infected with 109 S. aureus. Blinded individuals weighed the animals at indicated time points. 
= Perforin-2 wild-type …
Figure 7—figure supplement 3
Epicutaneous MRSA infection Day 6 organ load.
Mice were shaved, tape-stripped, and infected with 109 S. aureus. 
= Perforin-2 wild-type animals, 
= Perforin-2 heterozygous animals, • = Perforin-2 knockout animals. (A–D) Organs from seven …
Figure 8 with 3 supplements
Perforin-2 is required for in vivo survival after orogastric S. typhimurium challenge.
(A) Aggregated survival curves of 70 C57BL/6 × 129 × 1/SJV mice challenged with 105 S. typhimurium. (B) Aggregated survival curves of 45,129X1/SVJ mice challenged with 105 S. typhimurium. (C–F) …
Figure 8—figure supplement 1
Weight loss curves after in vivo orogastric S. typhimurium challenge.
Representative weight loss curves of 15 mice in each group challenged with 105 S. typhimurium. 
= MPEG1 (Perforin-2) wild-type (+/+) animals, 
= MPEG1 (Perforin-2) heterozygous (+/−) animals, •= M…
Figure 8—figure supplement 2
Equal Colonization in feces 12 hr following 105 S. typhimurium inoculation.
Feces was collected, homogenized, serially diluted, plated, and enumerated from 30 animals of each genotype 12 hr after S. typhimurium oral-gastric inoculation. No statistical differences were …
Figure 8—figure supplement 3
Organ load 60 hr after 105 S. typhimurium oral-gastric infection.
(A–D) Organs from 10 mice were collected 60 hr after S. typhimurium infection, weighed, homogenized, serially diluted, plated, and enumerated. Samples were normalized in weight to one another. (A) …
Tables
Table 1
Murine perforin-2 expression
| Cell type: | Perforin-2 expression: |
|---|---|
| Peritoneal macrophage | Constitutive |
| Bone marrow derived macrophage (BMDM) | Constitutive |
| Bone marrow derived dendritic cell (BMDC) | Constitutive |
| BV-2 microglia cell line | Constitutive |
| Raw264.7 macrophage cell line | Constitutive |
| J774A.1 macrophage cell line | Constitutive |
| Microglia | Constitutive |
| Neutrophil (peritoneum stimulation) | Constitutive |
| Neutrophil (bone marrow) | Constitutive |
| Gamma delta (γδ) T cell (from Skin) | Constitutive |
| Gamma delta (γδ) T cell (from Gut) | Constitutive |
| Gamma delta (γδ) T cell (from Vagina) | Constitutive |
| Marginal zone B cell | Constitutive |
| Keratinocyte (Back) | Constitutive |
| Intestinal epithelial cells | Constitutive |
| Splenocytes | Constitutive |
| OT1 CD8 T cell induced with TGFβ, RA, and IL2 | Constitutive |
| OT1 CD8 T cell | Inducible |
| CD4 T cell | Inducible |
| B cell | Inducible |
| Astrocyte | Inducible |
| Neuron | Inducible |
| Cath.a neuroblastoma cell line | Inducible |
| Neuro-2A neuroblastoma cell line | Inducible |
| Adult CNS fibroblast | Inducible |
| Embryonic fibroblast | Inducible |
| NIH 3T3 fibroblast cell line | Inducible |
| Balb/c 3T3 fibroblast cell line | Inducible |
| C2C12 myoblast cell line | Inducible |
| Neonatal ventricular myocytes | Inducible |
| CMT-93 rectal carcinoma cell line | Inducible |
| CT26 colon carcinoma cell line | Inducible |
| B16-F10 melanoma cell line | Inducible |
| B16-F0 melanoma cell line | Inducible |
| MOVCAR 5009 ovarian cancer cell line | Inducible |
| MOVCAR 5447 ovarian cancer cell line | Inducible |
| LL/2 Lewis lung carcinoma cell line | Inducible |
| ED-1 lung adenocarcinoma cell line | Inducible |
-
Italics: Ex vivo primary cells utilized for analysis.
Table 2
Human peforin-2 expression
| Cell type: | Perforin-2 expression: |
|---|---|
| Monocyte derived macrophage (MDM) | Constitutive |
| Monocyte derived dendritic cell (MDC) | Constitutive |
| PBMC isolated NK cell | Constitutive |
| Polymorphonuclear granulocyte (neutrophil) | Constitutive |
| HL-60 promyelocyte cell line RA differentiated to PMN | Constitutive |
| HL-60 cell line PMA differentiated to Macrophage | Constitutive |
| Fetal keratinocyte | Constitutive |
| Adult keratinocyte | Constitutive |
| PMA differentiated Thp-1 monocyte cell line | Constitutive |
| NK-92 cell line | Constitutive |
| Normal colon biopsy | Constitutive |
| Normal skin biopsy | Constitutive |
| Umbilical endothelial cell (HUVEC) | Inducible |
| HeLa cervical carcinoma cell line | Inducible |
| A2EN endocervical epithelial cell line | Inducible |
| UM-UC-3 bladder cancer cell line | Inducible |
| UM-UC-9 bladder cancer cell line | Inducible |
| CaCo-2 colorectal carcinoma cell line | Inducible |
| HEK293 embryonal kidney cell line | Inducible |
| MIA-PaCa-2 pancreatic cancer cell line | Inducible |
| Skin fibroblast | Inducible |
| Thp-1 monocyte cell line | Inducible |
| HL-60 promyelocyte cell line | Inducible |
| OVCAR3 ovarian carcinoma cell line | Inducible |
| A549 alveolar adenocarcinoma cell line | Inducible |
| U-1752 bronchiolar epithelial cell line | Inducible |
| Jeg-3 placental choriocarcinoma cell line | Inducible |
-
Italics: Ex vivo primary cells utilized for analysis.
Additional files
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Supplementary file 1
Type I and Type II interferon increase Perforin-2 message in murine non-hematopoietic cell lines. (A–D) Select murine cell lines from Table 1 indicating qPCR delta CT (Perforin-2 normalized to GAPDH) (five experimental replicates) following Type I (Interferon-α, β stimulation), Type II (Interferon-γ stimulation), or both Type I and II (Interferon-αβγ stimulation). (A) Ovarian cancer cell line MOVCAR 5009, (B) Cath.a neuroblastoma cell line, (C) C2C12 myoblast cell line, (D) B16-F10 melanoma cell line. (E–H) Interferon stimulation corresponds with an increase in Perforin-2 protein. (E) Ovarian Cancer MOVCAR 5009 and (F) C2C12 myoblast cell line. Densitometry analysis of five experimental replicates of (G) MOVCAR 5009 or (H) C2C12. (A–D) Statistical analysis was performed with one-way ANOVA with Tukey post-hoc multiple comparisons. (G, H) Statistical analysis was performed with Student's T-test. *p < 0.05.
- https://doi.org/10.7554/eLife.06508.032
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Supplementary file 2
Type I and Type II interferon increase Perforin-2 message in human non-hematopoietic cell lines. Select human cell lines from Table 2 analyzed by qPCR demonstrating delta CT (Perforin-2 normalized to GAPDH) (five experimental replicates) after Type I (Interferon-αβ stimulation), Type II (Interferon-γ stimulation), or both Type I and II (Interferon-αβγ stimulation). (A) Primary HUVEC cells, (B) HEK293 cell line, and (C) MIA-PaCa-2 pancreatic cancer cell line. Interferon stimulation also increased human Perforin-2 protein with (D) MIA-PaCa-2 and (E) HUVEC cell lines. Densitometry analysis of five experimental replicates of (F) MIA-PaCa-2 or (G) HUVEC. (A–C) Statistical analysis was performed with one-way ANOVA with Tukey post-hoc multiple comparisons. (F, G) Statistical analysis was performed with Student's T-test. *p < 0.05.
- https://doi.org/10.7554/eLife.06508.033
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Supplementary file 3
Perforin-2 significantly contributes to intracellular killing in murine non-hematopoietically derived cells. (A–C) One day prior to the experiment, cells were transfected with either a pool of scramble (□) or murine Perforin-2 specific (■) siRNA and 14 hr prior to the experiment induced with IFN-γ. (A) MOVCAR 5009 infected with S. typhimurium, (B) CT26 infected with MRSA, or (C) C2C12 infected with M. smegmatis. The above graphs contain 5 biologic replicates, and are representative of 3 independent experiments. Statistical analysis was performed utilizing multiple T-tests with correction for multiple comparisons using the Holm-Sidak method. *p < 0.05.
- https://doi.org/10.7554/eLife.06508.034
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Supplementary file 4
Perforin-2 siRNA knockdown is efficient in both murine and human cells. (A–D) Representative blots from knockdown of selected cells from Table 1, Table 2, and Figure 3. (A, C) Human HUVEC cells representing knockdown of human Perforin-2; (B, D) Murine C2C12 myoblast cell line-demonstrating knockdown of murine Perforin-2. Cells were transfected with either a pool of human or mouse Perforin-2 specific siRNA or scramble siRNA. Cells were induced for 14 hr with IFN-γ and 24 hr post-transfection lysed for protein quantification. Statistical analysis was conducted utilizing Student's T-test. *p < 0.05.
- https://doi.org/10.7554/eLife.06508.035
