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Neutrophils are resistant to Yersinia YopJ/P-induced apoptosis and are protected from ROS-mediated cell death by the type III secretion system.

Spinner JL, Seo KS, O'Loughlin JL, Cundiff JA, Minnich SA, Bohach GA, Kobayashi SD - PLoS ONE (2010)

Bottom Line: In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death.PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner.Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho, United States of America.

ABSTRACT

Background: The human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs) for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis) and YopP (Y. enterocolitica) rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined.

Methodology/principal findings: In this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM) and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death. PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5.

Conclusions: Our findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis.

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Role of YopJ and YopP in J774A.1 macrophage-like and human MDM cell death.Y. pestis strains with the virulence plasmid expressing YopJ (KIM5), YopP (KIM5 YopJ-YopP), or lacking YopJ (KIM5ΔyopJ), along with Y. enterocolitica 8081v and the virulence plasmid–deficient strain (KIM6) were grown at 37°C to induce expression of the TTSS and Yops. Yersinia strains were combined with J774A.1 and HMDM cells and compared to detergent lysed controls as described in Materials and Methods. The percentage of J774A.1 cell death following incubation alone or with Yersinia strains at the indicated time was determined by (A) LDH release into the supernatant and by (B) EthD-1 staining of J774A.1 cells. HMDM cell death was determined by measuring (C) LDH release and (D) EthD-1 uptake into HMDMs. The results are expressed as the mean ± SEM of at least three experiments. *, represents difference from J774A.1 or HMDM controls (P<0.05).
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pone-0009279-g001: Role of YopJ and YopP in J774A.1 macrophage-like and human MDM cell death.Y. pestis strains with the virulence plasmid expressing YopJ (KIM5), YopP (KIM5 YopJ-YopP), or lacking YopJ (KIM5ΔyopJ), along with Y. enterocolitica 8081v and the virulence plasmid–deficient strain (KIM6) were grown at 37°C to induce expression of the TTSS and Yops. Yersinia strains were combined with J774A.1 and HMDM cells and compared to detergent lysed controls as described in Materials and Methods. The percentage of J774A.1 cell death following incubation alone or with Yersinia strains at the indicated time was determined by (A) LDH release into the supernatant and by (B) EthD-1 staining of J774A.1 cells. HMDM cell death was determined by measuring (C) LDH release and (D) EthD-1 uptake into HMDMs. The results are expressed as the mean ± SEM of at least three experiments. *, represents difference from J774A.1 or HMDM controls (P<0.05).

Mentions: To evaluate a role for YopJ and YopP in induction of cell death, we measured LDH release and membrane integrity via EthD-1 staining in the murine J774A.1 cell line and HMDMs following interaction with Yersinia. Incubation of J774A.1 cells or HMDMs with KIM5, Y. enterocolitica, or KIM5 YopJ-YopP resulted in YopJ- or YopP-dependent LDH release and EthD-1 staining (Fig. 1); however, HMDM cell death was delayed between 24–48 h of incubation compared to J774A.1 cells (Fig. 1c and 1d). In contrast, KIM6 or KIM5ΔyopJ incubation with J774A.1 cells or HMDMs resulted in decreased LDH release and EthD-1 staining (Fig. 1, e.g., at 24 h KIM5 and Y. enterocolitica induced 98.2±3.2% and 98.2±1.9% of J774A.1 LDH release, respectively, compared to KIM5ΔyopJ and KIM6 which induced 42.5±11.4% and 43.9±13.0% of J774A.1 LDH release, respectively).


Neutrophils are resistant to Yersinia YopJ/P-induced apoptosis and are protected from ROS-mediated cell death by the type III secretion system.

Spinner JL, Seo KS, O'Loughlin JL, Cundiff JA, Minnich SA, Bohach GA, Kobayashi SD - PLoS ONE (2010)

Role of YopJ and YopP in J774A.1 macrophage-like and human MDM cell death.Y. pestis strains with the virulence plasmid expressing YopJ (KIM5), YopP (KIM5 YopJ-YopP), or lacking YopJ (KIM5ΔyopJ), along with Y. enterocolitica 8081v and the virulence plasmid–deficient strain (KIM6) were grown at 37°C to induce expression of the TTSS and Yops. Yersinia strains were combined with J774A.1 and HMDM cells and compared to detergent lysed controls as described in Materials and Methods. The percentage of J774A.1 cell death following incubation alone or with Yersinia strains at the indicated time was determined by (A) LDH release into the supernatant and by (B) EthD-1 staining of J774A.1 cells. HMDM cell death was determined by measuring (C) LDH release and (D) EthD-1 uptake into HMDMs. The results are expressed as the mean ± SEM of at least three experiments. *, represents difference from J774A.1 or HMDM controls (P<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2823771&req=5

pone-0009279-g001: Role of YopJ and YopP in J774A.1 macrophage-like and human MDM cell death.Y. pestis strains with the virulence plasmid expressing YopJ (KIM5), YopP (KIM5 YopJ-YopP), or lacking YopJ (KIM5ΔyopJ), along with Y. enterocolitica 8081v and the virulence plasmid–deficient strain (KIM6) were grown at 37°C to induce expression of the TTSS and Yops. Yersinia strains were combined with J774A.1 and HMDM cells and compared to detergent lysed controls as described in Materials and Methods. The percentage of J774A.1 cell death following incubation alone or with Yersinia strains at the indicated time was determined by (A) LDH release into the supernatant and by (B) EthD-1 staining of J774A.1 cells. HMDM cell death was determined by measuring (C) LDH release and (D) EthD-1 uptake into HMDMs. The results are expressed as the mean ± SEM of at least three experiments. *, represents difference from J774A.1 or HMDM controls (P<0.05).
Mentions: To evaluate a role for YopJ and YopP in induction of cell death, we measured LDH release and membrane integrity via EthD-1 staining in the murine J774A.1 cell line and HMDMs following interaction with Yersinia. Incubation of J774A.1 cells or HMDMs with KIM5, Y. enterocolitica, or KIM5 YopJ-YopP resulted in YopJ- or YopP-dependent LDH release and EthD-1 staining (Fig. 1); however, HMDM cell death was delayed between 24–48 h of incubation compared to J774A.1 cells (Fig. 1c and 1d). In contrast, KIM6 or KIM5ΔyopJ incubation with J774A.1 cells or HMDMs resulted in decreased LDH release and EthD-1 staining (Fig. 1, e.g., at 24 h KIM5 and Y. enterocolitica induced 98.2±3.2% and 98.2±1.9% of J774A.1 LDH release, respectively, compared to KIM5ΔyopJ and KIM6 which induced 42.5±11.4% and 43.9±13.0% of J774A.1 LDH release, respectively).

Bottom Line: In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death.PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner.Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho, United States of America.

ABSTRACT

Background: The human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs) for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis) and YopP (Y. enterocolitica) rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined.

Methodology/principal findings: In this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM) and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death. PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5.

Conclusions: Our findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis.

Show MeSH
Related in: MedlinePlus