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

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.

Figure 1. 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).

Figure 2. Induction of caspase activity is YopJ/YopP dependent but delayed in human MDMs compared to J774A.1 cells.

HMDMs and J774A.1 cells were incubated alone or with Y. pestis KIM5, KIM5 YopJ-YopP, KIM5ΔyopJ, KIM6 and Y. enterocolitica 8081v grown at 37°C. Caspase-3, -8, -9, and -2 activity was measured after 6 h of incubation as described in Materials and Methods and is expressed in relative fluorescence units (RFLU). Results are expressed as the mean ± SEM of three experiments. *, represents difference from HMDM and J774A.1 controls (P<0.05).

Figure 3. Role of YopJ and YopP in human PMN cell death.

PMNs were incubated alone or with Y. pestis KIM5, KIM5 YopJ-YopP, KIM5ΔyopJ, KIM6 and Y. enterocolitica 8081v grown at 37°C. The percentage of PMN cell death following incubation with each strain for the indicated time was determined by comparison to detergent lysed controls. Indicators of cell death measured were release of (A) LDH into the supernatant and (B) EthD-1 uptake into PMN cells as described in Materials and Methods . Results are expressed as the mean ± SEM of three experiments. *, represents difference from PMN controls (P<0.05).

Figure 4. Analysis of PMN phosphatidylserine (PS) externalization.

Y. pestis KIM5, KIM5 YopJ-YopP, KIM5ΔyopJ, KIM6 and Y. enterocolitica were grown at 37°C, combined with PMNs, and incubated for the times indicated. PS externalization was determined by annexin V-FITC and flow cytometry following interaction with Yersinia Results are expressed as the mean ± SEM of at least three experiments. *, represents difference from PMN controls (P<0.05).

Figure 5. Caspase-3, -8, -9, and -2 activity in PMNs following incubation with Yersinia.

PMNs were incubated alone or with Y. pestis KIM5, KIM5 YopJ-YopP, KIM5ΔyopJ, KIM6 and Y. enterocolitica 8081v grown at 37°C. Caspase-3, -8, -9, and -2 activity was measured after 3 h and 6 h of incubation as described in Materials and Methods and is expressed in relative fluorescence units. Results are expressed as the mean ± SEM of three experiments. *, represents difference from PMN controls (P<0.05).

Figure 6. Effects of Yersinia on neutrophil ROS production and cell death.

PMNs were incubated alone or with Y. pestis KIM5, KIM5 YopJ-YopP, KIM5ΔyopJ, KIM6 or Y. enterocolitica 8081v grown at 37°C. (A) PMN ROS production following incubation with the indicated Yersinia strains. (B) PMN ROS production with or without 10 µM DPI. PMN cell death following phagocytic interaction with Yersinia was assessed by (C) PMN release of LDH into the supernatant and (D) PMN uptake of EthD-1. Results are expressed as the mean ± SEM of three experiments. *, represents difference from PMN controls (P<0.05).