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Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection.

Tseng CW, Biancotti JC, Berg BL, Gate D, Kolar SL, Müller S, Rodriguez MD, Rezai-Zadeh K, Fan X, Beenhouwer DO, Town T, Liu GY - PLoS Pathog. (2015)

Bottom Line: Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide.However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology.PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Infectious Diseases and the Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

ABSTRACT
Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγ (NSG) mice engrafted with human CD34+ umbilical cord blood cells. These "humanized" NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL+ versus isogenic PVL- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL+ S. aureus but not PVL- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.

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Humanized NSG mice are more susceptible to PVL-induced dermonecrosis.PMN were isolated from the blood of human volunteers and bone marrow of humanized mice or BALB/c mice. (A) Percentage of human CD66B+ and murine Ly6G + cells in the PMN preparations (n = 4). (B) Percentage of hC5aR+ cells in the PMN preparations (n = 4). (C) The PMN preparations were incubated with 100 ng/mL rPVL. After 3h, the percentages of viable cells were calculated based on MTT values using untreated PMN isolated from the respective hosts as standards (n = 4). (D—F) Humanized and control mice (n = 5–6 per group) were infected on the left flank with 106 CFU WT S. aureus and on the right flank with 106 CFU PVL- isogenic mutant strain. The mice were sacrificed on d 3 post-infection. Shown are (D) skin lesion size and bacterial burden (H: humanized mice, B: BALB/c mice) and (E) MPO activity in infected humanized mice. (F) Visual representation of skin lesions induced by WT S. aureus (red arrow head) or the PVL- mutant (blue arrow head), *: p <0.05, **: p < 0.01, ***: p < 0.005, ****: p < 0.001.
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ppat.1005292.g002: Humanized NSG mice are more susceptible to PVL-induced dermonecrosis.PMN were isolated from the blood of human volunteers and bone marrow of humanized mice or BALB/c mice. (A) Percentage of human CD66B+ and murine Ly6G + cells in the PMN preparations (n = 4). (B) Percentage of hC5aR+ cells in the PMN preparations (n = 4). (C) The PMN preparations were incubated with 100 ng/mL rPVL. After 3h, the percentages of viable cells were calculated based on MTT values using untreated PMN isolated from the respective hosts as standards (n = 4). (D—F) Humanized and control mice (n = 5–6 per group) were infected on the left flank with 106 CFU WT S. aureus and on the right flank with 106 CFU PVL- isogenic mutant strain. The mice were sacrificed on d 3 post-infection. Shown are (D) skin lesion size and bacterial burden (H: humanized mice, B: BALB/c mice) and (E) MPO activity in infected humanized mice. (F) Visual representation of skin lesions induced by WT S. aureus (red arrow head) or the PVL- mutant (blue arrow head), *: p <0.05, **: p < 0.01, ***: p < 0.005, ****: p < 0.001.

Mentions: Published studies have established that human PMN are exquisitely sensitive to PVL-induced cytolysis, whereas murine PMN are relatively unresponsive to the toxin as determined by chemokine secretion and cytolysis [15,22]. This difference has been attributed to poor binding of the LukS-PV component of PVL to murine C5aR compared to binding to human C5aR [15]. We investigated the interaction of PVL with PMN isolated from humanized NSG mice, human volunteers, and BALB/c mice. First, PMN from the various sources were analyzed by flow cytometry for expression of the human neutrophil marker, hCD66B, and the murine granulocyte marker, Ly6G. As shown in Fig 2A, PMN prepared from the bone marrow of humanized mice yielded 45% human PMN based on hCD66B staining and 31% murine PMN based on Ly6G staining, which is consistent with published reports that NSG mice have a modest but significant number of murine PMN [23]. By contrast, PMN prepared from human volunteers were 87% positive for hCD66B and did not stain for Ly6G. PMN from the bone marrow of BALB/c mice yielded 67% Ly6G-positive cells and had an insignificant percentage of hCD66B-positive cells. Consistent with these findings, PMN obtained from the humanized mice were 44% positive for the PVL receptor hC5aR, compared to 0% positive for PMN from BALB/c mice (Fig 2B). When the cellular preps from the various sources were exposed to recombinant PVL (rPVL), murine PMN showed little loss of viability (Fig 2C) and minimal CXCL1 (IL-8 or KC) response (S3 Fig). In comparison, PMN preparations from humans and humanized mice exhibited significant sensitivity to rPVL as measured by cell viability (Fig 2C) and IL-8 production (S3 Fig). These data show that human PMN generated in humanized NSG mice, like human PMN, are responsive to PVL.


Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection.

Tseng CW, Biancotti JC, Berg BL, Gate D, Kolar SL, Müller S, Rodriguez MD, Rezai-Zadeh K, Fan X, Beenhouwer DO, Town T, Liu GY - PLoS Pathog. (2015)

Humanized NSG mice are more susceptible to PVL-induced dermonecrosis.PMN were isolated from the blood of human volunteers and bone marrow of humanized mice or BALB/c mice. (A) Percentage of human CD66B+ and murine Ly6G + cells in the PMN preparations (n = 4). (B) Percentage of hC5aR+ cells in the PMN preparations (n = 4). (C) The PMN preparations were incubated with 100 ng/mL rPVL. After 3h, the percentages of viable cells were calculated based on MTT values using untreated PMN isolated from the respective hosts as standards (n = 4). (D—F) Humanized and control mice (n = 5–6 per group) were infected on the left flank with 106 CFU WT S. aureus and on the right flank with 106 CFU PVL- isogenic mutant strain. The mice were sacrificed on d 3 post-infection. Shown are (D) skin lesion size and bacterial burden (H: humanized mice, B: BALB/c mice) and (E) MPO activity in infected humanized mice. (F) Visual representation of skin lesions induced by WT S. aureus (red arrow head) or the PVL- mutant (blue arrow head), *: p <0.05, **: p < 0.01, ***: p < 0.005, ****: p < 0.001.
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Related In: Results  -  Collection

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ppat.1005292.g002: Humanized NSG mice are more susceptible to PVL-induced dermonecrosis.PMN were isolated from the blood of human volunteers and bone marrow of humanized mice or BALB/c mice. (A) Percentage of human CD66B+ and murine Ly6G + cells in the PMN preparations (n = 4). (B) Percentage of hC5aR+ cells in the PMN preparations (n = 4). (C) The PMN preparations were incubated with 100 ng/mL rPVL. After 3h, the percentages of viable cells were calculated based on MTT values using untreated PMN isolated from the respective hosts as standards (n = 4). (D—F) Humanized and control mice (n = 5–6 per group) were infected on the left flank with 106 CFU WT S. aureus and on the right flank with 106 CFU PVL- isogenic mutant strain. The mice were sacrificed on d 3 post-infection. Shown are (D) skin lesion size and bacterial burden (H: humanized mice, B: BALB/c mice) and (E) MPO activity in infected humanized mice. (F) Visual representation of skin lesions induced by WT S. aureus (red arrow head) or the PVL- mutant (blue arrow head), *: p <0.05, **: p < 0.01, ***: p < 0.005, ****: p < 0.001.
Mentions: Published studies have established that human PMN are exquisitely sensitive to PVL-induced cytolysis, whereas murine PMN are relatively unresponsive to the toxin as determined by chemokine secretion and cytolysis [15,22]. This difference has been attributed to poor binding of the LukS-PV component of PVL to murine C5aR compared to binding to human C5aR [15]. We investigated the interaction of PVL with PMN isolated from humanized NSG mice, human volunteers, and BALB/c mice. First, PMN from the various sources were analyzed by flow cytometry for expression of the human neutrophil marker, hCD66B, and the murine granulocyte marker, Ly6G. As shown in Fig 2A, PMN prepared from the bone marrow of humanized mice yielded 45% human PMN based on hCD66B staining and 31% murine PMN based on Ly6G staining, which is consistent with published reports that NSG mice have a modest but significant number of murine PMN [23]. By contrast, PMN prepared from human volunteers were 87% positive for hCD66B and did not stain for Ly6G. PMN from the bone marrow of BALB/c mice yielded 67% Ly6G-positive cells and had an insignificant percentage of hCD66B-positive cells. Consistent with these findings, PMN obtained from the humanized mice were 44% positive for the PVL receptor hC5aR, compared to 0% positive for PMN from BALB/c mice (Fig 2B). When the cellular preps from the various sources were exposed to recombinant PVL (rPVL), murine PMN showed little loss of viability (Fig 2C) and minimal CXCL1 (IL-8 or KC) response (S3 Fig). In comparison, PMN preparations from humans and humanized mice exhibited significant sensitivity to rPVL as measured by cell viability (Fig 2C) and IL-8 production (S3 Fig). These data show that human PMN generated in humanized NSG mice, like human PMN, are responsive to PVL.

Bottom Line: Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide.However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology.PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Infectious Diseases and the Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.

ABSTRACT
Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγ (NSG) mice engrafted with human CD34+ umbilical cord blood cells. These "humanized" NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL+ versus isogenic PVL- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL+ S. aureus but not PVL- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.

Show MeSH
Related in: MedlinePlus