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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 show enhanced susceptibility to S. aureus-induced skin lesions.(A) Fluorescence confocal imaging of humanized mouse spleen showing separate mouse CD45 and human CD45-expressing cells (top) and flow cytometry contour plot showing % engraftment in humanized mice (bottom). (B-C) Humanized mice and control mice were infected s.c. with 105 to 108 CFU of S. aureus. On d 3 post-infection, (B) skin lesion size and (C) bacterial burden were analyzed. (D) Mice were infected with 106 CFU of S. aureus and lesion sizes on d 3 post-infection were plotted against % engraftment. Shown is the best fit linear regression line (R2 = 0.65). Red bar = mean, *: p <0.05, **: p < 0.01.
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ppat.1005292.g001: Humanized NSG mice show enhanced susceptibility to S. aureus-induced skin lesions.(A) Fluorescence confocal imaging of humanized mouse spleen showing separate mouse CD45 and human CD45-expressing cells (top) and flow cytometry contour plot showing % engraftment in humanized mice (bottom). (B-C) Humanized mice and control mice were infected s.c. with 105 to 108 CFU of S. aureus. On d 3 post-infection, (B) skin lesion size and (C) bacterial burden were analyzed. (D) Mice were infected with 106 CFU of S. aureus and lesion sizes on d 3 post-infection were plotted against % engraftment. Shown is the best fit linear regression line (R2 = 0.65). Red bar = mean, *: p <0.05, **: p < 0.01.

Mentions: Humanized NSG mice were generated using an established protocol [21], and engraftment was quantified by staining splenic cells with a monoclonal antibody specific for human nuclei and verified by immunostaining with anti-human and anti-mouse CD45 antibodies (Fig 1A and S1 Fig). Engraftment rates were 70.59 ± 4.02%, which are consistent with results reported by another group [21]. The percentages of human cell subsets in the spleen are shown in S1 Table and demonstrate a predominance of T and B cell subsets over myeloid subsets, consistent with published data [21], though in our study the percentage of CD3+ T cells was higher than the percentage of CD20+ B cells, unlike the previous study. For infection experiments, control mice consisted of either NSG mice engrafted with murine bone marrow cells (designated as murinized mice) or wild type (WT) BALB/c mice which are congenic with NSG mice. Mice with greater than 40% human CD45+ cell engraftment were used in all experiments except for the experiment shown in Fig 1D where engraftment efficiency is correlated to lesion size. To determine the susceptibility of the humanized NSG mice to S. aureus infection, we infected mice subcutaneously with inocula ranging from 1 x 105 to 1 x 108 CFU. Based on work from our lab and other groups, peak lesion size is documented on d 3 after infection [6,12]. We therefore sacrificed the animals on d 3 for various analyses. Using inocula of 1 x 105 and 1 x 106 CFU, all humanized mice exhibited visible skin lesions (Fig 1B). By comparison, murinized NSG or BALB/c mice exhibited minimal skin lesions at an inoculum of 1 x 105 CFU, while approximately 50% of infected control mice showed visible skin lesions at 1 x 106 CFU. Upon increasing the inocula to 1 x 107 and 1 x 108 CFU, all murinized mice and BALB/c mice showed dermonecrosis (Fig 1B). In spite of the large difference in susceptibility to lesion formation, humanized and control mice generally did not exhibit significant differences in bacterial burden across the range of inocula (Fig 1C). Because many pups injected with hCD34+ cells died from maternal neglect or cannibalism in our colony, some of the murinized or humanized NSG groups were small, and for those groups, the data need to be interpreted cautiously.


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 show enhanced susceptibility to S. aureus-induced skin lesions.(A) Fluorescence confocal imaging of humanized mouse spleen showing separate mouse CD45 and human CD45-expressing cells (top) and flow cytometry contour plot showing % engraftment in humanized mice (bottom). (B-C) Humanized mice and control mice were infected s.c. with 105 to 108 CFU of S. aureus. On d 3 post-infection, (B) skin lesion size and (C) bacterial burden were analyzed. (D) Mice were infected with 106 CFU of S. aureus and lesion sizes on d 3 post-infection were plotted against % engraftment. Shown is the best fit linear regression line (R2 = 0.65). Red bar = mean, *: p <0.05, **: p < 0.01.
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ppat.1005292.g001: Humanized NSG mice show enhanced susceptibility to S. aureus-induced skin lesions.(A) Fluorescence confocal imaging of humanized mouse spleen showing separate mouse CD45 and human CD45-expressing cells (top) and flow cytometry contour plot showing % engraftment in humanized mice (bottom). (B-C) Humanized mice and control mice were infected s.c. with 105 to 108 CFU of S. aureus. On d 3 post-infection, (B) skin lesion size and (C) bacterial burden were analyzed. (D) Mice were infected with 106 CFU of S. aureus and lesion sizes on d 3 post-infection were plotted against % engraftment. Shown is the best fit linear regression line (R2 = 0.65). Red bar = mean, *: p <0.05, **: p < 0.01.
Mentions: Humanized NSG mice were generated using an established protocol [21], and engraftment was quantified by staining splenic cells with a monoclonal antibody specific for human nuclei and verified by immunostaining with anti-human and anti-mouse CD45 antibodies (Fig 1A and S1 Fig). Engraftment rates were 70.59 ± 4.02%, which are consistent with results reported by another group [21]. The percentages of human cell subsets in the spleen are shown in S1 Table and demonstrate a predominance of T and B cell subsets over myeloid subsets, consistent with published data [21], though in our study the percentage of CD3+ T cells was higher than the percentage of CD20+ B cells, unlike the previous study. For infection experiments, control mice consisted of either NSG mice engrafted with murine bone marrow cells (designated as murinized mice) or wild type (WT) BALB/c mice which are congenic with NSG mice. Mice with greater than 40% human CD45+ cell engraftment were used in all experiments except for the experiment shown in Fig 1D where engraftment efficiency is correlated to lesion size. To determine the susceptibility of the humanized NSG mice to S. aureus infection, we infected mice subcutaneously with inocula ranging from 1 x 105 to 1 x 108 CFU. Based on work from our lab and other groups, peak lesion size is documented on d 3 after infection [6,12]. We therefore sacrificed the animals on d 3 for various analyses. Using inocula of 1 x 105 and 1 x 106 CFU, all humanized mice exhibited visible skin lesions (Fig 1B). By comparison, murinized NSG or BALB/c mice exhibited minimal skin lesions at an inoculum of 1 x 105 CFU, while approximately 50% of infected control mice showed visible skin lesions at 1 x 106 CFU. Upon increasing the inocula to 1 x 107 and 1 x 108 CFU, all murinized mice and BALB/c mice showed dermonecrosis (Fig 1B). In spite of the large difference in susceptibility to lesion formation, humanized and control mice generally did not exhibit significant differences in bacterial burden across the range of inocula (Fig 1C). Because many pups injected with hCD34+ cells died from maternal neglect or cannibalism in our colony, some of the murinized or humanized NSG groups were small, and for those groups, the data need to be interpreted cautiously.

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