Immunological Mechanisms Underlying the Genetic Predisposition to Severe Staphylococcus aureus Infection in the Mouse Model
2008; Elsevier BV; Volume: 173; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.080337
ISSN1525-2191
AutoresMaren von Köckritz‐Blickwede, Manfred Rohde, Sonja Oehmcke, Lloyd Miller, Ambrose L. Cheung, Heiko Herwald, Simon J. Foster, Eva Medina,
Tópico(s)Antimicrobial Resistance in Staphylococcus
ResumoHost genetic variations play a significant role in conferring predisposition to infection. In this study, we examined the immune mechanisms underlying the host genetic predisposition to severe Staphylococcus aureus infection in different mouse strains. Whereas C57BL/6 mice were the most resistant in terms of control of bacterial growth and survival, A/J, DBA/2, and BALB/c mice were highly susceptible and succumbed to infection shortly after bacterial inoculation. Other strains (C3H/HeN, CBA, and C57BL/10) exhibited intermediate susceptibility levels. Susceptibility of mice to S. aureus was associated with an inability to limit bacterial growth in the kidneys and development of pathology. Resistance to S. aureus in C57BL/6 mice was dependent on innate immune mechanisms because Rag2-IL2Rγ−/− C57BL/6 mice, which are deficient in B, T, and NK cells, were also resistant to infection. Indeed, neutrophil depletion or inhibition of neutrophil recruitment rendered C57BL/6 mice completely susceptible to S. aureus, indicating that neutrophils are essential for the observed resistance. Although neutrophil function is not inhibited in A/J mice, expression of neutrophil chemoattractants KC and MIP-2 peaked earlier in the kidneys of C57BL/6 mice than in A/J mice, indicating that a delay in neutrophil recruitment to the site of infection may underlie the increased susceptibility of A/J mice to S. aureus. Host genetic variations play a significant role in conferring predisposition to infection. In this study, we examined the immune mechanisms underlying the host genetic predisposition to severe Staphylococcus aureus infection in different mouse strains. Whereas C57BL/6 mice were the most resistant in terms of control of bacterial growth and survival, A/J, DBA/2, and BALB/c mice were highly susceptible and succumbed to infection shortly after bacterial inoculation. Other strains (C3H/HeN, CBA, and C57BL/10) exhibited intermediate susceptibility levels. Susceptibility of mice to S. aureus was associated with an inability to limit bacterial growth in the kidneys and development of pathology. Resistance to S. aureus in C57BL/6 mice was dependent on innate immune mechanisms because Rag2-IL2Rγ−/− C57BL/6 mice, which are deficient in B, T, and NK cells, were also resistant to infection. Indeed, neutrophil depletion or inhibition of neutrophil recruitment rendered C57BL/6 mice completely susceptible to S. aureus, indicating that neutrophils are essential for the observed resistance. Although neutrophil function is not inhibited in A/J mice, expression of neutrophil chemoattractants KC and MIP-2 peaked earlier in the kidneys of C57BL/6 mice than in A/J mice, indicating that a delay in neutrophil recruitment to the site of infection may underlie the increased susceptibility of A/J mice to S. aureus. 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Because of this, the laboratory mouse has been the experimental model of choice to study pathogenesis of infection, including innate and acquired host defense mechanisms. 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This attribute has been exploited to identify novel loci influencing resistance/susceptibility to infection and to provide new insight on host mechanisms involved in response to those pathogens that ultimately affect the onset, progression, and outcome of the infection. The aim of the study presented here is to uncover the immune mechanisms contributing to natural resistance to S. aureus using a murine model of infection. Identifying genetically regulated host immune responses is the first step in understanding the molecular targets and immunological mechanisms on which rational therapies to augment host resistance against pathogens can be developed. The rsbU+ derivative SH1000 of strain 8325-4, that has been shown to cause severe septic arthritis in NMRI mice and express low levels of exoproteins in a similar way to that observed for many clinical isolates was used in this study.29Jonsson IM Arvidson S Foster S Tarkowski A Sigma factor B and Rsbu are required for virulence in Staphylococcus aureus-induced arthritis and sepsis.Infect Immun. 2004; 72: 6106-6111Crossref PubMed Scopus (60) Google Scholar, 30Horsburgh MJ Aish JL White IJ Shaw L Lithgow JK Foster SJ Sigma factor B modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4.J Bacteriol. 2002; 184: 5457-5467Crossref PubMed Scopus (558) Google Scholar Additionally, for in vivo bioluminescence analysis the previously described genetically engineered bioluminescent S. aureus strain SH1000 ALC2906 was used.31Miller LS O'Connell RM Gutierrez MA Pietras EM Shahangian A Gross CE Thirumala A Cheung AL Cheng G Modlin RL MyD88 mediates neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus.Immunity. 2006; 24: 79-92Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar Bacteria were grown to the stationary phase (15 hours) at 37°C with shaking (125 rpm) in brain-heart infusion medium, collected by centrifugation for 10 minutes at 3000 rpm, washed twice with sterile phosphate-buffered saline (PBS), and adjusted to a concentration of 5 × 108 cfu/ml. Aliquots of the S. aureus-suspension were frozen at −80°C for further use. The bacterial suspensions were further diluted with PBS to the required concentration and the number of viable bacteria (cfu) was determined after serial diluting and plating on blood agar containing 5% sheep blood (Invitrogen, Karlsruhe, Germany). Naive inbred, specific pathogen-free (SPF status), 8- to 12-week-old mice were purchased from Harlan Winkelmann (Borchen, Germany): A/J OlaHsd (H-2a), BALB/c OlaHsd (H-2d), C57BL/6 JOlaHsd (H-2b), C57BL/10 ScSnOlaHsd (H-2b), C3H/HeNHsd (H-2k), CBA/J OlaHsd (H-2k), DBA/2 OlaHsd (H-2d). The B-, T-, and NK cell-deficient Rag2-IL-2Rγ−/− C57BL/6 mice were kindly provided by Werna Müller (Helmholtz Center for Infection Research, Braunschweig, Germany). These mice carry a deletion in Rag2 and the interleukin 2 receptor γ-chain (IL-2Rγ) rendering them deficient in B, T, and NK cell differentiation and function. Mice were maintained under standard conditions and according to institutional guidelines. All experiments were approved by the appropriate ethical board (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany). Mice were inoculated with 4 × 107 cfu of S. aureus in 0.2 ml of PBS via a lateral tail vein. For determination of bacterial loads (cfu), infected mice were sacrificed by CO2 asphyxiation at different times of infection and the amount of bacteria determined by preparing kidney homogenates in 5 ml of PBS and plating 10-fold serial dilutions on blood agar. Bacteria colonies were counted after incubation for 24 hours at 37°C. For collection of plasma or serum, citrated or untreated blood, respectively, were collected at time of sacrifice, centrifuged at 4000 rpm for 10 minutes, and frozen at −80°C until use for further analysis. Mice were intravenously infected with bioluminescent S. aureus strain SH1000 ALC2906,31Miller LS O'Connell RM Gutierrez MA Pietras EM Shahangian A Gross CE Thirumala A Cheung AL Cheng G Modlin RL MyD88 mediates neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus.Immunity. 2006; 24: 79-92Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar anesthetized via inhalation of Isoflurane (Isoba; Essex Tierarznei, München, Germany) and analyzed with the Xenogen Vivo Vision IVIS 200 system (Xenogen Corp., Hopkinton, MA). The amount of replicating S. aureus in infected mice was shown by the color scale overlaid on a gray-scale photograph of the infected animal. Kidneys or lungs of mice were fixed with 10% buffered neutral formalin solution, embedded in paraffin, and then cut into 3-μm-thick sections. Tissue sections were stained with hematoxylin and eosin (H&E) and then examined microscopically using an Olympus BX51 microscope (Olympus Europe GmbH, Hamburg, Germany) for pathological alterations. Serum levels of urea were measured using the analyzer Olympus AU400 (Olympus Europe GmbH) according to the manufacturer's instructions. Activation of the intrinsic coagulation system (contact activation) was measured by incubating 50 μl of citrated plasma with 50 μl of aPTT reagent (aPTT Automate; Diagnostica Stago, Asnieres, France) for 60 seconds at 37°C. Clotting was initiated by the addition of 50 μl of a 25-mmol/L CaCl2 solution and activated partial thromboplastin time (aPTT) was measured using an Amelung coagulometer (Amelung, Lemgo, Germany). Levels of bradykinin in plasma were determined by enzyme-linked immunosorbent assay (MARKIT-M-Bradykinin; Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) according to the recommendations of the manufacturer. For scanning electron microscopy samples were fixed with 5% formaldehyde and 2% glutaraldehyde in cacodylate buffer (0.1 mol/L cacodylate, 0.01 mol/L CaCl2, 0.01 mol/L MgCl2, pH 6.9) for 1 hour on ice and washed in TE buffer (20 mmol/L Tris, 1 mmol/L ethylenediaminetetraacetic acid, pH 7.0). Samples were dehydrated with a graded series of acetone (10, 30, 50, 70, 90, 100%) and critical-point dried with CO2 (CPD030; Bal-Tec, Liechtenstein). Dried samples were then fractured and sputter-coated with gold (SCD500, Bal-Tec) before examination in a field emission scanning electron microscope DSM982 Gemini (Zeiss, Oberkochen, Germany) at 5 kV using the Everhart-Thornley secondary electron detector and the in-lens secondary electron detector in a 50:50 ratio. Images were recorded onto a MO-disk and contrast and brightness was adjusted with Adobe Photoshop 9.0. Rat anti-mouse RB6 antibody specific for murine neutrophils and eosinophils32Rogers HW Unanue ER Neutrophils are involved in acute, nonspecific resistance to Listeria monocytogenes in mice.Infect Immun. 1993; 61: 5090-5096PubMed Google Scholar was a kind gift from Siegfried Weiss (HZI, Braunschweig, Germany). Depletion of neutrophils by using anti-mouse RB6 antibodies was performed as previously described.33Chromek M Slamová Z Bergman P Kovács L Podracká L Ehrén I Hökfelt T Gudmundsson GH Gallo RL Agerberth B Brauner A The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection.Nat Med. 2006; 12: 636-641Crossref PubMed Scopus (498) Google Scholar Briefly, mice received an intravenous injection of 100 μg of anti-RB6 antibodies 1 day before bacterial inoculation. Control mice received equivalent amounts of isotype control antibodies in sterile PBS. The neutropenia was confirmed by collecting citrated blood at 24 and 48 hours after inoculation of the antibody. After lysis of erythrocytes, absence of neutrophils in blood was confirmed by staining of cells with fluorescein isothiocyanate-conjugated anti-RB6 antibodies (Serotec, Oxford, UK) and subsequent flow cytometry analysis. For blockage of neutrophil migration from blood into infected tissue, mice were treated intravenously with 50 μg of both rat anti-mouse CD11b and anti-mouse CD18 antibodies (Leinco Technologies, Inc. St. Louis, MO) 4 hours before infection. Control mice received respective amounts of isotype control antibodies in sterile PBS. Blockage of neutrophil recruitment was verified by quantification of neutrophil recruitment into the peritoneal cavity after S. aureus challenge. For this purpose, mice were injected intraperitoneally with 4 × 107 cfu S. aureus SH1000 in 200 μl of PBS 4 hours before the analysis. Mice were then euthanized, the peritoneal cavity lavaged, collected exudates stained with fluorescein isothiocyanate-conjugated anti-RB6 antibodies, and subjected to flow cytometric analysis. To investigate the ability of peritoneal neutrophils to kill S. aureus, mice were injected intraperitoneally with 1 mg of carrageenan (type IV λ; Sigma, St. Louis, MO) 4 and 2 days before infection to induce macrophage depletion and infiltration of neutrophils in the peritoneal cavity. Mice were then intraperitoneally infected with 4 × 107 cfu of S. aureus SH1000 for 1 hour, sacrificed by CO2 inhalation and subjected to peritoneal lavage using Dulbecco's modified Eagle's medium. Peritoneal exudates were washed with sterile PBS, centrifuged at 1000 rpm for 10 minutes, resuspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 μg/ml of gentamicin, seeded in 24-well tissue culture plates at a density of 106 cells/well, and further incubated for 2 hours at 37°C and 5% CO2. Immediately (0 hours) or 1 hour thereafter, neutrophils were collected by centrifugation, washed with PBS, and disrupted with sterile water to release intracellular bacteria. The resulting suspension was serially diluted and bacterial numbers were determined after plating onto blood agar. Serum cytokine levels were determined by a solid phase sandwich immunoassay using the Mouse Cytokine Twenty Plex LMC0006 (Biosource Europe, Nivelles, Belgium) in conjunction with the 100 IS Total System (Luminex, Oosterhout, The Netherlands) according to the recommendations of the manufacturer. The levels of chemokines KC and MCP-1 were determined in kidney tissue after homogenization in lysis buffer (200 mmol/L NaCl, 5 mmol/L ethylenediaminetetraacetic acid, 10 mmol/L Tris, 10% glycerol, 1 mmol/L phenylmethyl sulfonyl fluoride, 1 μg/ml leupeptide, and 28 μg/ml aprotinin, pH 7.4) at a concentration of 50 mg of tissue/ml. Tissue homogenates were centrifuged twice at 1500 × g for 15 minutes at 4°C and supernatants were stored at −80°C until use for further analysis. KC and MIP-2 were determined using the respective R&D Systems DuoSet Elisa Development Systems (R&D Systems, Minneapolis, MN) according to the recommendations of the manufacturer. Total RNAs were isolated from kidney tissue of infected and uninfected A/J and C57BL/6 mice after homogenization in RLT buffer by using a RNeasy Midi kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. cDNA synthesis was performed using a Gibco RT-PCR kit (Invitrogen) following the manufacturer's instructions. The β-actin gene was used as an internal reference gene for RT-PCR. Rsp-9 was used as a housekeeping gene for quantitative real-time PCR. For the RT-PCR, the following oligonucleotides and annealing temperatures were used to generate the specific PCR products: for β-actin, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (58°C, 318 bp); for rsp-9, 5′-CTGGACGAGGGCAAGATGAAGC-3′ and 5′-TGACGTTGGCGGATGAGCACA-3′ (58°C, 143 bp); for KC 5′-GCTGGGATTCACCTCAAGAA-3′ and 5′-AGGTGCCATCAGAGCAGTCT-3′ (55°C, 206 bp); for MIP-2, 5′-ACCAACCACCAGGCTACAG-3′ and 5′-GCGTCACACTCAAGCTCT-3′ (54°C, 109 bp). Cycling conditions for PCR amplifications were 15 seconds at 94°C, 30 seconds annealing, and 45 seconds at 72°C for 32 cycles. For quantitative real-time PCR, amplification was performed using a LightCycler 480 real-time PCR system (Roche Applied Science, Mannheim, Germany) and SYBR green PCR master mix (Roche). For each gene, a standard curve was constructed by using plasmids into which the full-length cDNA was cloned and subsequently subcloned into TOPO PCR2.1 cloning vector (Invitrogen). The resulting plasmids were prepared using a QIAprep Spin miniprep kit (Qiagen) following the manufacturer's instructions and used for the generation of standard curves. Cycle threshold values for MIP-2 or KC were normalized to that of the housekeeping gene Rsp-9. Data are expressed as relative mRNA expression levels. Data were analyzed by using Excel 2000 (Microsoft Office; Microsoft, Redmond, WA) or GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). Unless otherwise specified, all data are presented as mean ± SD. Each in vitro experiment was performed independently at least three times and within each experiment all samples were processed in duplicate. Comparison between groups was made by use of a variance analysis (F-test). Comparison of survival time curves was performed by use of log rank test. P values ≤0.05 were considered as significant. To determine the influence of the genetic background in susceptibility of mice to S. aureus infection, the survival times of mice from seven different inbred strains inoculated with 4 × 107 cfu of S. aureus SH1000 was determined throughout a period of 14 days. As shown in Figure 1, A/J, DBA/2, and BALB/c mice were very susceptible to S. aureus, exhibiting 100% mortality within 1 week of infection. Among these strains, the A/J mice were the most susceptible and all animals died at 24 hours after bacterial inoculation. In contrast, C57BL/6 was the most resistant mouse strain with only sporadic mortality (15%) observed in long-term experiments (up to 4 weeks). C3H/HeN, CBA, and C57BL/10 mice exhibited intermediate susceptibility and ∼50% of the mice survived the infection. Differences in survival times were statistically significant (P < 0.05) between the resistant, intermediate, and susceptible strains but not among strains belonging to the same group. To determine whether the survival advantage of resistant C57BL/6 mice is related to a better capacity to control bacterial growth, C57BL/6 and highly susceptible A/J mice were infected with a genetically engineered bioluminescent strain of S. aureus SH1000 and bioluminescence of live, actively metabolizing bacteria was measured at 24 hours after bacteria inoculation using the Xenogen Vivo Vision IVIS 200 system. Whereas luminescence in C57BL/6 mice was below detection levels (Figure 2A), a very intense bioluminescent signal was recorded in kidneys of infected A/J mice (Figure 2B), the previously reported organ target of S. aureus in this model of infection.29Jonsson IM Arvidson S Foster S Tarkowski A Sigma factor B and Rsbu are required for virulence in Staphylococcus aureus-induced arthritis and sepsis.Infect Immun. 2004; 72: 6106-6111Crossref PubMed Scopus (60) Google Scholar, 34Tarkowski A Bokarewa M Collins LV Gjertsson I Hultgren OH Jin T Jonsson IM Josefsson E Sakiniene E Verdrengh M Current status of pathogenic mechanisms in staphylococcal arthritis.FEMS Microbiol Lett. 2002; 217: 125-132Crossref PubMed Google Scholar, 35Collins LV Tarkowski A Animal models of experimental Staphylococcus aureus infection.in: Fischetti VA Novick RP Ferretti JJ Portnoy DA Rood JI Gram-Positive Pathogens. 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Determination of the bacterial loads in the kidneys of several other inbred strains of mice at 24 hours after inoculation shows that susceptible or intermediate mouse strains had significantly higher bacterial burden in kidneys compared to resistant C57BL/6 mice (Figure 2D). These results suggest that resistance of C57BL/6 mice to S. aureus infection is associated with a superior capacity of these mice to control bacterial growth after intravenous inoculation. For further studies, A/J and C57BL/6 were chosen as representative susceptible and resistant mouse strains, respectively. Because the kidney was the organ target for S. aureus in this model of infection, we performed histopathological evaluation of this organ from infected C57BL/6 and A/J mice to determine the extent of S. aureus-induced organ damage. Kidney tissues from uninfected C57BL/6 (Figure 3A1) and A/J (Figure 3A3) mice were included for comparison. Examination of kidney sections obtained from S. aureus-infected mice at 24 hours after bacteria inoculation revealed n
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