Aberrant Inflammatory Response to Streptococcus pyogenes in Mice Lacking Myeloid Differentiation Factor 88
2009; Elsevier BV; Volume: 176; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2010.090422
ISSN1525-2191
AutoresTorsten G. Loof, Oliver Goldmann, André Gessner, Heiko Herwald, Eva Medina,
Tópico(s)Neonatal and Maternal Infections
ResumoSeveral in vitro studies have emphasized the importance of toll-like receptor/myeloid differentiation factor 88 (MyD88) signaling in the inflammatory response to Streptococcus pyogenes. Since the extent of inflammation has been implicated in the severity of streptococcal diseases, we have examined here the role of toll-like receptor/MyD88 signaling in the pathophysiology of experimental S. pyogenes infection. To this end, we compared the response of MyD88-knockout (MyD88−/−) after subcutaneous inoculation with S. pyogenes with that of C57BL/6 mice. Our results show that MyD88−/− mice harbored significantly more bacteria in the organs and succumbed to infection much earlier than C57BL/6 animals. Absence of MyD88 resulted in diminished production of inflammatory cytokines such as interleukin-12, interferon-γ, and tumor necrosis factor-α as well as chemoattractants such as monocyte chemotactic protein-1 (MCP-1) and Keratinocyte-derived chemokine (KC), and hampered recruitment of effector cells involved in bacterial clearance (macrophages and neutrophils) to the infection site. Furthermore, MyD88−/− but not C57BL/6 mice exhibited a massive infiltration of eosinophils in infected organs, which can be explained by an impaired production of the regulatory chemokines, gamma interferon-induced monokine (MIG/CXCL9) and interferon-induced protein 10 (IP-10/CXCL10), which can inhibit transmigration of eosinophils. Our results indicate that MyD88 signaling targets effector cells to the site of streptococcal infection and prevents extravasation of cells that can induce tissue damage. Therefore, MyD88 signaling may be important for shaping the quality of the inflammatory response elicited during infection to ensure optimal effector functions. Several in vitro studies have emphasized the importance of toll-like receptor/myeloid differentiation factor 88 (MyD88) signaling in the inflammatory response to Streptococcus pyogenes. Since the extent of inflammation has been implicated in the severity of streptococcal diseases, we have examined here the role of toll-like receptor/MyD88 signaling in the pathophysiology of experimental S. pyogenes infection. To this end, we compared the response of MyD88-knockout (MyD88−/−) after subcutaneous inoculation with S. pyogenes with that of C57BL/6 mice. Our results show that MyD88−/− mice harbored significantly more bacteria in the organs and succumbed to infection much earlier than C57BL/6 animals. Absence of MyD88 resulted in diminished production of inflammatory cytokines such as interleukin-12, interferon-γ, and tumor necrosis factor-α as well as chemoattractants such as monocyte chemotactic protein-1 (MCP-1) and Keratinocyte-derived chemokine (KC), and hampered recruitment of effector cells involved in bacterial clearance (macrophages and neutrophils) to the infection site. Furthermore, MyD88−/− but not C57BL/6 mice exhibited a massive infiltration of eosinophils in infected organs, which can be explained by an impaired production of the regulatory chemokines, gamma interferon-induced monokine (MIG/CXCL9) and interferon-induced protein 10 (IP-10/CXCL10), which can inhibit transmigration of eosinophils. Our results indicate that MyD88 signaling targets effector cells to the site of streptococcal infection and prevents extravasation of cells that can induce tissue damage. Therefore, MyD88 signaling may be important for shaping the quality of the inflammatory response elicited during infection to ensure optimal effector functions. Streptococcus pyogenes represents one of the most frequent causes of mild infection of the upper respiratory tract and the skin with the potential to cause life-threatening invasive diseases such as necrotizing fasciitis and septicemia.1Jaggi P Shulman ST Group A streptococcal infections.Pediatr Rev. 2006; 27: 99-105Crossref PubMed Scopus (22) Google Scholar Although severe streptococcal diseases occur in only a small percentage of cases, they are an important cause of morbidity and mortality. Specific manifestations of streptococcal infections are dictated by the complex interplay of bacterial virulence factors and the host immune components. Deciphering the complexities of the host response to invasive S. pyogenes will greatly facilitate the use of more appropriate targeted therapies for affected patients in the future. Pivotal components of the host innate immune response to pathogens are the toll-like receptors (TLRs). TLRs recognize the presence of microbial pathogens via the detection of pathogen-associated molecular patterns such as lipopolysaccharide, lipoproteins, peptidoglycans, double-stranded RNA, CpG DNA, and flagellin.2Medzhitov R Janeway Jr, C The Toll receptor family and microbial recognition.Trends Microbiol. 2000; 8: 452-456Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 3Takeda K Kaisho T Akira S Toll-like receptors.Annu Rev Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4744) Google Scholar, 4Hayashi F Smith KD Ozinsky A Hawn TR Yi EC Goodlett DR Eng JK Akira S Underhill DM Aderem A The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5.Nature. 2001; 410: 1099-1103Crossref PubMed Scopus (2808) Google Scholar, 5Hemmi H Takeuchi O Kawai T Kaisho T Sato S Sanjo H Matsumoto M Hoshino K Wagner H Takeda K Akira S A Toll-like receptor recognizes bacterial DNA.Nature. 2000; 408: 740-745Crossref PubMed Scopus (5372) Google Scholar, 6Takeuchi O Hoshino K Kawai T Sanjo H Takada H Ogawa T Takeda K Akira S Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components.Immunity. 1999; 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204: 2407-2422Crossref PubMed Scopus (334) Google Scholar The importance of the TLR signaling in human diseases was highlighted in recent clinical studies in which the authors examined patients lacking interleukin (IL)-1 receptor–associated kinase 4, which is selectively recruited to TLRs and IL-1 receptors by MyD88.14Picard C Puel A Bonnet M Ku CL Bustamante J Yang K Soudais C Dupuis S Feinberg J Fieschi C Elbim C Hitchcock R Lammas D Davies G Al-Ghonaium A Al-Raynes H Al-Jumaah S Al-Hajjar S Al-Mohsen IZ Frayha HH Rucker R Hawn TR Aderem A Tufenkeji H Haraguchi S Day NK Good RA Gougerot-Pocidalo MA Ozinsky A Casanova JL Pyogenic bacterial infections in humans with IRAK-4 deficiency.Science. 2003; 299: 2076-2079Crossref PubMed Scopus (777) Google Scholar, 15von Bernuth H Picard C Jin Z Pankla R Xiao H Ku CL Chrabieh M Mustapha IB Ghandil P Camcioglu Y Vasconcelos J Sirvent N Guedes M Vitor AB Herrero-Mata MJ Arostegui JI Rodrigo L Alsina L Ruiz-Ortiz E Juan M Fortuny C Yagüe J Anton J Pascal M Chang HH Janniere L Rose Y Garty BZ Chapel H Issekutz A Marodi L Rodriguez-Gallegoi C Banchereau J Abel L Li X Chaussabel D Puel A Casanova JL Pyogenic bacterial infections in humans with MyD88 deficiency.Science. 2008; 321: 691-696Crossref PubMed Scopus (626) Google Scholar, 16Ku CL von Bernuth H Picard C Zhang SY Chang HH Yang K Chrabieh M Issekutz AC Cunningham CK Gallin J Holland SM Roifman C Ehl S Smart J Tang M Barrat FJ Levy O McDonald D Day-Good NK Miller R Takada H Hara T Al-Hajjar S Al-Ghonaium A Speert D Sanlaville D Li X Geissmann F Vivier E Marodi L Garty BZ Chapel H Rodriguez-Gallego C Bossuyt X Abel L Puel A Casanova JL Selective predisposition to bacterial infections in IRAK-4-deficient children: IRAK-4-dependent TLRs are otherwise redundant in protective immunity.J Exp Med. 2007; 204: 2407-2422Crossref PubMed Scopus (334) Google Scholar MyD88-deficient patients exhibit an impaired response to most of TLRs ligands as well as to IL-1 and display a life-threatening but narrow and transient predisposition to infection, apparently restricted to pyogenic bacterial diseases.14Picard C Puel A Bonnet M Ku CL Bustamante J Yang K Soudais C Dupuis S Feinberg J Fieschi C Elbim C Hitchcock R Lammas D Davies G Al-Ghonaium A Al-Raynes H Al-Jumaah S Al-Hajjar S Al-Mohsen IZ Frayha HH Rucker R Hawn TR Aderem A Tufenkeji H Haraguchi S Day NK Good RA Gougerot-Pocidalo MA Ozinsky A Casanova JL Pyogenic bacterial infections in humans with IRAK-4 deficiency.Science. 2003; 299: 2076-2079Crossref PubMed Scopus (777) Google Scholar, 15von Bernuth H Picard C Jin Z Pankla R Xiao H Ku CL Chrabieh M Mustapha IB Ghandil P Camcioglu Y Vasconcelos J Sirvent N Guedes M Vitor AB Herrero-Mata MJ Arostegui JI Rodrigo L Alsina L Ruiz-Ortiz E Juan M Fortuny C Yagüe J Anton J Pascal M Chang HH Janniere L Rose Y Garty BZ Chapel H Issekutz A Marodi L Rodriguez-Gallegoi C Banchereau J Abel L Li X Chaussabel D Puel A Casanova JL Pyogenic bacterial infections in humans with MyD88 deficiency.Science. 2008; 321: 691-696Crossref PubMed Scopus (626) Google Scholar Although optimal stimulation of TLRs is critical for triggering inflammatory responses that result in pathogen eradication, in some cases, excessive TLR activation may instead contribute to detrimental inflammation. Thus, mice deficient in MyD88 expression have been reported to be more resistant than normal animals to lethal shock after administration of a high-dose of lipopolysaccharide17Kawai T Adachi O Ogawa T Takeda K Akira S Unresponsiveness of MyD88-deficient mice to endotoxin.Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1726) Google Scholar or after induction of polymicrobial sepsis.18Weighardt H Kaiser-Moore S Vabulas RM Kirschning CJ Wagner H Holzmann B Cutting edge: myeloid differentiation factor 88 deficiency improves resistance against sepsis caused by polymicrobial infection.J Immunol. 2002; 169: 2823-2827PubMed Google Scholar The beneficial effect of MyD88 ablation in this experimental setting seems to rely on the attenuation of the hyperinflammatory response and concomitant reduced tissue injury.17Kawai T Adachi O Ogawa T Takeda K Akira S Unresponsiveness of MyD88-deficient mice to endotoxin.Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1726) Google Scholar, 18Weighardt H Kaiser-Moore S Vabulas RM Kirschning CJ Wagner H Holzmann B Cutting edge: myeloid differentiation factor 88 deficiency improves resistance against sepsis caused by polymicrobial infection.J Immunol. 2002; 169: 2823-2827PubMed Google Scholar Furthermore, a dual role of MyD88 has been demonstrated in an experimental model of group B streptococcal infection where MyD88 was essential for antimicrobial defenses during the initial infection but it contributed to lethality in the presence of overwhelming sepsis.19Mancuso G Midiri A Beninati C Biondo C Galbo R Akira S Henneke P Golenbock D Teti G Dual role of TLR2 and myeloid differentiation factor 88 in a mouse model of invasive group B streptococcal disease.J Immunol. 2004; 172: 6324-6329PubMed Google Scholar In the particular case of S. pyogenes, we have recently demonstrated the absolute requirement of MyD88 for the up-regulation of maturation markers and the production of inflammatory cytokines in S. pyogenes-stimulated dendritic cells.20Loof TG Goldmann O Medina E Immune recognition of Streptococcus pyogenes by dendritic cells.Infect Immun. 2008; 76: 2785-2792Crossref PubMed Scopus (55) Google Scholar In addition, Gratz et al21Gratz N Siller M Schaljo B Pirzada ZA Gattermeier I Vojtek I Kirschning CJ Wagner H Akira S Charpentier E Kovarik P Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9.J Biol Chem. 2008; 283: 19879-19887Crossref PubMed Scopus (74) Google Scholar have also reported that production of proinflammatory cytokines by macrophages after in vitro stimulation with S. pyogenes was completely dependent on MyD88. In view of the critical importance of MyD88 signaling in the immune response to S. pyogenes, our objective for this study presented here was to obtain further insights into the functional significance of this central adaptor molecule in regulating the host response to S. pyogenes by using an experimental mouse model of skin infection. Our results demonstrate that MyD88 signaling is decisive for triggering a rapid innate immune response against S. pyogenes that operates at two levels: (1) by inducing the up-regulation of inflammatory mediators that triggers chemotaxis of cells critically involved in bacterial clearance such as neutrophils and macrophages to the site of infection; and (2) by promoting the expression of regulatory chemokines such as MIG/CXCL9 and IP-10/CXCL10, shown to inhibit the recruitment of other cell types with potential detrimental effects (eg, eosinophils). In the absence of MyD88, the inflammatory response elicited in response to S. pyogenes shifted from a protective neutrophil/macrophage-mediated to a destructive eosinophil-dominated cellular infiltrate. Our data highlight a role of MyD88 in shaping the quality of the inflammatory response elicited during S. pyogenes infection. The S. pyogenes strain KTL3 (M1 type) was initially isolated from the blood of a patient with streptococcal bacteremia. Stock cultures were maintained at −70°C and cultured at 37°C in Todd-Hewitt broth (Oxoid, Basingstoke, UK), supplemented with 1% yeast extract. Bacteria were collected in Mid-Log-phase, washed twice with sterile PBS, diluted to the required inoculums, and the number of viable bacteria was determined by counting colony-forming units (CFU) after plating on blood agar plates containing 5% sheep blood (GIBCO, Paisley, UK). C57BL/6 mice were purchased from Harlan-Winkelmann (Borchen, Germany). MyD88−/− mice with a C57BL/6 genetic background were obtained from either the Max Planck Institute for Infection Biology (Berlin, Germany) or from Lund University (Lund, Sweden). Mice were housed in a pathogen-free animal facility and maintained under standard conditions according to institutional guidelines. All experiments were approved by the appropriate ethical board (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany). MyD88−/− and C57BL/6 mice were injected subcutaneously with the indicated amount of S. pyogenes strain KTL3 as previously described.22Loof TG Rohde M Chhatwal GS Jung S Medina E The contribution of dendritic cells to host defenses against Streptococcus pyogenes.J Infect Dis. 2007; 196: 1794-1803Crossref PubMed Scopus (37) Google Scholar Briefly, mice were anesthetized with Isoba (Essex, Munich, Germany) and infected subcutaneously with S. pyogenes in 100 μl of PBS at the right front flank. Infected mice were monitored for survival or sacrificed by CO2 inhalation at the indicated time intervals to measure bacterial burdens in blood and systemic organs. For calculation of 50% lethal dose, groups of 10 mice were subcutaneously inoculated with different amounts of S. pyogenes, ranging from 104 to 5 × 107 CFU and mortality was scored over a period of 15 days. The 50% lethal dose was calculated by using the method of Reed and Muench.23Reed LJ Muench H A simple method for estimating fifty percent end points.Am J Hyg. 1938; 27: 493-497Google Scholar The plasma levels of alanine aminotransferase, aspartate aminotransferase, creatin phosphokinase, and lactate dehydrogenase were determined by using the analyzer Olympus AU400 (Olympus Europe GmbH, Hamburg, Germany) according to the manufacturer's instructions. Serum cytokines profile was determined at 6, 12, and 20 hours of infection by using the Mouse Cytokine Twenty Plex LMC0006 (Biosource Europe, Nivelles, Belgium) in conjunction with the Luminex 100 IS Total System (Oosterhout, The Netherlands) according to the manufacturer's instructions. Bone marrow-derived macrophages were generated as previously described24Goldmann O Sastalla I Wos-Oxley M Rohde M Medina E Streptococcus pyogenes induces oncosis in macrophages through the activation of an inflammatory programmed cell death pathway.Cell Microbiol. 2009; 11: 138-155Crossref PubMed Scopus (71) Google Scholar and infected with S. pyogenes at a multiplicity of infection of 10 bacteria per macrophage for 1 hour. Monolayers were washed to remove unbound bacteria and further incubated in the presence of gentamicin (100 μg/ml) at 37°C in 5% CO2 to kill extracellular bacteria. After different periods of time, cells were washed and disrupted with dH2O to release intracellular bacteria. The resulting suspension was serially diluted and bacterial numbers were determined after plating onto blood agar. Control experiments were performed to confirm that gentamicin did not penetrate inside the macrophages during the incubation time (Supplemental Figure S1, see http://ajp.amjpathol.org). For this purpose, bone marrow-derived macrophages were seeded onto four-well tissue culture plates and incubated for 24 hours in medium alone or in the presence of 100 μg/ml of either gentamicin or erythromycin. Erythromycin has been previously described to be capable to permeabilize eukaryotic cells. After removing the culture supernatant, macrophages were thoroughly washed with sterile PBS, resuspended in 100 μl of ice-cold 0.1% Triton X-100 and incubated for 10 minutes at −20°C. After centrifugation, 5 μl of the resulting supernatants was dropped onto blood agar plates on which a lawn of S. pyogenes was seeded. The plates were incubated in 5% CO2 for 18 hours at 37°C. Mice were euthanized by CO2 asphyxiation, and their femurs and tibias were flushed with PBS. After hypotonic lysis of erythrocytes, cells were resuspended in HBSS, and the cell suspension was layered over a Percoll (Sigma, St. Louis, MO) gradient and centrifuged for 30 minutes at 1300 × g. The resulting neutrophil pellet was washed in PBS and suspended in Dulbecco's modified Eagle's medium. Neutrophils were counted on a Neubauer chamber, and the purity of the fractionated cell population was confirmed by flow cytometry examination. For determination of phagocytosis and killing of S. pyogenes, 5 × 105 neutrophils were preincubated with 1 μg/ml of PMA (Sigma) for 30 minutes and were washed and infected with 5 × 106 bacteria for 1 hour at 37°C, 5% CO2. Gentamicin (100 μg/ml) was added to kill extracellular bacteria and infected neutrophils were centrifuged 1 hour, 3 hours, and 5 hours thereafter. The cell pellet was then extensively washed and disrupted with dH2O plus Triton X-100 (0.1%) to release intracellular bacteria. The resulting suspension was serially diluted and the bacterial numbers were determined after plating onto blood agar. To generate air pouches, animals were anesthetized and 0.9 ml of air plus 100 μl of a suspension containing 5 × 107 CFU of S. pyogenes were injected subcutaneously on the back of the mouse. After 14 hours, mice were sacrificed, the air pouches were washed after incision of the skin, and the total number of cells was counted by using a hemocytometer. Cells were then stained with antibodies against Gr-1, CD19, CD11c, CD11b, CD4 or CD8a (PharMingen, San Diego, CA) and analyzed in a FACScalibur (Becton Dickinson, San Jose, CA). Tissues were fixed in 4% formaldehyde solution, embedded in paraffin, and then cut into 3-μm-thick sections. Sections were stained with H&E and were analyzed by using an Olympus Bx51 microscope. The degree of tissue eosinophilia was determined as previously described.25Bhattacharyya N Vyas DK Fechner FP Gliklich RE Metson R Tissue eosinophilia in chronic sinusitis: quantification techniques.Arch Otolaryngol Head Neck Surg. 2001; 127: 1102-1105Crossref PubMed Scopus (56) Google Scholar Briefly, stained tissue sections were examined at ×100 magnification, with a 10 × 10-mm reticulate present in the eyepiece. The total number of eosinophils present within this grid was determined as the eosinophil counts per high-power field. A total of five high-power fields were assessed by three independent individuals. Indirect quantification of eosinophils in tissue was performed by using an eosinophil peroxidase (EPO) assay as previously described.26Strath M Warren DJ Sanderson CJ Detection of eosinophils using an eosinophil peroxidase assay: its use as an assay for eosinophil differentiation factors.J Immunol Methods. 1985; 83: 209-215Crossref PubMed Scopus (257) Google Scholar Briefly, lungs were flushed through the pulmonary artery, homogenized in 2 ml of ice-cold 0.05 M Tris-HCl buffer (pH = 8) containing 0.1% Triton X-100, exposed to five freeze-thaw cycles, and centrifuged at 1600 × g for 5 minutes at 4°C. The EPO activity in the supernatant was measured based on the oxidation of o-phenylenediamine by EPO in the presence of hydrogen peroxide. It has been previously reported that o-phenylenediamine is a specific substrate for EPO, and that the assay is not affected by neutrophil myeloperoxidase in murine systems.26Strath M Warren DJ Sanderson CJ Detection of eosinophils using an eosinophil peroxidase assay: its use as an assay for eosinophil differentiation factors.J Immunol Methods. 1985; 83: 209-215Crossref PubMed Scopus (257) Google Scholar The substrate solution consists of 10 mmol/L o-phenylenediamine (Sigma) in 0.05 M Tris-HCl buffer (pH = 8) and 4 mmol/L hydrogen peroxide (Sigma). Substrate solution (100 μl) was added to lung supernatant samples (50 μl) in a 96-well microplate and incubated at room temperature for 60 minutes before the reaction was stopped by addition of 50 μl of 4 M sulfuric acid. The absorbance was then measured at 492 nm. For harvesting cells from the brochoalveolar lavage, mice were terminally anesthetized, the thoracic cavity was opened, and the lungs were exposed. A tube was inserted in the trachea and bronchoalveolar lavage was conducted with 2 × 0.5 ml of PBS containing complete protease inhibitor cocktail (Roche, Basel, Switzerland). The lavage was centrifuged and the supernatants were stored at −80°C until cytokine analysis. Data were analyzed by using Excel 2000 (Microsoft Office; Microsoft, Redmond, WA) or GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). Survival data were analyzed with Kaplan-Meier survival plots, followed by Long-rank test. The significance of differences between the values of an experimental group and those of the control group was determined by use of a variance analysis (F test). Significance levels were set at P < 0.05. It has been previously shown that MyD88 is required for the recognition of S. pyogenes by innate immune cells in vitro.20Loof TG Goldmann O Medina E Immune recognition of Streptococcus pyogenes by dendritic cells.Infect Immun. 2008; 76: 2785-2792Crossref PubMed Scopus (55) Google Scholar, 21Gratz N Siller M Schaljo B Pirzada ZA Gattermeier I Vojtek I Kirschning CJ Wagner H Akira S Charpentier E Kovarik P Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9.J Biol Chem. 2008; 283: 19879-19887Crossref PubMed Scopus (74) Google Scholar Here, we have evaluated the functional role of MyD88 signaling in the pathogenesis of S. pyogenes in vivo by using a previously described22Loof TG Rohde M Chhatwal GS Jung S Medina E The contribution of dendritic cells to host defenses against Streptococcus pyogenes.J Infect Dis. 2007; 196: 1794-1803Crossref PubMed Scopus (37) Google Scholar murine model of skin infection. In this experimental infection model, S. pyogenes is applied subcutaneously at the right front flank and disseminate from the skin to systemic organs. C57BL/6 and MyD88−/− mice were subcutaneously infected with 5 × 107 CFU of S. pyogenes and monitored for survival. As shown in Figure 1A, all MyD88−/− mice rapidly succumbed to infection, dying between 1 and 2 days after bacterial inoculation, whereas the median survival time of C57BL/6 animals was 5 days. Even when the mice were inoculated with a lower bacterial dose (104 CFU), MyD88−/− mice were still much more susceptible than C57BL/6 mice to S. pyogenes infection (Supplemental Figure S2, see http://ajp.amjpathol.org). Using the method published by Reed and Muench,23Reed LJ Muench H A simple method for estimating fifty percent end points.Am J Hyg. 1938; 27: 493-497Google Scholar we determined the 50% lethal dose to be approximately 2 × 106 CFU for C57BL/6 mice and less than 104 CFU for MyD88−/− mice. To determine whether the reason for the accelerated mortality of MyD88−/− mice was due to uncontrolled bacterial multiplication, bacterial burdens were measured in blood, livers, and lungs of S. pyogenes-infected mice at increasing times after bacterial inoculation. MyD88−/− mice exhibited a profound deficiency in their ability to control bacterial growth, with approximately 104 higher amount of bacteria in blood (Figure 1B), livers (Figure 1C), and lungs (Figure 1D) compared with the bacterial burdens in C57BL/6 animals at 20 hours of infection. In addition, significantly higher plasma levels of alanine aminotransferase (Figure 2A), aspartate aminotransferase (Figure 2B), creatin phosphokinase (Figure 2C), and lactate dehydrogenase (Figure 2D) were detected in MyD88−/− than in C57BL/6 mice at 20 hours after inoculation with S. pyogenes. The results suggest a higher degree of liver (alanine aminotransferase/aspartate aminotransferase), heart/lung (lactate dehydrogenase), and muscle (creatin phosphokinase) damage in MyD88−/− animals than in C57BL/6 mice. Therefore, ablation of MyD88 signaling enhanced significantly the severity of infection caused by S. pyogenes in mice. MyD88 signaling has been reported to be required for the production of certain cytokines by innate immune cells such as dendritic cells20L
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