Chemokine Receptor CXCR2 Mediates Bacterial Clearance Rather Than Neutrophil Recruitment in a Murine Model of Pneumonic Plague
2011; Elsevier BV; Volume: 178; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2010.11.067
ISSN1525-2191
AutoresNicholas A. Eisele, Hanni Lee-Lewis, Cynthia Besch‐Williford, Charles R. Brown, Deborah M. Anderson,
Tópico(s)Pharmacological Effects of Natural Compounds
ResumoPulmonary infection by Yersinia pestis causes pneumonic plague, a necrotic bronchopneumonia that is rapidly lethal and highly contagious. Acute pneumonic plague accompanies the up-regulation of pro-inflammatory cytokines and chemokines, suggesting that the host innate immune response may contribute to the development of disease. To address this possibility, we sought to understand the consequences of neutrophil recruitment during pneumonic plague, and we studied the susceptibility of C3H-HeN mice lacking the CXC chemokine KC or its receptor CXC receptor 2 (CXCR2) to pulmonary Y. pestis infection. We found that without Kc or Cxcr2, disease progression was accelerated both in bacterial growth and development of primary bronchopneumonia. When examined in an antibody clearance model, Cxcr2−/− mice were not protected by neutralizing Y. pestis antibodies, yet bacterial growth in the lungs was delayed in a manner associated with a neutrophil-mediated inflammatory response. After this initial delay, however, robust neutrophil recruitment in Cxcr2−/− mice correlated with bacterial growth and the development of fulminant pneumonic and septicemic plague. In contrast, attenuated Y. pestis lacking the conserved pigmentation locus could be cleared from the lungs in the absence of Cxcr2 indicating virulence factors within this locus may inhibit CXCR2-independent pathways of bacterial killing. Together, the data suggest CXCR2 uniquely induces host defense mechanisms that are effective against virulent Y. pestis, raising new insight into the activation of neutrophils during infection. Pulmonary infection by Yersinia pestis causes pneumonic plague, a necrotic bronchopneumonia that is rapidly lethal and highly contagious. Acute pneumonic plague accompanies the up-regulation of pro-inflammatory cytokines and chemokines, suggesting that the host innate immune response may contribute to the development of disease. To address this possibility, we sought to understand the consequences of neutrophil recruitment during pneumonic plague, and we studied the susceptibility of C3H-HeN mice lacking the CXC chemokine KC or its receptor CXC receptor 2 (CXCR2) to pulmonary Y. pestis infection. We found that without Kc or Cxcr2, disease progression was accelerated both in bacterial growth and development of primary bronchopneumonia. When examined in an antibody clearance model, Cxcr2−/− mice were not protected by neutralizing Y. pestis antibodies, yet bacterial growth in the lungs was delayed in a manner associated with a neutrophil-mediated inflammatory response. After this initial delay, however, robust neutrophil recruitment in Cxcr2−/− mice correlated with bacterial growth and the development of fulminant pneumonic and septicemic plague. In contrast, attenuated Y. pestis lacking the conserved pigmentation locus could be cleared from the lungs in the absence of Cxcr2 indicating virulence factors within this locus may inhibit CXCR2-independent pathways of bacterial killing. Together, the data suggest CXCR2 uniquely induces host defense mechanisms that are effective against virulent Y. pestis, raising new insight into the activation of neutrophils during infection. The mammalian respiratory tract has limited host defense mechanisms against pathogenic microbes. Upon infection, alveolar macrophages, fibroblasts, and endothelial and epithelial cells produce chemokines to attract immune effector cells, such as neutrophils, macrophages, and NK cells, to the lungs.1Hamilton T. Novotny M. Datta S. Mandal P. Hartupee J. Tebo J. Li X. 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Replication of Yersinia pestis in interferon γ-activated macrophages requires ripA, a gene encoded in the pigmentation locus.Proc Natl Acad Sci U S A. 2005; 102: 12909-12914Crossref PubMed Scopus (89) Google Scholar Furthermore, antibody-mediated phagocytosis of Y. pestis does not lead to its destruction inside activated macrophages in vitro, leaving the mechanism of immunity uncertain.47Noel B. Lilo S. Capurso D. Hill J. Bliska J. Yersinia pestis can bypass protective antibodies to LcrV and activation with gamma interferon to survive and induce apoptosis in murine macrophages.Clin Vaccine Immunol. 2009; 16: 1457-1466Crossref PubMed Scopus (16) Google Scholar In this work, we addressed the role of chemokines and neutrophils in plague, both during disease and in clearance after treatment with protective LcrV antibodies that broadly stimulate innate immune activation. We studied mice defective in signaling through CXC chemokines and characterized host responses to pulmonary Y. pestis challenge. We found that CXCR2 provided protective responses in both models, but had minimal impact on neutrophil recruitment to infected sites. Early containment of the infection was seen in anti-LcrV-treated Cxcr2−/− mice in a manner that was associated with neutrophil recruitment and little to no damaged lung tissue. Despite these seemingly protective responses, however, Cxcr2−/− mice later developed systemic disease and acute bronchopneumonia that were indistinguishable from untreated mice, suggesting that other, seemingly redundant, pathways of neutrophil recruitment and activation may be ineffective against Y. pestis. Systemic ablation of neutrophils also resulted in loss of antibody protection that mimicked the loss of protection seen in Cxcr2−/− mice consistent with CXCR2 as the primary mechanism for inducing bacterial clearance. Deletion of the pigmentation (pgm) locus rendered Y. pestis sensitive to CXCR2-independent clearance, indicating the pgm locus encodes virulence factors that likely inhibit activation of neutrophils. Together the data support a model whereby CXCR2 signaling of neutrophils is necessary to destroy virulent Y. pestis, whereas other chemotactic pathways stimulate recruitment of neutrophils that are inactivated by products of the pgm locus, allowing rapid bacterial replication and fulminant disease. All Y. pestis culture strains used were taken from frozen stocks and streaked for isolation onto heart infusion agar plates. The plates used for Y. pestis CO92 were supplemented with 0.005% Congo red and 0.2% galactose to screen for bacteria that retain the pigmentation locus.48Surgalla M. Beesley E. Congo red-agar plating medium for detecting pigmentation in Pasteurella pestis.Appl Microbiol. 1969; 18: 834-837PubMed Google Scholar For the pneumonic plague challenge, a single, red-pigmented colony was used to inoculate heart infusion broth supplemented with 2.5 mmol/L CaCl2 and grown 18 to 24 hours at 37°C at 120 rpm. All handling of samples containing live Y. pestis CO92 was performed in a select agent authorized BSL3 facility under protocols approved by the University of Missouri Institutional Biosafety Committee. Nonpigmented Y. pestis CO92 and KIM D27 were routinely grown fresh from frozen stock on heart infusion agar, followed by aerobic growth at 27°C in heart infusion broth overnight before use in experiments.33Lee-Lewis H. Anderson D. Absence of inflammation and pneumonia during infection with non-pigmented Yersinia pestis reveals new role for the pgm locus in pathogenesis.Infect Immun. 2010; 78: 220-230Crossref PubMed Scopus (32) Google Scholar, 42Brubaker R. Beesley E. Surgalla M. Pasteurella pestis: Role of Pesticin I and iron in experimental plague.Science. 1965; 149: 422-424Crossref PubMed Scopus (93) Google Scholar All animal procedures were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Missouri Animal Care and Use Committee. C3H-HeNCrl mice were used for studies on host responses to plague and were the parent strain of the Kc−/− and Cxcr2−/− mice. Wild-type C3H mice were purchased from Charles River Laboratories. Knockout mice were bred and housed in barrier containment facilities at the University of Missouri. Males and females, ranging from 15 to 50 g were used for challenge experiments. During challenge, mice were maintained in select agent-approved animal containment facilities at the University of Missouri. All infected mice were monitored regularly by daily weighing and assignment of health scores. Animals that survived to the end of the 14-day observation period or were identified as moribund (defined by pronounced ataxia, sometimes accompanied by severe dyspnea) were euthanized by CO2 asphyxiation, followed by bilateral pneumothorax; these methods are approved by the American Veterinary Medical Association Guidelines on Euthanasia. Y. pestis CO92, grown as previously described at 37°C, were diluted in sterile PBS to 400, 4000, or 6000 colony forming units (CFU)/0.02 ml just before use for challenge experiments. Actual dose and retention of the pigmentation locus were determined by plating in triplicate on heart infusion agar with Congo red. Where indicated, for some intranasal infections involving nonpigmented Y. pestis strains, mice were given 500 μg FeCl2 by intraperitoneal injection just before challenge. All animals intranasally infected with Y. pestis were first lightly anesthetized by isoflurane inhalation. Animals were observed for recovery from anesthesia and returned to housing. Rabbit polyclonal LcrV was produced as previously described; serum antibody titer to recombinant LcrV was 105 for all experiments.39Eisele N. Anderson D. Dual-function antibodies to Yersinia pestis LcrV required for pulmonary clearance of plague.Clin Vacc Immunol. 2009; 16: 1720-1727Crossref PubMed Scopus (16) Google Scholar For purification, rabbit serum containing α-LcrV antibodies was applied to a protein G column and purified according to the manufacturer's protocol (Sigma, St. Louis, MO). Samples were then applied to a PD-10 buffer exchange column (GE Healthcare, Buckinghamshire, UK) and eluted in PBS. Total IgG was quantified using bovine IgG as a standard in a BCA protein assay (Pierce, Rockford, IL). For pneumonic plague challenges, 400 μg/0.4 ml purified antibody was given by intraperitoneal injection 60 minutes before infection. Untreated control mice were given 400 μl sterile PBS by intraperitoneal injection 60 minutes before challenge. In some experiments, mice were given rat anti-mouse Gr-1 (RB6.8C5) monoclonal antibodies (BD Pharmingen, San Jose, CA) intraperitoneally in 100-μg (100 μg/100 μl) or 200-μg (200 μg/200 μl) doses at the times indicated. Control mice in these experiments were given equivalent volumes of PBS. Immediately after euthanasia, blood was collected directly from the heart by cardiac puncture. Lungs, spleens, and livers were collected aseptically and homogenized in 1 ml sterile PBS. Serial dilutions of the blood and homogenized tissues were then plated onto heart infusion agar plates for quantification of bacterial load (CFU/ml or CFU/organ, res
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