Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Contribution of NK Cells to the Innate Phase of Host Protection Against an Intracellular Bacterium Targeting Systemic Endothelium

2012; Elsevier BV; Volume: 181; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2012.03.020

ISSN

1525-2191

Autores

Rong Fang, Nahed Ismail, David H. Walker,

Tópico(s)

Autoimmune and Inflammatory Disorders Research

Resumo

We investigated the mechanisms by which natural killer (NK) cells mediate innate host defense against infection with an endothelium-targeting intracellular bacterium, Rickettsia. We found that a robust Rickettsia-induced innate response in resistant mice cleared the bacteria early in the infection and was associated with significantly higher frequencies of splenic interferon (IFN)-γ (+) CD8+ T cells and cytotoxic NK cells compared with susceptible mice. More importantly, NK cell-deficient Rag−/−γc−/− animals displayed significantly increased susceptibility to Rickettsia infection compared with NK cell-sufficient Rag−/− mice, as evidenced by impaired bacterial clearance, early development of severe thrombosis in the liver, and a decreased serum level of IFN-γ. Furthermore, the lack of NK cells also impaired host resistance of CB-17 scid mice to Rickettsia, similar to what was observed in Rag−/−γc−/− mice. Interestingly, perforin deficiency in Rag−/−Prf1−/− mice resulted in greater thrombosis and insignificantly different systemic levels of IFN-γ compared with Rag−/− mice, suggesting that perforin, which is mainly produced by NK cells, is involved in the prevention of vascular damage. Together, these findings reveal that NK cells mediate the innate phase of host protection against infection with rickettsiae, most likely via IFN-γ production. Furthermore, NK cells are involved in preventing rickettsial infection-induced endothelial cell damage, possibly via perforin production. We investigated the mechanisms by which natural killer (NK) cells mediate innate host defense against infection with an endothelium-targeting intracellular bacterium, Rickettsia. We found that a robust Rickettsia-induced innate response in resistant mice cleared the bacteria early in the infection and was associated with significantly higher frequencies of splenic interferon (IFN)-γ (+) CD8+ T cells and cytotoxic NK cells compared with susceptible mice. More importantly, NK cell-deficient Rag−/−γc−/− animals displayed significantly increased susceptibility to Rickettsia infection compared with NK cell-sufficient Rag−/− mice, as evidenced by impaired bacterial clearance, early development of severe thrombosis in the liver, and a decreased serum level of IFN-γ. Furthermore, the lack of NK cells also impaired host resistance of CB-17 scid mice to Rickettsia, similar to what was observed in Rag−/−γc−/− mice. Interestingly, perforin deficiency in Rag−/−Prf1−/− mice resulted in greater thrombosis and insignificantly different systemic levels of IFN-γ compared with Rag−/− mice, suggesting that perforin, which is mainly produced by NK cells, is involved in the prevention of vascular damage. Together, these findings reveal that NK cells mediate the innate phase of host protection against infection with rickettsiae, most likely via IFN-γ production. Furthermore, NK cells are involved in preventing rickettsial infection-induced endothelial cell damage, possibly via perforin production. Rickettsiae are obligately intracellular α-proteobacteria that primarily target the microvascular endothelium.1Walker D.H. Rocky Mountain spotted fever: a seasonal alert.Clin Infect Dis. 1995; 20: 1111-1117Crossref PubMed Scopus (102) Google Scholar, 2Woods M. Olano J. Host defenses to Rickettsia rickettsii infection contribute to increased microvascular permeability in human cerebral endothelial cells.J Clin Immunol. 2008; 28: 174-185Crossref PubMed Scopus (43) Google Scholar The main pathogenic mechanism involved in rickettsial disease is increased systemic microvascular permeability leading to edema, hypovolemia, and hypotension. The severity of rickettsial infection in both human and animal models is dependent on bacterial virulence, host factors, and bacterial dose.3Walker D.H. Hawkins H.K. Hudson P. Fulminant Rocky Mountain spotted fever Its pathologic characteristics associated with glucose-6-phosphate dehydrogenase deficiency.Arch Pathol Lab Med. 1983; 107: 121-125PubMed Google Scholar, 4Walker D.H. The role of host factors in the severity of spotted fever and typhus rickettsioses.Ann NY Acad Sci. 1990; 590: 10-19Crossref PubMed Scopus (41) Google Scholar, 5Walker D.H. Popov V.L. Wen J. Feng H.M. Rickettsia conorii infection of C3H/HeN mice A model of endothelial-target rickettsiosis.Lab Invest. 1994; 70: 358-368PubMed Google Scholar, 6Feng H.M. Walker D.H. Cross-protection between distantly related spotted fever group rickettsiae.Vaccine. 2003; 21: 3901-3905Crossref PubMed Scopus (25) Google Scholar Rocky Mountain spotted fever caused by Rickettsia rickettsii and Mediterranean spotted fever caused by R. conorii are considered to be important due to their wide geographic distribution and a potentially fatal outcome in severe cases.7Weinberger M. Keysary A. Sandbank J. Zaidenstein R. Itzhaki A. Strenger C. Leitner M. Paddock C.D. Eremeeva M.E. Fatal Rickettsia conorii subsp. israelensis infection.Israel Emerg Infect Dis. 2008; 14: 821-824Crossref PubMed Scopus (20) Google Scholar, 8Aliaga L. Sánchez-Blázquez P. Rodríguez-Granger J. Sampedro A. Orozco M. Pastor J. Mediterranean spotted fever with encephalitis.J Med Microbiol. 2009; 58: 521-525Crossref PubMed Scopus (23) Google Scholar, 9Papa A. Dalla V. Petala A. Maltezou H.C. Maltezos E. Fatal Mediterranean spotted fever in Greece.Clin Microbiol Infect. 2010; 16: 589-592Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 10de Sousa R. Nobrega S.D. Bacellar F. Torgal J. Mediterranean spotted fever in Portugal risk factors for fatal outcome in 105 hospitalized patients.Ann NY Acad Sci. 2003; 990: 285-294Crossref PubMed Scopus (120) Google Scholar On the other hand, other rickettsial diseases, such as African tick bite fever caused by R. africae, present as a mild disease. In addition, the severity of rickettsial diseases is in large part determined by host factors.3Walker D.H. Hawkins H.K. Hudson P. Fulminant Rocky Mountain spotted fever Its pathologic characteristics associated with glucose-6-phosphate dehydrogenase deficiency.Arch Pathol Lab Med. 1983; 107: 121-125PubMed Google Scholar, 4Walker D.H. The role of host factors in the severity of spotted fever and typhus rickettsioses.Ann NY Acad Sci. 1990; 590: 10-19Crossref PubMed Scopus (41) Google Scholar Fulminant Rocky Mountain spotted fever often occurs in African-American males with glucose-6-phosphate dehydrogenase deficiency associated with an overwhelming bacterial load, extensive endothelial damage, and thrombosis.3Walker D.H. Hawkins H.K. Hudson P. Fulminant Rocky Mountain spotted fever Its pathologic characteristics associated with glucose-6-phosphate dehydrogenase deficiency.Arch Pathol Lab Med. 1983; 107: 121-125PubMed Google Scholar, 4Walker D.H. The role of host factors in the severity of spotted fever and typhus rickettsioses.Ann NY Acad Sci. 1990; 590: 10-19Crossref PubMed Scopus (41) Google Scholar Furthermore, we have previously established several murine models of spotted fever rickettsiosis with different mouse strains and different bacterial inocula5Walker D.H. Popov V.L. Wen J. Feng H.M. Rickettsia conorii infection of C3H/HeN mice A model of endothelial-target rickettsiosis.Lab Invest. 1994; 70: 358-368PubMed Google Scholar, 11Feng H.M. Wen J. Walker D.H. Rickettsia australis infection: a murine model of a highly invasive vasculopathic rickettsiosis.Am J Pathol. 1993; 142: 1471-1482PubMed Google Scholar, 12Walker D.H. Popov V.L. Feng H.M. Establishment of a novel endothelial target mouse model of a typhus group rickettsiosis: evidence for critical roles for gamma interferon and CD8 T lymphocytes.Lab Invest. 2000; 80: 1361-1372Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar. For example, C3H mice are genetically susceptible to a high, but not a low dose, of R. conorii, whereas C57BL/6 (B6) mice are highly resistant to both inocula.13Fang R. Ismail N. Shelite T. Walker D.H. Differential interaction of dendritic cells with Rickettsia conorii: impact on host susceptibility to murine spotted fever rickettsiosis.Infect Immun. 2007; 75: 3112-3123Crossref PubMed Scopus (48) Google Scholar Using these murine models of spotted fever rickettsiosis, we found that protective adaptive immunity during primary infection correlates with induction of strong cell-mediated immunity, including effector CD8+ CTLs, Th1 cells, and production of inflammatory cytokines, such as interferon (IFN)-γ and tumor necrosis factor-α.14Feng H.M. Walker D.H. Interferon-gamma and tumor necrosis factor-alpha exert their antirickettsial effect via induction of synthesis of nitric oxide.Am J Pathol. 1993; 143: 1016-1023PubMed Google Scholar, 15Feng H.M. Popov V.L. Walker D.H. Depletion of gamma interferon and tumor necrosis factor alpha in mice with Rickettsia conorii-infected endothelium: impairment of rickettsicidal nitric oxide production resulting in fatal, overwhelming rickettsial disease.Infect Immun. 1994; 62: 1952-1960Crossref PubMed Google Scholar, 16Feng H. Popov V.L. Yuoh G. Walker D.H. Role of T lymphocyte subsets in immunity to spotted fever group Rickettsiae.J Immunol. 1997; 158: 5314-5320Crossref PubMed Google Scholar, 17Valbuena G. Feng H.M. Walker D.H. Mechanisms of immunity against rickettsiae New perspectives and opportunities offered by unusual intracellular parasites.Microbes Infect. 2002; 4: 625-633Crossref PubMed Scopus (57) Google Scholar The mechanisms involved in the innate phase of host responses against rickettsial infection in resistant and susceptible murine hosts, however, remain ill-defined. Natural killer (NK) cells are essential effectors of the innate immune system against infections as they mediate elimination of a variety of pathogens through secretion of IFN-γ and perforin/granzyme-mediated killing.18Korbel D.S. Finney O.C. Riley E.M. Natural killer cells and innate immunity to protozoan pathogens.Int J Parasitol. 2004; 34: 1517-1528Crossref PubMed Scopus (90) Google Scholar, 19Bancroft G.J. The role of natural killer cells in innate resistance to infection.Curr Opin Immunol. 1993; 5: 503-510Crossref PubMed Scopus (282) Google Scholar, 20Scharton-Kersten T.M. Sher A. Role of natural killer cells in innate resistance to protozoan infections.Curr Opin Immunol. 1997; 9: 44-51Crossref PubMed Scopus (103) Google Scholar, 21Lodoen M.B. Lanier L.L. Natural killer cells as an initial defense against pathogens.Curr Opin Immunol. 2006; 18: 391-398Crossref PubMed Scopus (349) Google Scholar, 22Paust S. Senman B. von Andrian U.H. Adaptive immune responses mediated by natural killer cells.Immunol Rev. 2010; 235: 286-296Crossref PubMed Scopus (111) Google Scholar, 23French A.R. Yokoyama W.M. Natural killer cells and viral infections.Curr Opin Immunol. 2003; 15: 45-51Crossref PubMed Scopus (232) Google Scholar There is increasing evidence, however, that cytotoxic granules produced by NK or CD8+ T cells are involved in development of immunopathology after infections with certain pathogens, such as lymphocytic choriomeningitis virus and Epstein-Barr virus.24de Saint Basile G.F.A. The role of cytotoxicity in lymphocyte homeostasis.Curr Opin Immunol. 2001; 13: 549-554Crossref PubMed Scopus (72) Google Scholar, 25Matloubian M. Suresh M. Glass A. Galvan M. Chow K. Whitmire J.K. Walsh C.M. Clark W.R. Ahmed R. A role for perforin in downregulating T-cell responses during chronic viral infection.J Virol. 1999; 73: 2527-2536Crossref PubMed Google Scholar, 26Jordan M.B. Hildeman D. Kappler J. Marrack P. An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder.Blood. 2004; 104: 735-743Crossref PubMed Scopus (514) Google Scholar Other studies suggest that infection-induced immunopathology can be restricted by perforin-dependent negative regulation of cytotoxic T lymphocytes responses.25Matloubian M. Suresh M. Glass A. Galvan M. Chow K. Whitmire J.K. Walsh C.M. Clark W.R. Ahmed R. A role for perforin in downregulating T-cell responses during chronic viral infection.J Virol. 1999; 73: 2527-2536Crossref PubMed Google Scholar, 27Kägi D. Odermatt B. Mak T.W. Homeostatic regulation of CD8+ T cells by bperforin.Eur J Immunol. 1999; 29: 3262-3272Crossref PubMed Scopus (137) Google Scholar, 28Badovinac V.P. Hamilton S.E. Harty J.T. Viral infection results in massive CD8+ T cell expansion and mortality in vaccinated perforin-deficient mice.Immunity. 2003; 18: 463-474Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar In the present work, we used various mouse strains with different susceptibilities to infection with R. conorii, and we studied the contributions of NK and T cells to host immunity, particularly during the early phase. We found that rapid bacterial clearance at the early phase of infection in the resistant host was associated with an increased production level of IFN-γ by CD8 T cells, and enhanced levels of activation and cytotoxic activity of NK cells. Further genetic manipulation of resistant hosts with a high rickettsial challenge dose proved our hypothesis that NK cells contribute greatly to the early phase of host protection, independent of acquired T-cell responses, through effective bacterial elimination, as well as preventing infection-induced pathology. Rickettsia conorii (Malish 7 strain) was obtained from the ATCC (VR 613; Manassas, VA). For animal inoculation, rickettsiae were cultivated in specific pathogen-free embryonated chicken eggs. After homogenization, rickettsiae were diluted in a 10% suspension of sucrose-phosphate-glutamate buffer (0.218 mmol/L sucrose, 3.8 mmol/L KH2PO4, 7.2 mmol/L K2HPO4, 4.9 mmol/L monosodium glutamic acid, pH 7.0). The concentration of rickettsiae from yolk sac was determined by plaque assay and quantitative real-time PCR, described as follows. The rickettsial stock was stored at −80°C until used. Plaque assay for testing the quantity of viable rickettsiae in the infected tissue was performed as previously described.5Walker D.H. Popov V.L. Wen J. Feng H.M. Rickettsia conorii infection of C3H/HeN mice A model of endothelial-target rickettsiosis.Lab Invest. 1994; 70: 358-368PubMed Google Scholar Wild-type (WT) female C3H/HeN mice, NK cell-deficient-scid mice on CB-17 background and scid mice on CB-17 background were purchased from Harlan Laboratories (Indianapolis, IN) and used at 6 to 10 weeks of age. Age- and sex-matched WT CB-17 mice, B6 mice, and T-cell- and B-cell-deficient Rag−/− mice, NK cell-deficient-Rag−/− mice (Rag−/−γc−/−), and perforin-deficient- Rag−/− mice (Rag−/−Prf1−/−) were purchased from Jackson Laboratories (Bar Harbor, Maine) and Taconic Farms Inc. (Hudson, NY). Mice were housed in a biosafety level 3 facility at the University of Texas Medical Branch, Galveston, TX. All experiments and procedures were approved by the University of Texas Medical Branch Animal Care and Use Committee, and mice were used according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Different mouse strains were infected intravenously with R. conorii at different doses as follows: WT C3H and B6 mice were inoculated with a low dose (3 × 104 plaque forming units) and a high dose (3 × 105 plaque forming units); Rag−/−, NK cell-depleted Rag−/−, Rag−/−γc−/−, and Rag−/−Prf1−/− mice were inoculated with a high dose (3 × 105 plaque forming units). Negative control mice were inoculated with sucrose-phosphate-glutamate buffer or 10% uninfected yolk sac processed in the same way as infected yolk sac, as previously described. Mice were monitored daily for signs of illness. For NK cell depletion, a nonactivating polyclonal antibody against asialo-GM1 (Wako Chemicals, Inc., Richmond, VA) was used as previously described.29Kang S.J. Liang H.E. Reizis B. Locksley R.M. Regulation of hierarchical clustering and activation of innate immune cells by dendritic cells.Immunity. 2008; 29: 819-833Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar Rag−/− mice on B6 background were inoculated i.v. with 50 μL of 1:80 dilution of anti-asialo-GM1 antibody on days 0, 2, and 5 of infection. Depletion of NK cells was approximately 93% efficient, as determined by flow cytometric analysis of the number of DX5 (+) CD3 (−) NK cells in the spleen of depleted mice compared to the sham-depleted mice. Rickettsial burdens in the livers of infected mice were determined using an iCycler IQ from BioRad (Hercules, CA). Primers (Sigma-Genosys, St. Louis, MO) and probes (Biosearch Technologies, Novato, CA) targeting R. conorii ompB and mouse GAPDH genes were used as previously described.30Valbuena G. Bradford W. Walker D.H. Expression analysis of the T-cell-targeting chemokines CXCL9 and CXCL10 in mice and humans with endothelial infections caused by rickettsiae of the spotted fever group.Am J Pathol. 2003; 163: 1357-1369Abstract Full Text Full Text PDF PubMed Scopus (79) Google ScholarThe results were normalized to and expressed as ompB copy number per 106 copies of GAPDH. For NK cell cytotoxicity assays, splenocytes were isolated from infected and uninfected C3H and B6 mice. Target YAC-1 cells (ATCC, Manassas, VA), mouse lymphoma cells which are the optimal target for mouse NK cells, were stained with 3, 3′-dioctadecyloxacarbocyanine using the LIVE/DEAD Cell-Mediated Cytotoxicity Kit (Molecular Probes, Inc., Eugene, OR). Effector spleen cells were isolated and co-cultured with YAC-1 cells (ATCC, Manassas, VA) at an effector: target cell ratio of 100:1. Cells were then collected, washed, and stained with Live/DEAD Fixable Violet Dead Cell Stain Kits (Life Technologies, Grand Island, NY), according to the manufacturer's instructions. The cells were analyzed by flow cytometry after 4 hours co-culture. The percent specific lysis was determined as follows: 100 × (experimental lysis–spontaneous lysis)/(maximum lysis–spontaneous lysis). The percent specific lysis was normalized to the number of NK cells in the spleen. Infected mice were sacrificed on day 2 postinfection (p.i.), and the spleen and serum were collected. Splenocytes were cultured in 96-well round bottom plates containing 5 × 105 cells/well, or 24-well plates containing 1.5 × 106 cells/well with or without rickettsial antigen stimulation. The culture supernatants were collected after 72 hours. The concentrations of cytokines and chemokines in the culture supernatant and sera were determined by quantitative ELISA kit (R&D Systems, Minneapolis, MN) or microsphere multiplexed cytokine immunoassays (Bio-Plex Cytokine Assay, Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Spleen cells were isolated and stimulated with or without rickettsial antigens or phorbol 12-myristate 13-acetate (10 ng/mL) and ionomycin (400 ng/mL) in the presence of Golgi stop (BD Bioscience, San Diego, CA). Then the cells were suspended in fluorescence activated cell sorter buffer (PBS containing 0.1% bovine serum albumin and 0.01% NaN3). Fc receptors were blocked with anti-CD16/32 (clone 2.4G2). The following fluorescein isothiocyanate-, phycoerythrin (PE)-, peridinin chlorophyll protein Cy5.5 (PerCP-Cy5.5)-, and allophycocyanin (APC)-conjugated antibodies were purchased from BD Bioscience unless indicated otherwise: APC- or PE–anti-CD3 (clone 145-2C11), PE–anti-IFN-γ (clone XMG1.2), PE–anti-IL-12R β1 chain (clone 114), PE–anti-CD69 (clone HI.2F3), PercP– or APC–anti-CD8 (clone 53–6.7), and PercP– or APC–anti-CD4 (clone RM4-5). Isotype control antibodies included fluorescein isothiocyanate-, PE-, PercP-Cy5.5- and APC-conjugated hamster IgG1 (clone A19-3), rat IgG1 (clone R3-34), and rat IgG2a (clone R35-95). Specific antibodies including PE–anti-granzyme B (clone 16G6), fluorescein isothiocyanate–anti- CD49b (clone DX5, Pan-NK cells), and isotype control antibodies, including rat IgG2b and rat IgM were purchased from eBioscience (San Diego, CA); 20,000 events were collected using the FACSCalibur or FACSCanto system (BD Biosciences, Franklin Lakes, NJ). Data were analyzed with FlowJo software version 7.6.1 (TreeStar Inc., Ashland, OR). Formalin-fixed, paraffin-embedded liver and lung samples were sectioned and stained with H&E. The quantity of pathological foci in livers in 10 high-power fields was determined using MetaMorph for Olympus (Olympus America Inc., Center Valley, PA). Thrombi were confirmed by staining with anti-mouse fibrinogen (Abbiotec, LLC., San Diego, CA) polyclonal antibody using Vectastain ABC reagents and Vector Red substrate (Vector Laboratories Inc., Burlingame, CA). For comparison of mean values of different experimental groups, the one-way analysis of variance or paired t-test was determined using GraphPad Prism software version 5.01. Posthoc group pairwise comparisons were conducted using the Bonferroni procedure and overall α level of significance of 0.05. For testing the difference in survival between different mouse groups, data were analyzed by the product limit (Kaplan-Meier) method using GraphPad Prism software version 5.01. A difference in mean values was deemed significant when P < 0.05. We first determined the bacterial loads in the liver of susceptible and resistant mice during the course of infection. Rickettsiae replicated progressively in susceptible mice and reached a peak before mice died, whereas bacteria were substantially cleared in fewer than 4 days in resistant B6 mice (Figure 1A). Using plaque assay, the decreased bacterial burden detected by real-time PCR in the liver of resistant B6 mice correlated with the presence of live rickettsiae in the lung of B6 mice at both 6 and 24 hours after infection with a high dose of R. conorii (Figure 1B). In addition, infected liver from B6 mice contained multiple inflammatory foci at 2 and 5 days p.i., even though rickettsiae were completely cleared at these time points (Figure 1C). Next we determined the acquired T-cell response in C3H and B6 mice infected with low and high doses of R. conorii. All B6 mice survived both doses of rickettsiae, whereas all C3H mice died on inoculation of the high dose after approximately 6 to 7 days, as shown in the previous studies.5Walker D.H. Popov V.L. Wen J. Feng H.M. Rickettsia conorii infection of C3H/HeN mice A model of endothelial-target rickettsiosis.Lab Invest. 1994; 70: 358-368PubMed Google Scholar, 13Fang R. Ismail N. Shelite T. Walker D.H. Differential interaction of dendritic cells with Rickettsia conorii: impact on host susceptibility to murine spotted fever rickettsiosis.Infect Immun. 2007; 75: 3112-3123Crossref PubMed Scopus (48) Google Scholar Greater bacterial expansion in susceptible C3H mice resulted in a significantly higher number of IFN-γ-producing CD4+ T cells (Figure 2, A and B), but not IFN-γ-producing CD8+ T cells (Figure 2, C and D) on day 9 p.i., compared to resistant B6 mice with the same inocula. Collectively, these results suggested that R. conorii established infection and initiated adaptive immune responses in the resistant host. Furthermore, these data suggest that innate responses play an important role in mediating host control of rickettsial infection.Figure 2Adaptive immune response was initiated by infection with R. conorii in vivo in both resistant B6 mice and susceptible C3H mice. WT C3H and B6 mice were inoculated with a low dose and a high dose of R. conorii (as described in Materials and Methods). Mice were sacrificed on day 9 postinfection (p.i.). Splenocytes were stimulated with antigen in vitro (as described in Materials and Methods). The percentage and absolute number of IFN-γ- producing CD4+ CD3+ T cells (A, B) and CD8+ CD3+ T cells (C, D) were determined by flow cytometric analysis. Flow cytometry dot plots (A, C) reveal the percentage of IFN-γ producing cells, which are quantified in (B, D). Each group includes five mice, and the results shown represent two independent experiments. *P < 0.05; ns, not statistically significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next we examined the response of T- and B-cell deficient (ie, Rag−/−) mice to an infection with a high dose of R. conorii. Similar to our previous data, all WT C3H mice succumbed to infection, whereas WT B6 and Rag−/− mice survived infection (Figure 3A). No Rag−/− mice succumbed to infection under observation until day 35 (data not shown). Interestingly, lack of T and B lymphocytes in Rag−/− mice resulted in a fourfold greater bacterial burden in the spleen compared to WT B6 mice on day 2 p.i. (Figure 3B), whereas no significant difference in bacterial burden between the two groups of mice was observed on day 5 p.i. These data suggested that although acquired immunity is dispensable for host survival after infection with Rickettsia in these animals, T and B lymphocytes contributed to early elimination of rickettsiae. Induction of IFN-γ producing Th1-type responses is critical for protection against infections with intracellular pathogens including rickettsiae.15Feng H.M. Popov V.L. Walker D.H. Depletion of gamma interferon and tumor necrosis factor alpha in mice with Rickettsia conorii-infected endothelium: impairment of rickettsicidal nitric oxide production resulting in fatal, overwhelming rickettsial disease.Infect Immun. 1994; 62: 1952-1960Crossref PubMed Google Scholar Thus, we examined whether the robust innate response in the resistant host is mediated by IFN-γ. At the single cell level, protective immunity in resistant B6 mice was associated with a significantly higher frequency of IFN-γ-producing splenocytes in B6 mice compared to C3H mice on day 2 p.i. (Figure 4A). Efficient bacterial clearance at the early stage of infection in B6 mice was associated with increased frequency of IFN-γ-producing splenic CD8+ T cells (Figure 4, B and C) compared to susceptible mice. These CD8+ T cells expressed memory phenotype markers (CD62Llow CD44high), suggesting effector memory CD8+ T cells (data not shown). These results suggested that IFN-γ produced by activated memory CD8+ T cells contributed to a host control of rickettsial infection at the early stage. Excessive systemic or local production of IFN-γ and other proinflammatory cytokines can be detrimental to the host immune responses against infection.31Feng C.G. Kaviratne M. Rothfuchs A.G. Cheever A. Hieny S. Young H.A. Wynn T.A. Sher A. NK cell-derived IFN-gamma differentially regulates innate resistance and neutrophil response in T cell-deficient hosts infected with Mycobacterium tuberculosis.J Immunol. 2006; 177: 7086-7093Crossref PubMed Scopus (172) Google Scholar, 32Stevenson H.L. Estes M.D. Thirumalapura N.R. Walker D.H. Ismail N. Natural killer cells promote tissue injury and systemic inflammatory responses during fatal Ehrlichia-induced toxic shock-like syndrome.Am J Pathol. 2010; 177: 766-776Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Our data show that splenocytes from uninfected C3H mice produced a significantly higher level of IL-10 compared to resistant mice, but no difference in the levels of IFN-γ production (Figure 5A). In contrast, splenocytes of infected C3H mice produced a suppressed level of IFN-γ and an enhanced level of IL-10 after antigen stimulation compared to uninfected C3H mice and infected B6 mice (Figure 5A). Furthermore, infection of susceptible hosts with R. conorii increased the serum level of IFN-γ, but not IL-12p40, compared to resistant mice (Figure 5B). Resistance of B6 mice against fatal Rickettsia infection was associated with significantly lower serum levels of monocyte chemoattractant protein-1 (Figure 5B) compared to susceptible mice. These results suggested that enhanced susceptibility to rickettsial infection was associated with a suppressed type 1 response in the spleen but increased systemic production of monocyte chemoattractant protein-1. Next we examined whether differential host susceptibility to Rickettsia infection is associated with altered activation and cytotoxic activity of NK cells. Our data showed that infection of B6 mice with R. conorii significantly enhanced activation of NK cells, as evidenced by greater expression of CD69 and IL-12p40 receptor β1 on NK cells when compared to susceptible C3H mice (Figure 6A). Furthermore, the frequency of granzyme-B-expressing splenic NK cells (Figure 6B) and NK cell cytotoxic activity, measured by the percentage of YAC-1 cell killing at an effector: target cell ratio of 100: 1 (Figure 6C), was significantly higher in infected resistant mice compared to susceptible mice on days 1 and/or 2 p.i. (Figure 6B). Interestingly, protective immunity in B6 mice was associated with significantly lower frequencies of IFN-γ-producing NK cells when compared to susceptible C3H mice on day 2 p.i. (Figure 6, D–F). These data do not exclude a potential protective role of IFN-γ produced by NK cells in host defense against Rickettsia, although it suggests that NK cell-mediated cytotoxic killing of infected target cells plays a pivotal role in effective rickettsial elimination. To determine whether genetic background influences the effector function of NK cells against R. conorii, we examined the outcome of infection and host responses in NK cell competent or deficient CB-17 scid mice and Rag−/− mice. Similar to highly resistant B6 mice and Rag−/− mice, relatively resistant WT CB-17 mice and CB-17 scid mice survived the same dose of infection with R. conorii (Figure 7A). Interestingly, NK cell-deficient CB-17 scid mice were more susceptible to fatal disease, as demonstrated by ∼50% survival compared to 100% survival of CB-17 scid mice (Figure 7A). On day 2 p.i., R. conorii-infected NK cell-deficient CB-17 scid mice had a negligible percentage (ie, <1%) of IFN-γ- and granzyme B-expressing NK cells compared to CB-17 scid mice (data not shown).

Referência(s)