Role of p47phox in Antigen-Presenting Cell-Mediated Regulation of Humoral Immunity in Mice
2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.02.038
ISSN1525-2191
AutoresSam Vasilevsky, Qi Liu, Sherry Koontz, Robin J. Kastenmayer, Katherine Shea, Sharon H. Jackson,
Tópico(s)Immune cells in cancer
ResumoMicrobial-induced inflammation is important for eliciting humoral immunity. Genetic defects of NADPH oxidase 2–based proteins interrupt phagocyte superoxide generation and are the basis for the human immunodeficiency chronic granulomatous disease (CGD). Hyperinflammation is also a significant clinical manifestation of CGD. Herein, we evaluated humoral immunity in the phagocyte oxidase p47phox-deficient model of CGD and found that UV-inactivated Streptococcus pneumoniae and Listeria monocytogenes (Lm) elicited higher specific antibody (Ab) titers in p47phox-/- mice than wild-type (WT) mice. Both organisms elicited robust and distinct antigen-presenting cell maturation phenotypes, including IL-12 hypersecretion, and higher major histocompatibility complex II and costimulatory protein expression in Lm-stimulated p47phox-/- dendritic cells (DCs) relative to WT DCs. Furthermore, p47phox-/- DCs pulsed with Lm and adoptively transferred into naïve WT mice elicited Ab titers, whereas Lm-pulsed WT DCs did not elicit these titers. The observed robust p47phox-/- mouse humoral response was recapitulated with live Lm and sustained in vivo in p47phox-/- mice. Notably, anti–serum samples from p47phox-/- mice that survived secondary Lm infection were protective in WT and p47phox-/- mice that were rechallenged with secondary lethal Lm infection. These findings demonstrate a novel benefit of NADPH oxidase 2 deficiency (ie, dependent inflammation in antigen-presenting cell–mediated humoral immunity) and that anti-Lm Ab can be protective in an immunodeficient CGD host. Microbial-induced inflammation is important for eliciting humoral immunity. Genetic defects of NADPH oxidase 2–based proteins interrupt phagocyte superoxide generation and are the basis for the human immunodeficiency chronic granulomatous disease (CGD). Hyperinflammation is also a significant clinical manifestation of CGD. Herein, we evaluated humoral immunity in the phagocyte oxidase p47phox-deficient model of CGD and found that UV-inactivated Streptococcus pneumoniae and Listeria monocytogenes (Lm) elicited higher specific antibody (Ab) titers in p47phox-/- mice than wild-type (WT) mice. Both organisms elicited robust and distinct antigen-presenting cell maturation phenotypes, including IL-12 hypersecretion, and higher major histocompatibility complex II and costimulatory protein expression in Lm-stimulated p47phox-/- dendritic cells (DCs) relative to WT DCs. Furthermore, p47phox-/- DCs pulsed with Lm and adoptively transferred into naïve WT mice elicited Ab titers, whereas Lm-pulsed WT DCs did not elicit these titers. The observed robust p47phox-/- mouse humoral response was recapitulated with live Lm and sustained in vivo in p47phox-/- mice. Notably, anti–serum samples from p47phox-/- mice that survived secondary Lm infection were protective in WT and p47phox-/- mice that were rechallenged with secondary lethal Lm infection. These findings demonstrate a novel benefit of NADPH oxidase 2 deficiency (ie, dependent inflammation in antigen-presenting cell–mediated humoral immunity) and that anti-Lm Ab can be protective in an immunodeficient CGD host. Antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages, are important cellular mediators of inflammation. APCs also bridge innate and humoral immunity to combat microbial infection. Bacteria induce APC maturation. In turn, mature APCs instruct adaptive immunity by presenting bacteria-derived peptides, along with costimulatory signals, to T cells and secreting inflammatory cytokines that drive T-cell activation and consequent T-cell–mediated and/or humoral immunity. Both Listeria monocytogenes (Lm), a facultative intracellular bacterium, and Streptococcus pneumoniae (Pn), an anaerobic extracellular bacterium, have been used to study the fundamental components of innate and adaptive immunity.1Zenewicz L.A. Shen H. Innate and adaptive immune responses to Listeria monocytogenes: a short overview.Microbes Infect. 2007; 9: 1208-1215Crossref PubMed Scopus (144) Google Scholar, 2Pamer E.G. Immune responses to Listeria monocytogenes.Nat Rev Immunol. 2004; 4: 812-823Crossref PubMed Scopus (661) Google Scholar, 3AlonsoDeVelasco E. Verheul A.F. Verhoef J. Snippe H. Streptoccocus pneumoniae: virulance factors, pathogenesis, and vaccines.Microbiol Rev. 1995; 59: 591-603Crossref PubMed Google Scholar Lm and Pn each elicit strong APC-mediated inflammatory and cellular responses that are important for initiating protective immune responses. Pn elicits antigen-specific antibody (Ab) production and anti-Pn humoral immunity.3AlonsoDeVelasco E. Verheul A.F. Verhoef J. Snippe H. Streptoccocus pneumoniae: virulance factors, pathogenesis, and vaccines.Microbiol Rev. 1995; 59: 591-603Crossref PubMed Google Scholar In contrast, early Lm research4Mackaness G.B. Cellular resistance to infection.J Exp Med. 1962; 116: 381-406Crossref PubMed Scopus (829) Google Scholar indicated that Lm-induced Ab production was not specific for Lm resistance, and experimental evidence1Zenewicz L.A. Shen H. Innate and adaptive immune responses to Listeria monocytogenes: a short overview.Microbes Infect. 2007; 9: 1208-1215Crossref PubMed Scopus (144) Google Scholar, 2Pamer E.G. Immune responses to Listeria monocytogenes.Nat Rev Immunol. 2004; 4: 812-823Crossref PubMed Scopus (661) Google Scholar showed that T-cell–mediated immunity is most critical for eliminating Lm. However, subsequent studies5Edelson B.T. Cossart P. Unanue E.R. Paradigm revisited: antibody provides resistance to Listeria infection.J Immun. 1999; 163: 4087-4090PubMed Google Scholar, 6Edelson B.T. Unanue E.R. Intracellular antibody neutralizes Listeria growth.Immunity. 2001; 14: 503-512Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar have shown that humoral immunity can play a significant role in the elimination of Listeria infection. The local oxidative environment, reactive oxygen species (ROS), and free radical responses are widely postulated to promote inflammation as part of the adaptive response to restoring tissue homeostasis after acute infection and tissue injury. However, recent observations that phagocytes, and nonphagocytic cells, generate ROS as they orchestrate adaptive immune responses raise questions about the source and relative role of ROS in modulating inflammatory responses that are important for eliciting humoral immunity.7Bokoch G.M. Knaus U.G. NADPH oxidases: not just for leukocytes anymore!.Trends Biochem Sci. 2003; 28: 502-508Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 8Geiszt M. Leto T.L. The Nox family of NAD(P)H oxidases: host defense and beyond.J Biol Chem. 2004; 279: 51715-51718Crossref PubMed Scopus (381) Google Scholar Patients with chronic granulomatous disease (CGD) have heterogeneous genetic defects of phagocytic oxidase NADPH oxidase 2 (Nox2)–based proteins and an absent or reduced phagocyte respiratory burst.9Heyworth P.G. Cross A.R. Curnutte J.T. Chronic granulomatous disease.Curr Opin Immunol. 2003; 15: 578-584Crossref PubMed Scopus (342) Google Scholar, 10Williams D.A. Tao W. Yang F. Kim C. Gu Y. Mansfield P. Levine J.E. Petryniak B. Derrow C.W. Harris C. Jia B. Zheng Y. Ambruso D.R. Lowe J.B. Atkinson S.J. Dinauer M.C. Boxer L. Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.Blood. 2000; 96: 1646-1654PubMed Google Scholar, 11Ambruso D.R. Knall C. Abell A.N. Panepinto J. Kurkchubasche A. Thurman G. Gonzalez-Aller C. Hiester A. deBoer M. Harbeck R.J. Oyer R. Johnson G.L. Roos D. Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.Proc Natl Acad Sci U S A. 2000; 97: 4654-4659Crossref PubMed Scopus (369) Google Scholar, 12Matute J.D. Arias A.A. Wright N.A. Wrobel I. Waterhouse C.C. Li X.J. Marchal C.C. Stull N.D. Lewis D.B. Steele M. Kellner J.D. Yu W. Meroueh S.O. Nauseef W.M. Dinauer M.C. A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity.Blood. 2009; 114: 3309-3315Crossref PubMed Scopus (321) Google Scholar CGD is a multifaceted clinical disease that manifests clinically as life-threatening bacterial and fungal infections.9Heyworth P.G. Cross A.R. Curnutte J.T. Chronic granulomatous disease.Curr Opin Immunol. 2003; 15: 578-584Crossref PubMed Scopus (342) Google Scholar, 13Assari T. Chronic granulomatous disease: fundamental stages in our understanding of CGD.Med Immunol. 2006; 5: 4Crossref PubMed Scopus (68) Google Scholar Interestingly, noninfectious hyperinflammation is also a common occurrence in patients with CGD.14Rosenzweig S.D. Inflammatory manifestations in chronic granulomatous disease (CGD).J Clin Immunol. 2008; 28: S67-S72Crossref PubMed Scopus (114) Google Scholar Because one of the clinical manifestations of CGD is increased inflammation, we investigated the ability of p47phox (Ncf1)–deficient (p47phox-/-) APCs to secrete cytokines and up-regulate receptors important for initiating humoral immunity against Lm and Pn. We demonstrate that Pn and Lm stimulation leads to dissimilar p47phox-/- DC maturation. We also show that, although Pn predictably induces humoral immunity, including memory Ab production in p47phox-/- mice, anti-Pn humoral immunity is enhanced in p47phox-/- mice compared with wild-type (WT) control mice. Interestingly, we found that Lm similarly elicits enhanced and protective humoral immunity in p47phox-/- mice. Nox p47phox-deficient (p47phox-/-) mice have been described.15Jackson S.H. Gallin J.I. Holland S.M. The p47phox mouse knock-out model of chronic granulomatous disease.J Exp Med. 1995; 182: 751-758Crossref PubMed Scopus (406) Google Scholar, 16Liu Q. Cheng L.I. Yi L. Zhu N. Wood A. Changpriroa C.M. Ward J.M. Jackson S.H. p47phox deficiency induces macrophage dysfunction resulting in progressive crystalline macrophage pneumonia.Am J Pathol. 2009; 174: 153-163Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Congenic p47phox-/- mice on a C57BL/6NTac background were generated by backcrossing over 10 generations with WT C57BL/6NTac. gp91phox-/-/Nox2-/- B6.129S6-Cybbtm1Din/J mice17Pollock J.D. Williams D.A. Gifford M.A. Li L.L. Du X. Fisherman J. Orkin S.H. Doerschuk C.M. Dinauer M.C. Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production.Nat Genet. 1995; 9: 202-209Crossref PubMed Scopus (763) Google Scholar were obtained from The Jackson Laboratory (Bar Harbor, ME). Animal care was provided in accordance with Institutional Animal Care and Use Committee procedures, approved by the National Institute of Allergy and Infectious Diseases, NIH (Bethesda, MD). All mice used were between the ages of 3 and 6 weeks. Frozen stocks of nonencapsulated variant (strain R36A) of virulent Pn capsular type 2 (strain D39) were thawed and subcultured on BBL agar plates (VWR International, West Chester, PA). Similarly, recombinant Lm strain 10403S expressing ovalbumin; a gift from Dr. Hao Chen18Foulds K.E. Rotte M.J. Seder R.A. IL-10 is required for optimal CD8 T cell memory following Listeria monocytogenes infection.J Immunol. 2006; 177: 2565-2574PubMed Google Scholar (University of Pennsylvania School of Medicine, Philadelphia, PA) was subcultured on Difco Brain Heart Infusion Agar (BD, Franklin Lakes, NJ). Isolated colonies were collected and UV inactivated (UVi) (UV Stratalinker 1880; Artisan Scientific, Champagne, IL) at 1000 mJ for 1 hour. Sterility was confirmed by subculture on blood agar plates for Pn and Brain Heart Infusion Agar for Lm. After extensive washings, the bacterial suspensions were adjusted with PBS to provide an OD of 0.6, which corresponded to 109 colony-forming units (CFUs)/mL of Pn; and 0.1, which corresponded to 108 CFUs/mL of Lm. Bacteria were then divided at 1010 CFUs/mL and frozen at -80°C until their use. Bone marrow–derived DCs (BMDCs) were prepared as previously described.19Vasilevsky S. Colino J. Puliaev R. Canaday D.H. Snapper C. Macrophages pulsed with Streptococcus pneumoniae elicit a T cell-dependant antibody response upon transfer into naive mice.J Immunol. 2008; 181: 1787-1797PubMed Google Scholar Briefly, bone marrow was flushed with PBS, resuspended in 1 mL ACK Lysing Buffer (BioWhittaker, Walkersville, MD), and incubated at room temperature for 5 minutes to eliminate red cells. The single-cell suspension was filtered through a 40-μm cell strainer (BD Falcon/BD Biosciences, San Jose, CA), resuspended at a density of 1.25 × 106 cells/mL (24-well plates) in RPMI 1640 plus 5% fetal bovine serum, 10,000 IU penicillin, 10 mg/mL streptomycin, 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, and 25 mmol/L HEPES (culture medium), supplemented with 10 ng/mL murine recombinant granulocyte macrophage colony-stimulating factor (M-CSF) (Sigma-Aldrich, St. Louis, MO). After 7 days of culture, nonadherent cells were harvested. Bone marrow–derived macrophages (BMMs) were obtained using a similar approach as used for BMDCs, with slight modifications. Bone marrow cells were cultured at 1 × 106 cells/mL in cell culture medium supplemented with 10 ng/mL murine M-CSF (Sigma-Aldrich). Cells were plated in six-well plates in a volume of 4 to 5 mL per well. On days 3 and 5, three-fourths culture medium was removed; and fresh culture medium was added. On day 7, BMMs were harvested by washing plates with sterile PBS to remove nonadherent cells. Cells were detached from the plate as previously described.19Vasilevsky S. Colino J. Puliaev R. Canaday D.H. Snapper C. Macrophages pulsed with Streptococcus pneumoniae elicit a T cell-dependant antibody response upon transfer into naive mice.J Immunol. 2008; 181: 1787-1797PubMed Google Scholar Briefly, 2 mL of detachment buffer (4 mg/mL lidocaine, 5 mmol/L EDTA, and PBS) was added for 3 to 5 minutes. After incubation, detachment buffer was pipetted until the adherent cells detached. Recombinant pneumococcal surface protein A (PspA; family 1, sero clade 2) was provided by Clifford M. Snapper (Uniformed Services University of the Health Sciences, Bethesda). Purified listeriolysin-O (LLO) was purchased from Abcam (Cambridge, MA). A necropsy of all mice was performed at the ages noted. All tissues were examined grossly, and most were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned. Slides containing formalin-fixed, paraffin-embedded tissue sections (3 to 4 μm) were deparaffinized in xylene and rehydrated by processing them through alcohols. Pretreatment of tissues before incubation with the Listeria primary Ab consisted of bleaching with Peroxidazed 1 (Biocare Medical, Concord, CA) for 5 minutes, digesting with proteinase K (Dako, Carpentaria, CA) for 5 minutes, and pageing with Background Sniper (Biocare Medical) for 10 minutes. Sections were incubated with a goat polyclonal Ab against Listeria (KPL, Gaithersburg, MD) for 60 minutes at a dilution of 1:1500. The bound Ab was detected using a goat polymer detection system (Biocare Medical) and Vulcan Fast Red chromogen (Biocare Medical). Sections were counterstained with CAT hematoxylin (Biocare Medical), air dried, and mounted using Permount mounting medium (Fisher Scientific, Pittsburgh, PA). Negative controls included replacing the primary Ab with normal goat serum at a comparable protein concentration and testing noninfected tissues with the primary Ab. Slides were imaged using Aperio ScanScope software (Aperio Technologies Inc., Vista, CA). All steps were performed on ice. Fc receptors were pageed with 10 μg/mL purified rat anti-mouse CD16/CD32 mouse Fc page (clone 2.4G2). Cells were stained for 30 minutes with fluorescein isothiocyanate–mouse IgG2a and κ anti-mouse major histocompatibility complex (MHC) class IIb (clone AF6-120.1), phosphatidylethanolamine-mouse IgG2a and κ anti-mouse CD40 (clone 3/23), phosphatidylethanolamine-mouse IgG2a and κ anti-mouse CD86 (clone GL1), and Armenian hamster IgG2 and κ anti-mouse CD80 (clone 16-10A1). All monoclonal antibodies were purchased from BD Pharmingen (Franklin Lakes, NJ). Irrelevant isotype- and species-matched monoclonal antibodies (Abs) were used as staining controls. Cells were analyzed on a BD FacsCanto flow cytometer. BMDCs, cultured in granulocyte M-CSF, and BMMs, cultured in M-CSF, were pulsed in vitro with UVi Pn (108 CFUs) or UVi Lm (108 CFUs) overnight. IL-6, IL-12, and tumor necrosis factor (TNF)-α were measured from supernatant by sandwich enzyme-linked immunosorbent assay (ELISA). WT, p47phox-/-, and/or Nox2-/- mice were immunized by i.p. injection with 2 × 108 CFUs of UVi Pn or Lm on day 0; on day 14, a secondary challenge of UVi bacteria was given to assess the potential generation of memory. Serum was harvested 7, 14 (before rechallenge), and 21 days after bacterial challenge. For live Lm infection, WT and p47phox-/- mice were infected i.v. with 5 × 104 CFUs (0.1 LD50) of Lm. ELISA plates (Immunolon 4) were coated with 5 μg/mL (50 μL/well) PspA, 3 μg/mL (50 μL/well) LLO, and 3 μg/mL (50 μL/well) UVi Pn (107 CFUs/well) or UVi Lm (107 CFUs/well) in PBS overnight at 4°C. Plates were pageed with PBS plus 1% bovine serum albumin (BSA) for 30 minutes at 37°C and washed three times with PBS plus 0.1% Tween 20. Threefold dilutions of serum samples, starting at a 1:50 serum dilution, in PBS plus 0.05% Tween 20 were then added and left overnight at 4°C. Plates were washed three times with PBS plus 0.1% Tween 20. Alkaline phosphatase–conjugated polyclonal goat anti-mouse IgM or IgG Abs (200 ng/mL final concentration in PBS plus 0.05% Tween 20) were then added, and plates were incubated for 37°C for 1 hour. Plates were washed five times with PBS plus 0.1% Tween 20. Substrate (p-nitrophenyl phosphate, disodium) at 1 mg/mL in TM buffer [1 mol/L Tris plus 0.3 mmol/L MgCl2 (pH 9.8)] was then added for 30 minutes at room temperature for color development. Color was read at an absorbance of 405 nm. Concentrations of specific cytokines released into the medium of cell cultures were measured using optimized standard sandwich ELISA. ELISA (Immunolon 4) plates were coated with IL-6 (2 μg/mL), IL-12 (6 μg/mL), and TNF-α (10 μg/mL) capture Ab in PBS overnight at 4°C. Plates were pageed with PBS plus 1% BSA for 30 minutes at 37°C and washed three times with PBS plus 0.1% Tween 20. Twofold dilutions of supernatant samples and standards, including recombinant (r) IL-6 (4 ng/mL), rIL-12 (8 ng/mL), or rTNF-α (8 ng/mL) in PBS plus 0.05% Tween 20, were then added and left overnight at 4°C. Plates were washed three times with PBS plus 0.1% Tween 20. Secondary IL-6 (1 μg/mL), IL-12 (1 μg/mL), and TNF-α (1 μg/mL) antibodies with PBS plus 1% BSA were added for 1 hour at 37°C and washed three times with PBS plus 0.1% Tween 20. Streptavidin-alkaline phosphatase with PBS plus 1% BSA was added at a 1:1000 concentration for 1 hour at 37°C. Substrate (p-nitrophenyl phosphate, disodium; at 1 mg/mL in TM buffer [1 mol/L Tris plus 0.3 mmol/L MgCl2 (pH 9.8)] was then added for 30 minutes at room temperature for color development. Color was read at an absorbance of 405 nm. Twenty WT and p47phox-/- mice were i.v. infected with 5 × 104 Lm, then were rechallenged with 5 × 106 Lm 28 days later. Serum was harvested from the surviving WT (n = 20) and p47phox-/- (n = 16) mice 6 days after reinfection and was adoptively transferred i.v. into a second set of WT and p47phox-/- recipients that were infected with 5 × 104 Lm 28 days previously. One day after immune serum transfer, WT and p47phox-/- mice were reinfected with 5 × 106 Lm and monitored for survival. Differences between the group means were analyzed by the Student's t-test (Prism 5; GraphPad Software, Inc., San Diego, CA). P ≤ 0.05 was considered statistically significant. For initial investigations, we used UVi Lm or Pn to compare proinflammatory cytokine induction in WT and p47phox-/- BMMs and BMDCs propagated in vitro. In contrast to heat-killed inactivation, which can cause bacterial rupture, UV-irradiated bacteria are replication incompetent20Zavilgesky G.B. Gurzadyan G.G. Nikogosyan D.N. Pyrimidine dimers, single-strand breaks and crosslinks induced DNA by powerful laser UV irradiation.Photochem Photobiol. 1984; 8: 175-187Google Scholar and remain structurally intact. As illustrated in Table 1, Pn-pulsed p47phox-/- APCs hypersecrete IL-6 and TNF-α; in contrast, Lm-stimulated p47phox-/- APCs hypersecrete IL-12 relative to WT APCs. Interestingly, although Lm-pulsed p47phox-/- DCs secrete less TNF-α than WT DCs, p47phox-/- macrophages propagated in M-CSF secreted at least twofold more IL-6, IL-12, and TNF-α in response to both Pn and Lm relative to WT macrophages (Table 1).Table 1APC Cytokine Secretion from WT and p47phox-/- Mice Cultured with 1 × 108 CFUs/mL of UVi Pn or Lm for 24 Hours in VitroData are from Vasilevsky et al.19Vasilevsky S. Colino J. Puliaev R. Canaday D.H. Snapper C. Macrophages pulsed with Streptococcus pneumoniae elicit a T cell-dependant antibody response upon transfer into naive mice.J Immunol. 2008; 181: 1787-1797PubMed Google ScholarVariableIL-6IL-12TNF-αWTp47phox-/-WTp47phox-/-WTp47phox-/-BMDCs Pn5.68 ± 0.6310.27 ± 0.88⁎P = 0.04.1.25 ± 0.041.37 ± 0.06ND0.89 ± 0.03Lm24.39 ± 0.6321.24 ± 0.883.12 ± 0.144.82 ± 0.03†P = 0.0004.5.25 ± 0.303.62 ± 0.50 Medium0.625 ± 0.030.646 ± 0.0020.263 ± 0.0010.237 ± 0.57ND0.393 ± 0.17BMMs (M-CSF)Pn0.75 ± 0.092.05 ± 0.08†P = 0.0004.1.40 ± 0.094.70 ± 0.10†P = 0.0004.ND1.84 ± 0.09Lm2.81 ± 0.395.16 ± 0.33⁎P = 0.04.1.89 ± 0.076.43 ± 0.12†P = 0.0004.1.50 ± 0.203.87 ± 0.14†P = 0.0004. MediumNDNDNDNDND1.42 ± 0.38Data are given as mean ± SEM (in ng/mL). n = 4. Concentrations of IL-6, IL-12, and TNF-α in the culture supernatant were determined by ELISA. The limit of detection for IL-6 was <31 pg/mL; IL-12, <62 pg/mL; and TNF-α, <250 pg/mL.ND, not detectable. P = 0.04.† P = 0.0004. Open table in a new tab Data are given as mean ± SEM (in ng/mL). n = 4. Concentrations of IL-6, IL-12, and TNF-α in the culture supernatant were determined by ELISA. The limit of detection for IL-6 was <31 pg/mL; IL-12, <62 pg/mL; and TNF-α, <250 pg/mL. ND, not detectable. Next, we compared MHCs with costimulatory molecule expression in Lm- and Pn-stimulated APCs (Table 2). After overnight culture with UVi microorganisms, p47phox-/- DCs expressed higher surface levels of MHC class II and CD80 in media alone and in response to Lm stimulation compared with WT DCs (Figure 1A). In addition, surface CD40 and CD86 is significantly up-regulated in Lm-stimulated p47phox-/- DCs relative to similarly treated WT DCs. In contrast, WT DCs expressed higher surface levels of MHC class II, CD86, and CD40 in response to Pn stimulation compared with p47phox-/- DCs (Figure 1B). WT and p47phox-/- macrophages propagated in M-CSF and pulsed with Lm or Pn similarly up-regulated MHC class II, CD80, CD86, and CD40 (Figure 1, C and D). Thus, in addition to finding that p47phox differentially regulates proinflammatory cytokine secretion in Pn- and Lm-stimulated APCs, these observations show that Pn and Lm induce distinct maturation immunophenotypes in p47phox-/- and WT APCs. Notably, only Lm-pulsed p47phox-/- DCs expressed higher levels of MHC class II, CD80, CD86, and CD40 compared with WT, suggesting a role for p47phox in DC antigen presentation.Table 2APC MFI ValuesVariableBMDCsBMMsWTp47phox-/-WTp47phox-/-gMFINo. of cellsgMFINo. of cellsgMFINo. of cellsgMFINo. of cellsMHC Class II Medium725481123574923031953423970Pn123463010069761241637718776590Lm1414568210646695872428957418 Control56652160711936641403437518CD80 Medium50345501033647746249553845017Pn62159001147638355858247217163Lm51160591656574345165505827501 Control1306461127784332482072907375CD86 Medium3365590266684827844,8793514280Pn7516129309734443949695297198Lm42744381035585967774907257516 Control114580786852224541402327375CD40 Medium2085764130667382135864464513Pn379612225574942142669920287040Lm355443575560692592735313746979 Control11458071277843737375757375APCs from WT and p47phox-/- mice were cultured with 1 108 CFUs/mL of UVi Pn or Lm for 24 hours in vitro.gMFI, geometric mean fluorescence intensity. Open table in a new tab APCs from WT and p47phox-/- mice were cultured with 1 108 CFUs/mL of UVi Pn or Lm for 24 hours in vitro. gMFI, geometric mean fluorescence intensity. Although Pn-elicited humoral immunity in mice is well characterized,3AlonsoDeVelasco E. Verheul A.F. Verhoef J. Snippe H. Streptoccocus pneumoniae: virulance factors, pathogenesis, and vaccines.Microbiol Rev. 1995; 59: 591-603Crossref PubMed Google Scholar, 21Colino J. Shen Y. Snapper C.M. Dendritic cells pulsed with intact Streptococcus pneumoniae elicit both protein- and polysaccharide-specific immunoglobulin isotype responses in vivo through distinct mechanisms.J Exp Med. 2002; 195: 1-13Crossref PubMed Scopus (119) Google Scholar few investigations5Edelson B.T. Cossart P. Unanue E.R. Paradigm revisited: antibody provides resistance to Listeria infection.J Immun. 1999; 163: 4087-4090PubMed Google Scholar, 6Edelson B.T. Unanue E.R. Intracellular antibody neutralizes Listeria growth.Immunity. 2001; 14: 503-512Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar have shown a benefit for Lm-induced humoral immunity and Ab production. Next, to clarify whether p47phox deficiency affected anti-Lm Ab production, we used ELISA to compare specific Ab titers for intact bacteria and Pn and Lm virulence factors, PspA and LLO, respectively, after UVi Pn or Lm challenge. Although LLO is a heat-liable protein,22Bhunia A.K. Antibodies to Listeria monocytogenes.Crit Rev Microbiol. 1997; 23: 77-107Crossref PubMed Scopus (58) Google Scholar we exploited our UVi challenge model to examine this parameter. For these investigations, we immunized p47phox-/- and WT mice with UVi bacteria on day 0; on day 14, a secondary challenge of UVi bacteria was given to assess the generation of memory Ab production. As illustrated in Figure 2A, Pn predictably induced potent Ab production in both WT and p47phox-/- mice. However, the p47phox-/- anti-Pn IgM titers were twofold higher than WT 7 and 14 days after UVi Pn challenge. In addition, p47phox-/- anti-Pn IgG titers were threefold higher on day 7 before rechallenge and twofold higher than WT after UVi Pn rechallenge (Figure 2A, day 21). Similarly, p47phox-/- mice had higher IgM and IgG isotype PspA-specific titers than WT mice after UVi Pn challenge (Figure 2C). Notably, serum from UVi-Lm–challenged p47phox-/- mice also exhibited increased anti-Lm IgM (twofold to threefold higher on days 7 and 14) and IgG (33-fold higher on day 14 and 15-fold higher on day 21) titers relative to WT mice (Figure 2B). Moreover, p47phox-/- anti-LLO IgM titers were elevated 11-fold on day 21 and anti-LLO IgG titers were elevated twofold higher than WT on day 14; serum anti-LLO IgG titers in WT and p47phox-/- mice were similar on day 21 after a secondary UVi Lm boost on day 14 (Figure 2C). Collectively, these results demonstrate that humoral immunity is enhanced in inflammation-prone p47phox-/- mice and thereby reveal a complex and unforeseen benefit of phagocyte oxidase deficiency for enhancing anti-Pn and Lm humoral immunity. Previous investigations23Dinauer M.C. Deck M.B. Unanue E.R. Mice lacking reduced nicotinamide adenine dinucleotide phosphate oxidase activity show increased susceptibility to early infection with Listeria monocytogenes.J Immunol. 1997; 158: 5581-5583PubMed Google Scholar, 24Shiloh M.U. MacMicking J.D. Nicholson S. Brause J.E. Potter S. Marino M. Fang F. Dinauer M. Nathan C. Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase.Immunity. 1999; 10: 29-38Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar showed that, although gp91phox-deficient (Nox2-/-) mice have increased susceptibility to Lm infection and that Nox2-/- macrophages cannot kill virulent Lm, primary Lm infection is not fatal in p47phox-/- mice.25Endres R. Luz A. Schulze H. Neubauer H. Futterer A. Holland S.M. Wagner H. Pfeffer K. Listeriosis in p47(phox-/-) and TRp55-/- mice: protection despite absence of ROI and susceptibility despite presence of RNI.Immunity. 1997; 7: 419-432Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Thus, to discern the role of p47phox independently or as part of the multicomponent phagocytic Nox in Lm-elicited humoral immunity, we examined Lm-induced Ab production in Nox2-/- catalytic subunit mice. As shown in Figure 2B, compared with p47phox-/- mice, anti-Lm IgM-specific titers were not elevated from Lm-challenged Nox2-/- mice. However, anti–Lm-specific IgG titers were elevated 2.5-fold on day 14 in Nox2-/- mice compared with Lm-challenged WT mice and twofold and 31-fold compared with p47phox-/- and WT mice, respectively, on day 21 after a secondary Lm boost on day 14 (Figure 2B). These data show that, although the kinetics of anti–Lm-specific IgG induction are different in Nox2-/- and p47phox-/- mice, Lm challenge elicits an equally robust recall IgG Ab response in Nox2-/- mice as in p47phox-/- mice, therefore indicating that Nox2 enzymatic activity is important for controlling Lm-elicited Ab production. These observations lead us to question the relevance of the observed enhanced Lm-induced p47phox-/- DC maturation phenotype to the robust UVi Lm-elicited humoral response in p47phox-/- mice and whether the anti-Lm humoral immune response may be protective in p47phox-/- mice. Therefore, to further investigate these parameters, we adoptively transferred 1 × 106 Lm-pulsed p47phox-/- and WT BMDCs into naïve WT recipient mice. As controls, unpulsed p47phox-/- and WT DCs were also transferred into a separate
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