Activation of the SHP-1 Phosphatase in Macrophages Through Camp

Activation of the SHP-1 Phosphatase in Macrophages Through Camp

The Journal of Immunology Bordetella pertussis Adenylate Cyclase Toxin Blocks Induction of Bactericidal Nitric Oxide in Macrophages through cAMP-Dependent Activation of the SHP-1 Phosphatase Ondrej Cerny, Jana Kamanova,1 Jiri Masin, Ilona Bibova, Karolina Skopova, and Peter Sebo The adenylate cyclase toxin–hemolysin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates comple- ment receptor 3–expressing phagocytes and catalyzes uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by complement-dependent oxidative burst and opsonophagocytic mechanisms. We show that cAMP signaling through the protein kinase A (PKA) pathway activates Src homology domain 2 containing protein tyrosine phosphatase (SHP) 1 and suppresses production of bactericidal NO in macrophage cells. Selective activation of PKA by the cell-permeable analog N6-benzoyladenosine-39,59-cyclic monophosphate interfered with LPS-induced inducible NO synthase (iNOS) expression in RAW264.7 macrophages, whereas inhibition of PKA by H-89 largely restored the production of iNOS in CyaA-treated murine macrophages. CyaA/cAMP signaling induced SHP phosphatase–dependent dephosphorylation of the c-Fos subunit of the transcription factor AP-1 and thereby inhibited TLR4- triggered induction of iNOS gene expression. Selective small interfering RNA knockdown of SHP-1, but not of the SHP-2 phosphatase, rescued production of TLR-inducible NO in toxin-treated cells. Finally, inhibition of SHP phosphatase activity by NSC87877 abrogated B. pertussis survival inside murine macrophages. These results reveal that an as yet unknown cAMP- activated signaling pathway controls SHP-1 phosphatase activity and may regulate numerous receptor signaling pathways in leukocytes. Hijacking of SHP-1 by CyaA action then enables B. pertussis to evade NO-mediated killing in sentinel cells of innate immunity. The Journal of Immunology, 2015, 194: 4901–4913. he whooping cough agent Bordetella pertussis produces cells (7). The AC catalyzes uncontrolled conversion of cytosolic an RTX (Repeats in ToXin) adenylate cyclase (AC) toxin– ATP to the key second messenger molecule cAMP and interferes T hemolysin (CyaA, ACT, or AC-Hly) that paralyzes bac- with signaling of opsonins through receptors of complement and tericidal functions of myeloid phagocytes (1–6). The 1706- Igs on neutrophils, macrophages, or dendritic cells (7). As a result, residue-long bifunctional toxin binds the complement receptor 3 production of bactericidal reactive oxygen species (ROS) and (aMb2 integrin, CD11b/CD18, or Mac-1). The toxin forms small formation of neutrophil extracellular traps is rapidly inhibited (8– cation-selective (hemolytic) pores in cellular membrane and its 10). In macrophage cells, the produced cAMP causes transient main cytotoxic activity consists in delivery of the N-terminal inhibition of RhoA GTPase activity and elicits massive actin calmodulin-activated AC enzyme domain into the cytosol of cytoskeleton rearrangements and unproductive membrane ruff- ling (11). As a result, phagocytosis and killing of complement- opsonized bacteria by neutrophils and macrophages are blocked Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology of the ASCR, v.v.i., Czech Academy of Sciences, 142 20, Prague 4, Czech Republic near-instantaneously (8, 11, 12). Moreover, CyaA/cAMP signaling also skews the TLR-triggered maturation of dendritic cells and 1Current address: Yale University School of Medicine, New Haven, CT. impairs their capacity to present Ags to T cells for induction of Received for publication November 24, 2014. Accepted for publication March 10, 2015. adaptive cellular immune responses (13). This work was supported by Czech Science Foundation Grants P302/12/0460 (to J.M.), In the absence of opsonization, however, a fraction of internalized 13-14547S (to P.S.), and the Institutional Reasearch Project No. RVO61388971. O.C. B. pertussis bacteria was repeatedly found to escape killing and to is a doctoral student of the Faculty of Science of the Charles University in Prague, survive within primary human macrophages for several days (14, Czech Republic; I.B. and K.S. are doctoral students of the Institute of Chemical Technology in Prague, Czech Republic. 15). Because inducible NO production belongs to the major bacte- Address correspondence and reprint requests to Dr. Peter Sebo, Institute of Micro- ricidal mechanisms in macrophages, this indicated that B. pertussis biology of the ASCR, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic. might possess an as yet unknown mechanism that enables it to es- E-mail address: [email protected] cape killing by NO produced by the inducible NO synthase (iNOS, The online version of this article contains supplemental material. NOS2). iNOS expression is, indeed, induced in macrophages upon Abbreviations used in this article: AC, adenylate cyclase; BGA, Bordet-Gengou agar; activation by bacterial LPS, or IFN-g or TNF-a (16). Production of 6 BMDM, bone marrow–derived macrophage; 6-Bnz-cAMP, N -benzoyladenosine- the highly active Ca2+-independent iNOS enzyme in macrophage 39,59-cyclic monophosphate; 8-CPT-cAMP, 8-(4-chlorophenylthio)-29-O-methylade- nosine-39,59-cyclic monophosphate; CyaA, AC toxin–hemolysin; DAF-FM, 4-amino- cells results, indeed, in production of toxic amounts of NO, and this 5-methylamino-29,79-difluorofluorescein diacetate; db-cAMP, N6,29-O-dibutyrylade- plays an important role in host defense against pathogens, including nosine-39,59- cyclic monophosphate; Epac, exchange protein directly activated by cAMP; IBMX, 3-isobutyl-1-methylxanthine; iNOS, NOS2, inducible NO synthase; B. pertussis (17, 18). Regulation of NO production in monocytic MOI, multiplicity of infection; PKA, protein kinase A; PT, pertussis toxin; ROS, cells then largely depends on regulation of iNOS gene expression reactive oxygen species; siRNA, small interfering RNA; STAT1, signal transducer level (19). This results from the interplay of transcription factors, and activator of transcription 1. such as NF-kB, the signal transducer and activator of transcription 1 Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 (STAT1), and the AP-1, respectively (20, 21). Involvement of the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402941 4902 iNOS EXPRESSION ABLATION BY cAMP SIGNALING CyaA TOXIN transcription factors NF-kB and AP-1 in iNOS expression further DMEM containing 10% (v/v) FCS in the presence of 10% (v/v) conditioned appears to be modulated by the activity of the SHP-1, which was media of L929 CMG cells and antibiotic–antimycotic solution (0.1 mg/ml also shown to modulate the activity of the transcription factor streptomycin, 100 U/ml penicillin, and 0.25 mg/ml amphotericin; Sigma- Aldrich) for 7 d at 37˚C in a humidified air–CO2 (5%) atmosphere. STAT1 and of the IRF-1 (22, 23). RAW264.7 mouse macrophage cells (ATCC cat. no. TIB 71) were grown in Production of the iNOS enzyme was previously observed to play RPMI 1640 medium supplemented with 10% (v/v) FCS. Prior to assays, the a role in host defense against B. pertussis infection, indicating that phosphate-buffered RPMI 1640 medium was replaced with HEPES-buffered 2+ the pathogen may be sensitive to NO-mediated killing. Indeed, DMEM medium with 10% (v/v) FCS, which contains 1.9 mM Ca , and the cells were allowed to rest in DMEM for 2 h before toxin addition. naive iNOS-deficient mice were found to be more susceptible to B. pertussis respiratory challenge than were wild-type mice, and Determination of ROS production upon immunization by a whole-cell pertussis vaccine, the iNOS- ROS production was measured using a luminol-based assay as described deficient mice were less efficiently protected from infection than (28). Briefly, 5 3 105 RAW264.7 macrophages in HBSS supplemented their wild-type littermates (17). It has, however, not been explored with 1% glucose and 2 mM CaCl2 were incubated for 3 min at 37˚C with whether B. pertussis uses manipulation of iNOS expression in 150 mM luminol, before cells were transferred to wells containing the macrophages as part of its virulence strategy. We report that al- given activator (e.g., human complement–opsonized zymosan at 10 mg/ml, complement-opsonized B. pertussis at multiplicity of infection [MOI] ready low CyaA doses inhibit iNOS expression and NO produc- 10:1, or unopsonized B. pertussis at an MOI 10:1, respectively). Lumi- tion in mouse macrophages and that the AC enzyme activity of the nescence was recorded using a Safire2 microplate reader (Tecan), and data CyaA toxin is essential for extended survival of B. pertussis in output is given in relative light units integrated over 1 h of measurement. macrophage cells. We show that cAMP signaling leads to acti- Opsonization of bacteria with human complement vation of tyrosine phosphatase SHP-1 and that this blocks TLR- induced NO production. Randomized fresh human blood was purchased at the transfusion unit of the Thomayer Hospital in Prague, and complete human sera were obtained by centrifugation at 1200 rpm, 20 min, 17˚C. Prior to use, the sera were Materials and Methods controlled by ELISA for the absence of any detectable amounts of Abs Abs and reagents recognizing the PT, CyaA, and FHA Ags of B. pertussis (data not shown). For opsonization by human complement, 1 3 108 heat-killed B. pertussis Escherichia coli 0111:B4 LPS, 3-isobutyl-1-methylxanthine (IBMX), cells (70˚C, 30 min), or 1 mg of zymosan, were incubated with 50% human and N6,29-O-dibutyryladenosine-39,59- cyclic monophosphate (db-cAMP) serum for 30 min at 37˚C under gentle shaking, and the suspensions were were obtained from Sigma-Aldrich. N6-benzoyladenosine-39,59-cyclic washed twice with serum-free HBSS. monophosphate (6-Bnz-cAMP) and 8-(4-chlorophenylthio)-29-O-methyl- adenosine-39,59-cyclic monophosphate (8-CPT-cAMP) were from NO assay BIOLOG Life Science Institute.

View Full Text

Details

  • File Type
    pdf
  • Upload Time
    -
  • Content Languages
    English
  • Upload User
    Anonymous/Not logged-in
  • File Pages
    13 Page
  • File Size
    -

Download

Channel Download Status
Express Download Enable

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

  • Not to be reproduced or distributed without explicit permission.
  • Not used for commercial purposes outside of approved use cases.
  • Not used to infringe on the rights of the original creators.
  • If you believe any content infringes your copyright, please contact us immediately.

Support

For help with questions, suggestions, or problems, please contact us