SHP-1 inhibits LPS-mediated TNF and iNOS production in murine macrophages

The adenylate cyclase toxin–hemolysin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates complement 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-3′,5′-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.

Section: Discussionmentioning

confidence: 73%

Bordetella pertussis Adenylate Cyclase Toxin Blocks Induction of Bactericidal Nitric Oxide in Macrophages through cAMP-Dependent Activation of the SHP-1 Phosphatase

Černý

Kamanová

Mašín

et al. 2015

“…Recently, we have shown a crucial and positive role for SHP-1 in LPS-mediated IL-6 production in SHP-1-null bone marrow-derived macrophages from motheaten (me/me) mice (41). Loss of SHP-1 function in me/ me mice results in enhanced production of TNF-a, contributing to profound inflammatory disorders (42)(43)(44). However, the involvement of SHP-1 and PTKs in regulation of TNF-a and IL-10 production is not known.…”

mentioning

confidence: 99%

SHP-1–Pyk2–Src Protein Complex and p38 MAPK Pathways Independently Regulate IL-10 Production in Lipopolysaccharide-Stimulated Macrophages

Okenwa

Kumar

Rego

et al. 2013

The role of tyrosine phosphatase Src homology region 2 domain-containing phosphatase (SHP)-1 in LPS-activated cytokine production and inflammation was investigated by determining TNF-α and IL-10 production in splenic macrophages employing SHP-1–null (me/me) mouse model. LPS-stimulated me/me splenic macrophages secreted significantly less IL-10 with concomitantly elevated levels of TNF-α compared with wild-type (WT) macrophages irrespective of LPS dose and duration of stimulation. IL-10 significantly inhibited LPS-induced TNF-α production in both me/me and WT macrophages. The critical requirement for SHP-1 in regulating LPS-induced IL-10 and TNF-α production was confirmed by interfering with SHP-1 expression in WT macrophages and by reconstituting me/me macrophages with the SHP-1 gene. To delineate the role of SHP-1 in positive regulation of LPS-induced IL-10 production, signaling proteins representing SHP-1 targets were examined. The results reveal that tyrosine kinases Src and proline-rich tyrosine kinase 2 (Pyk2) regulate SHP-1–dependent LPS-induced IL-10 production and infer that optimal LPS-induced IL-10 production requires an assembly of a protein complex consisting of SHP-1–Pyk2–Src proteins. Moreover, LPS-induced IL-10 production also requires activation of the p38 MAPK independent of SHP-1 function. Overall, to our knowledge our results show for the first time that SHP-1 acts as a positive regulator of LPS-induced IL-10 production in splenic macrophages through two distinct and independent SHP-1–Pyk2–Src and p38 MAPK pathways.

“…In macrophages, SHP-1 participates in the regulation of various processes, including proinflammatory and innate immune responses (17)(18)(19)(20). SHP-1 has previously been shown to associate with phagosomes (21,22), thereby suggesting a role in the biogenesis of phagolysosomes.…”

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confidence: 99%

The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis

Gómez

Shio

Duplay

et al. 2012

The process of phagocytosis and phagosome maturation involves the recruitment of effector proteins that participate in phagosome formation and in the acidification and/or fusion with various endocytic vesicles. In the current study, we investigated the role of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) in phagolysosome biogenesis. To this end, we used immortalized bone marrow macrophages derived from SHP-1–deficient motheaten mice and their wild-type littermates. We found that SHP-1 is recruited early and remains present on phagosomes for up to 4 h postphagocytosis. Using confocal immunofluorescence microscopy and Western blot analyses on purified phagosome extracts, we observed an impaired recruitment of lysosomal-associated membrane protein 1 in SHP-1–deficient macrophages. Moreover, Western blot analyses revealed that whereas the 51-kDa procathepsin D is recruited to phagosomes, it is not processed into the 46-kDa cathepsin D in the absence of SHP-1, suggesting a defect in acidification. Using the lysosomotropic agent LysoTracker as an indicator of phagosomal pH, we obtained evidence that in the absence of SHP-1, phagosome acidification was impaired. Taken together, these results are consistent with a role for SHP-1 in the regulation of signaling or membrane fusion events involved in phagolysosome biogenesis.