Molecular control of PDPNhi macrophage subset induction by ADAP as a host defense in sepsis
Pengchao Zhang, Xinning Wang, Xiaodong Yang, Hebin Liu
Pengchao Zhang, Xinning Wang, Xiaodong Yang, Hebin Liu
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Research Article Immunology Inflammation

Molecular control of PDPNhi macrophage subset induction by ADAP as a host defense in sepsis

Abstract

Induction of podoplanin (PDPN) expression is a critical response of macrophages to LPS stimulation or bacterial infection in sepsis, but how this key process of TLR4-stimulated PDPN upregulation is regulated and the effect of PDPN expression on macrophage function remain elusive. Here, we determined how this process is regulated in vitro and in vivo. PDPN failed to be upregulated in TLR4-stimulated macrophages deficient in adhesion and degranulation-promoting adapter protein (ADAP), which could be rescued by the reconstitution of ADAP. A distinct PDPNhi peritoneal macrophage (PM) subset, which exhibited an M2-like phenotype and enhanced phagocytic activity, was generated in WT but not in ADAP-deficient septic mice. The blockade of PDPNhi PMs mimicked the effect of ADAP deficiency, which exacerbated sepsis. Mechanistically, Bruton’s tyrosine kinase–mediated (BTK-mediated) tyrosine phosphorylation of ADAP at Y571 worked together with mTOR to converge on STAT3 activation for the transactivation of the PDPN promoter. Moreover, agonist activation of STAT3 profoundly potentiated the PDPNhi PM subset generation and alleviated sepsis severity in mice. Together, our findings reveal a mechanism whereby ADAP resets macrophage function by controlling the TLR4-induced upregulation of PDPN as a host innate immune defense during sepsis.

Authors

Pengchao Zhang, Xinning Wang, Xiaodong Yang, Hebin Liu

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Figure 6

BTK-mediated tyrosine phosphorylation of ADAP at Y571 is indispensable for TLR4-induced PDPN upregulation in macrophages.

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BTK-mediated tyrosine phosphorylation of ADAP at Y571 is indispensable f...
(A–D) WT PMs were pretreated with resatorvid (0.1, 0.5, and 1 μM), IKK-16 (0.1, 0.3, and 0.5 μM), Bay 11-7085 (0.5, 1, and 5 μM), SB203580 (1, 5, and 10 μM), or SP600125 (1, 10, and 20 μM) for 1 hour, followed by LPS stimulation (100 ng/mL) for 24 hours (A, C, and D) or 12 hours (B). ADAP and PDPN expression was analyzed by Western blotting and qPCR (B, n = 3 each, 1-way ANOVA, Tukey’s multiple-comparison test). Relative mRNA levels were normalized to Hprt. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels (D). (E) RAW264.7 cells transduced with ADAP were treated with LPS (100 ng/mL, 24 hours) in the presence of IKK-16 (0.5 μM). ADAP and PDPN expression was analyzed by Western blotting. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels. (F) Schematic of kinase inhibitors screening and summary of inhibitors blocking LPS-induced PDPN upregulation. (G) WT PMs pretreated with ibrutinib (1, 5, and 10 μM) or dasatinib (0.01, 0.1, and 1 μM) for 1 hour were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was assessed by Western blotting. (H) MS confirmed LPS-induced (100 ng/mL, 1 hour) ADAP phosphorylation at Y571 in PMs. MS/MS spectra of the phosphorylated peptide (TTAVEIDYDSLKR) are shown. (I) ADAPKD RAW264.7 cells reconstituted with empty vector (EV), ADAP-WT, or ADAP (Y571F) were stimulated with LPS (100 ng/mL, 24 hours). PDPN and ADAP expression was analyzed by Western blotting. (J) CD11b+F4/80+PDPNhi (C2) and CD11b+F4/80+PDPNlo (C1) PMs were sorted from WT septic mice 18 hours after E. coli injection (2 × 107 CFU, i.p.). Lysates were immunoprecipitated with anti-ADAP and immunoblotted for p-Tyr100 and ADAP.