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 7

mTOR-mediated STAT3 phosphorylation potentiates ADAP-STAT3–dependent PDPN transcription in response to TLR4 stimulation.

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mTOR-mediated STAT3 phosphorylation potentiates ADAP-STAT3–dependent PDP...
(A) PDPN expression in PMs pretreated with BP-1-102 (0.5, 1, and 5 μM) or fludarabine (10, 50, and 100 μM) for 1 hour and stimulated with LPS (100 ng/mL, 24 hours), analyzed by Western blotting and densitometry normalized to α-tubulin (n = 6 each, 1-way ANOVA, Tukey’s multiple-comparison test). (B) STAT3 binding motifs in the Pdpn promoter and CUT & RUN qPCR showing STAT3 enrichment at site 2 in untreated or LPS-treated PMs (100 ng/mL, 1 hour) (n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). (C) Luciferase activity of Pdpn-WT-Luc or Pdpn-Mut-Luc in WT or ADAPKD RAW264.7 cells following LPS stimulation (100 ng/mL, 6 hours) (n = 3 each, 2-way ANOVA, Tukey’s multiple-comparison test). (D) EMSA showing STAT3-DNA binding in nuclear extracts of WT or ADAPKD RAW264.7 cells after LPS stimulation (100 ng/mL, 1 hour). (E) Flow cytometry of CD11b+F4/80+PDPNhi PMs in WT mice treated with colivelin (1 mg/kg, i.p.) after E. coli infection (2 × 107 CFU, i.p.) (n = 5 each, unpaired t test). (F and G) Western blot of PDPN, p-STAT3, and STAT3 in LPS-treated PMs (100 ng/mL) with or without mTOR inhibitors or rapamycin (1 μM). (H) LPS-stimulated PMs (100 ng/mL, 1 hour) pretreated with rapamycin (1 μM, 1 hour) were immunoprecipitated with anti-ADAP and immunoblotted for STAT3. (I) PDPN expression in WT and Adap–/– PMs treated with LPS (10 ng/mL) and MHY1485 (10 μM, 24 hours) was analyzed by Western blotting. (J and K) Kaplan-Meier survival curves and bacterial burden in WT mice treated with colivelin (1 mg/kg, i.p.) after E. coli infection (5 × 107 CFU, i.p.), showing survival (n = 15 each, log-rank test) and bacterial load in peritoneal lavage fluid and blood at 18 hours (n = 5 each; unpaired t test).