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 3

ADAP is indispensable for the induction of PDPN expression in macrophages in response to LPS stimulation or bacterial infection during sepsis.

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ADAP is indispensable for the induction of PDPN expression in macrophage...
(A) Gating strategy for FACS of CD11b+F4/80+ and CD11b+F4/80– cells from the peritoneal cells of WT and Adap–/– mice 18 hours after E. coli injection (2 × 107 CFU, i.p.). RNA-Seq analysis identified DEGs (log2[fold change] ≤ −1/ ≥ 1, 5% FDR) in sorted cell populations. (B) Schematic of RNA-Seq analysis of WT and Adap–/– PMs exposed to LPS (100 ng/mL) for 12 hours in vitro (upper panel). Venn diagram of DEGs in WT versus Adap–/– PMs from E. coli–infected mice (209 DEGs) and WT versus Adap–/– PMs treated with LPS (166 DEGs), revealing 13 overlapping DEGs (lower panel). (C) Heatmap and hierarchical clustering of the 13 overlapping DEGs from B. (D–F) PDPN expression in WT and Adap–/– PMs with or without LPS stimulation (100 ng/mL) was analyzed by Western blotting (D), flow cytometry (E), and qPCR (F, n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt. (G) Immunoblot analysis of PDPN in WT or ADAP-knockdown (ADAPKD) RAW264.7 cells stimulated with or without LPS (100 ng/mL) for the indicated time points. (H) Pdpn mRNA expression in WT and Adap–/– iBMMs with or without LPS stimulation (100 ng/mL, 12 hours), analyzed by qPCR (n = 3 each, 2-way ANOVA, Šidák’s multiple-comparison test). Relative mRNA levels were normalized to Hprt. (I) ADAPKD RAW264.7 cells were reconstituted via lentiviral transduction with ADAP to induce overexpression (OE) or with an empty vector (EV) as a control, followed by mock treatment or stimulation with LPS (100 ng/mL) for 24 hours. Whole cell lysates were prepared and subjected to Western blot analysis of PDPN. α-Tubulin blots are derived from the same samples run contemporaneously in parallel gels.