Fig. 4

PLXNC1 alters immune response to intracellular LPS through a mechanism based on cAMP metabolism and interaction with adenylate cyclase 4 (ADCY4). Next-generation sequencing (NGS) was performed on BMDMs from PLXNC1^-/- mice and their littermate controls after LPS transfection. Analysis of A the inflammasome pathway on the mRNA expression levels of inflammasome-related genes. B The cAMP-mediated signaling pathway in BMDMs from PLXNC1^-/- mice; notably, the mRNA expression of ADCY4 (red frame) in PLXNC1-/- cells under baseline conditions, and upon LPS differs. C PKA activity upon LPS transfection in BMDMs from PLXNC1-/- and wild type BMDMs. D Human monocytes/macrophages were transfected with LPS, and Co-immunoprecipitation was performed: samples were precipitated for PLXNC1, and blotted for ADCY4, as well as precipitated for PLXNC1 and blotted for ADCY4 (Input: a sample of the total protein lysate taken before the immunoprecipitation step; respective molecular weight of the proteins have been added to the left of the blot). E Digitonin assay with wild-type BMDMs revealed the presence of intracellular PLXNC1, which was enriched upon LPS transfection. F PLXNC1 interacts with ADCY4 to inhibit caspase-11 activation through a mechanism based on cAMP-metabolism and PKA activity (The data are presented as the means ± SEMs; n = 4–11 mice/group; Western blots, n = 3 mice/group; *p < 0.05; **p < 0.01 and ***p < 0.001 as indicated). The illustration for Fig. 4F was created using biorender.com. ADCY4, Adenylate cyclase 4; cAMP, cyclic adenosine monophosphate; CSP1/11, Caspase-1/-11; GsdmD, Gasdermin D; IL-1β, Interleukin-1β/-18; LPS, lipopolysaccharide; mM, mus musculus; Rb, rabbit