CXCL4 and CXCL4L1 activate human macrophage via TLR4 signaling to modulate inflammatory responses

The chemokine CXCL4 activates human monocytes/macrophages and contributes to the pathogenesis of inflammatory and fibrotic diseases. However, the receptor of CXCL4, CXCR3, is not expressed in human monocytes/macrophages. Thus, signaling pathways and receptors that mediate human monocytes/macrophages response to CXCL4 alone are not well characterized. Using human primary monocytes and mouse bone marrow-derived macrophages, we reported that CXCL4 activated NF-κB and a TBK1-JNK-AP1 signaling axis that drove the expression of inflammatory, fibrotic and neutrophil chemokine genes, and also a TRIF-RIPK3 axis-dependent necroptosis in primary human monocytes. Surprisingly, multiple evidence targeting TLR4 expression and function suggested a role for TLR4 in CXCL4 responses. Further, we show that CXCL4 and its natural variant CXCL4L1 interact with TLR4/MD-2 complex in human monocytes and CXCL4L1 induced a transcriptomic profile divergent to LPS. Ectopic expression of human CXCL4L1 using adeno-associated virus (AAV) system effectively mitigated papain-induced asthma in wild-type (WT) mice. Notably, the therapeutic impact of CXCL4L1 was absent in <i>Tlr4</i> ^-/- mice. Our findings indicate that CXCL4 and its variant CXCL4L1 modulate inflammatory and fibrotic gene expression, as well as necroptosis in human monocytes, and regulating lung inflammation through TLR4-dependent signaling, highlighting TLR4's critical role in the pathophysiological response to CXCL4/CXCL4L1.