Apoptosis signal-regulating kinase 1 (ASK1) inhibition reduces endothelial cytokine production without improving permeability after toll-like receptor 4 (TLR4) challenge

Abstract

Sepsis represents a life-threatening event often mediated by the host's response to pathogens such as gram-negative organisms, which release the proinflammatory lipopolysaccharide (LPS). Within the endothelium, the mitogen-activated protein kinase (MAPK) pathway is an important driver of endothelial injury during sepsis, of which oxidant-sensitive apoptosis signal-regulating kinase 1 (ASK1) is postulated to be a critical upstream regulator. We hypothesized that ASK1 would play a key role in endothelial inflammation during bacterial challenge. Utilizing RNA sequencing data from patients and cultured human microvascular endothelial cells (HMVECs), ASK1 expression was increased in sepsis and after LPS challenge. Two ASK1 inhibitors, GS444217 and MSC2023964A, reduced cytokine production in HMVECs following LPS stimulation, but had no effect on permeability as measured by transendothelial electrical resistance and intercellular space. MAPKs are known to interact with endothelial nitric oxide synthase (eNOS) and ASK1 expression levels correlated with eNOS expression in patients with septic shock. In addition, eNOS physically interacted with ASK1, though this interaction was not altered by ASK1 inhibition, nor did inhibition alter MAPK p38 activity. Instead, among MAPKs, ASK1 inhibition only impaired LPS-induced JNK phosphorylation. The reduction in JNK activation caused by ASK1 inhibition impaired JNK-mediated cytokine production without affecting permeability. Thus, LPS triggers JNK-dependent cytokine production that requires ASK1 activation, but both its effects on permeability and activation of p38 are ASK1-independent. These data demonstrate how distinct MAPK signaling pathways regulate endothelial inflammatory outputs during acute infectious challenge.

Keywords: MAPK kinase kinase (MAPKKK); apoptosis signal-regulating kinase 1 (ASK1); c-Jun N-terminal kinase (JNK); endothelial nitric oxide synthase (eNOS); extracellular signal-regulated kinase (ERK); human microvascular endothelial cells (HMVECs); lipopolysaccharide (LPS); mitogen-activated protein kinase (MAPK); pathogen-associated molecular patterns (PAMPs); proximity ligation assays (PLA); toll-like receptor (TLR); transendothelial electrical resistance (TEER); tumor necrosis factor alpha (TNFα).