Neutrophil extracellular traps impair intestinal barrier functions in sepsis by regulating TLR9-mediated endoplasmic reticulum stress pathway

Increased neutrophil extracellular traps (NETs) formation has been found to be associated with intestinal inflammation, and it has been reported that NETs may drive the progression of gut

dysregulation in sepsis. However, the biological function and regulation of NETs in sepsis-induced intestinal barrier dysfunction are not yet fully understood. First, we found that both

circulating biomarkers of NETs and local NETs infiltration in the intestine were significantly increased and had positive correlations with markers of enterocyte injury in abdominal sepsis

patients. Moreover, the levels of local citrullinated histone 3 (Cit H3) expression were associated with the levels of BIP expression. To further confirm the role of NETs in sepsis-induced

intestinal injury, we compared peptidylarginine deiminase 4 (PAD4)-deficient mice and wild-type (WT) mice in a lethal septic shock model. In WT mice, the Cit H3-DNA complex was markedly

increased, and elevated intestinal inflammation and endoplasmic reticulum (ER) stress activation were also found. Furthermore, PAD4 deficiency alleviated intestinal barrier disruption and

decreased ER stress activation. Notably, NETs treatment induced intestinal epithelial monolayer barrier disruption and ER stress activation in a dose-dependent manner in vitro, and ER stress

inhibition markedly attenuated intestinal apoptosis and tight junction injury. Finally, TLR9 antagonist administration significantly abrogated NETs-induced intestinal epithelial cell death

through ER stress inhibition. Our results indicated that NETs could contribute to sepsis-induced intestinal barrier dysfunction by promoting inflammation and apoptosis. Suppression of the

TLR9–ER stress signaling pathway can ameliorate NETs-induced intestinal epithelial cell death.

Sepsis is defined as life-threatening organ dysfunction caused by the dysregulation of the host response secondary to infection, which remains a leading cause of high mortality in the

intensive care unit [1, 2]. The gastrointestinal tract is the most easily and frequently involved organ in the process of sepsis and the gut has been deemed the motor of sepsis [3, 4]. The

breakdown of the gut barrier can result in many bacteria and toxins entering the internal environment, driving lethal sepsis and even multiple organ dysfunction syndrome (MODS) [5]. Hence, a

more thorough understanding of the inflammatory mechanism involved in intestinal barrier dysfunction is pivotal for developing more efficient treatments against sepsis and MODS.

Since neutrophil extracellular traps (NETs) were firstly described by Brinkman in 2004, excessive NETs formation has been shown to be involved in the pathophysiology of sepsis [6, 7]. NETs

are web-like structures protruding from the membrane of activated neutrophils, comprising decondensed DNA fibers accompanied by intracellular proteins, including histones, myeloperoxidase

(MPO), and other antimicrobial proteins [6]. Histone citrullination induced by peptidylarginine deiminase 4 (PAD4) plays a pivotal role in chromatin decondensation, which is one of the most

crucial processes in NETs extrusion [8]. NETs can trap and kill a broad range of pathogens, including bacteria and viruses [9]. However, uncontrolled NETs formation is generally considered a

double-edged sword [10]. Excessive NETs formation has been indicated to have a role in both infectious and noninfectious diseases, including but not limited to thrombosis, diabetes,

vasculitis, and cancer [11, 12]. NETs are widely recognized as endogenous damage-related molecular patterns (DAMPs) that can be recognized by TLR receptors [13]. Strategies targeting NETs

formation have shown therapeutic and can improve survival in animal models of sepsis [14]. Our preliminary research detected increased NETs infiltration in the intestines, and NETs

disruption ameliorated intestinal injury in endotoxin mice [15]. However, the biological function and downstream signaling pathway of NETs in sepsis-induced intestinal barrier dysfunction

are not yet fully understood.

The endoplasmic reticulum (ER) is the primary cellular organelle in which protein synthesis, maturation, folding, modification, and degradation take place [16]. If the optimal balances of

protein folding are interrupted in the ER, a condition known as “ER stress” may occur [17]. Sustained ER stress can initiate inflammation through various mechanisms, including the production

of reactive oxygen species (ROS) [18]. Quillard et al. reported that TLR2 stimulation followed by NETs participation may render smooth muscle cell-rich plaques susceptible to superficial

erosion and thrombotic complications by inducing ER stress and ROS production [19]. ER stress is implicated in the progression of intestinal barrier impairments in inflammatory bowel

diseases [20]. Endoplasmic reticulum stress can be regulated by TLR receptors and has a role in the process of sepsis-induced intestinal injury [21, 22].

We hypothesized that in sepsis, NETs can induce TLR receptor–ER stress–ROS signaling and activation of the inflammatory response, increasing intestinal permeability and bacterial

translocation. In the current study, to our knowledge, increased local NET infiltration in the intestine in abdominal sepsis patients was firstly found, and the levels of local citrullinated

histone 3 (Cit H3) were associated with the level of ER stress activation. We further determined that, in vivo, NETs depletion by PAD4 deficiency alleviated intestinal inflammation and

decreased ER stress activation in a lipopolysaccharide (LPS)-induced lethal septic shock model and that, in vitro, NETs treatment induced ER stress activation and intestinal epithelial

monolayer barrier disruption in a dose-dependent manner, which confirmed the detrimental effect of NETs on intestinal barrier functions. Notably, we also observed that NETs-induced

intestinal barrier dysfunction was mediated by ER stress, which is regulated by Toll-like receptor 9 (TLR9).

To determine NETs performance and ER stress levels in sepsis and their possible pathological impact on intestinal barrier dysfunction, we compared the relative expression in serum and

relative protein levels in intestinal samples of healthy and abdominal sepsis patients. We firstly investigated whether the expression of NETs is altered in the peripheral system of these

patients. Serum cell-free DNA (cf-DNA), a rough biomarker of NETs, and serum Cit H3-DNA complex, a specific biomarker of NETs, were significantly elevated in abdominal sepsis patients

compared with healthy controls (Fig. 1A). In addition, the levels of serum D-lactate and intestinal fatty-acid binding protein (I-FABP), biomarkers of intestinal damage, were markedly

increased in abdominal sepsis patients (Fig. 1B). Moreover, there were significant correlations between NETs markers and intestinal damage markers in serum (Fig. 1C).

A Circulating cf-DNA and Cit H3-DNA complexes in abdominal sepsis patients (n = 5) and healthy controls (n = 5) were analyzed by ELISA. B Serum I-FABP and D-lactate were analyzed by ELISA. C

Correlation between circulating NETs biomarkers (cf-DNA and Cit H3-DNA complex) and circulating intestinal injury biomarkers (I-FABP and D-lactate). NETs neutrophil extracellular traps,

cf-DNA cell-free DNA, Cit H3 citrullinated histone 3, ELISA enzyme-linked immunosorbent assay, I-FABP intestinal fatty-acid binding protein. Data are expressed as the means ± SD. *P