TLRs are trans-membrane proteins expressed in innate immune cells especially the neutrophils, which play a central role in detecting invading microbes or endogenous danger signals and initiating corresponding immune responses (Kawai & Akira, 2010). Ten TLRs (i.e. TLR1-TLR10) have been identified in humans, while different TLRs could recognize various ligands, including bacterial cell wall components and viral double-stranded RNA(Xing, Li, Cao, & Huang, 2007). There were five housekeeping genes and 84 genes related to TLR-mediated signal transduction. These genes were divided into different groups according to their functions: TLR, adaptor and interacting proteins, effectors, regulation of the adaptive immunity group, downstream pathways and target genes. In this study, we found a stepwise down-regulation of TLR3 in neutrophils from patients with cirrhosis to those with ACLF. These results were consistent with a recent study showing defective TLR3 expression in ACLF patients(Bernsmeier et al., 2017). As previously described, TLR3 could recognize retroviral dsRNA, which then triggered the activation of transcription factor interferon regulatory factor-3 (IRF3) in a MyD88-independent manner. This allowed the induction of IFNs (Doyle et al., 2002). Thus, defective TLR3 signaling may lead to down-regulation of type I and II interferons, interferon regulatory factor TBK1 and interferon-stimulated gene (ISG) CXCL10 expression in immune cells of ACLF patients. Although TLR4 engagement also induced IFNs in immune cells (Fairfax et al., 2014), TLR4 signaling was not suppressed in ACLF patients. Therefore, defective TLR3 signaling may be a major contributor to the downregulation of interferons and their ISGs in ACLF patients. Along with the IFNs, genes regulating adaptive immunity, including IL-2 and IL-12A, were significantly down-regulated. It had been shown that enhanced activity of type I IFN signaling pathways was associated with excessive inflammation and tissue damages in mouse models of endotoxin shock (Karaghiosoff et al., 2003) and autoimmune diseases (Ronnblom, 2016). Thus, down-regulation of type I interferon regulatory signaling may be protective against excessive inflammation in ACLF patients. In parallel, the anti-inflammatory cytokine IL-10 was significantly up-regulated in neutrophils, and pro-inflammatory IL-1B was down-regulated specifically in neutrophils of patients with ACLF. Taken together, these findings suggested that peripheral neutrophils in ACLF patients showed defective expression of TLR3 and IFN, together with up-regulation of the anti-inflammatory cytokine IL-10 at the transcriptional level.
The TLR signaling transcriptional landscape of immune cells in ACLF was almost completely distinct from that in cirrhosis patients, which supported the clinical and experimental data that ACLF patients showed different immune dysfunctions (Albillos, Lario, & Alvarez-Mon, 2014). The transcriptional alterations in immune cells may have significant clinical implications. The prevailing theory hypothesized a shift from systemic inflammatory response syndrome (SIRS) to compensatory anti-inflammatory response syndrome (CARS) during the natural history of ACLF, which led to immune paralysis and increased susceptibility to infections (Jalan et al., 2012). Indeed, several clinical studies had confirmed that ACLF patients showed a higher incidence of bacterial infection than the non-ACLF counterparts (Fernandez et al., 2018; Shi et al., 2015). In human and animal studies, there was a link between impaired cellular immune function and an increased risk of bacterial infection, loss of infection control and infection-associated mortality (Bernsmeier et al., 2017; Hackstein et al., 2017; Vergis et al., 2017). Our findings revealed that a reduction in TLR signaling may impair TLR-driven neutrophils responses, which therefore represented a potential therapeutic target. A recent study demonstrated that the TLR-3 agonist poly(I:C) effectively reconstituted innate immune function in ACLF, which may serve as a promising TLR-based immunotherapy for ACLF (Bernsmeier et al., 2017).
In fact, our study has some limitations. First, further validation was required for the PCR array results. Second, the expression of genes could not represent the entire biological response. Thus, in vitro and animal studies are required to investigate the feasibility of crucial roles in the TLR3 signaling pathway that may serve as a target for the treatment of ACLF.
In summary, genes in the TLR signaling pathway were differentially expressed in neutrophils through different stages of cirrhosis. In addition, we identified a dysregulated key TLR pathway in the neutrophils of ACLF patients. In future, further investigation is required to test its potential as a therapeutic target.