Expression of NALPs in adipose and the fibrotic progression of non-alcoholic fatty liver disease in obese subjects

The prevalence of obesity in United States has remained unabated over the past 10 years with ~35% prevalence among adults [1]. A pro-inflammatory state (also referred to as low-grade chronic inflammation/meta-inflammation/sterile inflammation) associated with visceral obesity, has been also shown to be strongly correlated with the development of non-alcoholic fatty liver disease (NAFLD) [2]. This non-pathogen associated pro-inflammatory state can be stimulated and propagated by a variety of damage-associated molecular patterns (DAMPs) that originate from damaged tissue and/or tissue under stress [3]-[5]. Once released, DAMPs bind to pattern-recognition receptors (PRRs) to elicit an immune response by promoting the release of pro-inflammatory mediators and by recruiting the immune cells to the tissue.

Among the five main classes of vertebrate PRRs, the largest receptor family is comprised by nucleotide oligomerization and binding domain (NOD)-like receptors (NLRs) with a conserved NOD motif [6]. NLRs are believed to be the most evolutionarily ancient family of PRRs [7]. NACHT, LRR and PYD domains-containing proteins (NLRPs/NALPs) belong to the NOD-like receptor (NLR) family that differs from other NLRs by their N-terminal PYD domains [8],[9].

Recently NALPs have attracted attention as PRRs that link recognition of DAMPs with the regulation of inflammatory response [7],[10]. Human genome encodes 14 NALPs, some of which are required for inflammasome formation [9]. The inflammasomes are large, signal-induced multiprotein complexes responsible for the proteolytic cleavage and activation of procaspases-1. In turn, activated caspase-1 promotes maturation of the pro-inflammatory cytokines interleukin-18 (IL-18), interleukin-1β (IL-1β) and interleukin-33 (IL-33) [10],[11],[12]. The NALP1 inflammasome is composed of NALP1, apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC), caspase-1, and caspase-5, whereas the NALP2/3 inflammasome contains NALP2 or NALP3, CARDINAL, ASC, and caspase-1 [13].

The downstream targets of inflammasomes include known regulators of inflammation and immunity- IL-1β and IL-18. IL-1β triggers the production of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), two cytokines that elicits immune cell migration and infiltration into tissue [14], and promote the generation and maintenance of interferon-gamma (IFN-γ) and interleukin-17 (IL-17) producing T cells [15]. Interleukin-18 (IL-18) incites immune cell recruitment and activation [11] and influences natural killer (NK) cell and T cell effector responses [16],[17].

A large number of recent studies have shown adipose tissue to be the major source of inflammatory molecules in obesity [18]-[21]. The expression patterns of NALPs have not been previously explored in visceral adipose. It is reasonable to speculate that NALPs may be activated in adipose tissue of obese individuals and that these NALPs may contribute to the release of pro-inflammatory IL-1β and IL-18 from adipocytes, the development of the systemic inflammation and the obesity-associated progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH).

With this in mind, we profiled visceral adipose expression of 14 NALPs as well as caspase-1, ASC, IL-18 and IL-1β in 45 obese subjects (BMI ≥ 35) with biopsy proven NAFLD. Expression levels of NALPs were correlated to the serum indicators of system-wide inflammation IL-1β and IL-18 and to various parameters describing the underlying liver pathology.

Systemic inflammation is a common finding in both visceral obesity and chronic liver disease [25],[26]. Inflammasome activation has been recently recognized to play an increasingly important role in the development of liver disease [27]-[29]. However, the tissue source and mechanisms of inflammasome mediated liver damage in obesity associated NAFLD remain disputed [29],[30]. Activation of inflammasomes involves two separate steps, the priming of tissue to inflammatory response that results in transcriptional activation of NALPs, IL18 and IL1B genes, and the activation of the inflammasome complex that culminates in the secretion of active pro-inflammatory IL-1β and IL-18 cytokines. To better understand the role of adipose specific inflammasome components in NAFLD, we assessed the adipose tissue mRNA for inflammasome components and serum levels of its downstream targets in 45 morbidly obese patients (BMI ≥ 35) with various liver conditions of NAFLD spectrum.

Previous studies have shown that the NALP3 inflammasome and NALP3-dependent caspase-1 activation are central to the inflammation and the development of the liver fibrosis [31]. In our study, expression levels of NALP3 mRNA in adipose were positively correlated with serum levels of liver enzyme AST(r = 0.32; p = 0.03) and the levels of mRNA encoding caspase-1 were positively correlated with Kupffer cell hypertrophy and the hepatic lobular inflammation scores. However, to our surprise, the levels of mRNA encoding for NALP 4, 5, 7, 8, 9, 10, 11, 13 and IL-1B were significantly downregulated in adipose tissue samples collected from NASH cohort as compared to patients without NASH (Table 2). Moreover, the expression patterns of mRNA for many NALPs, especially for non-inflammasome forming NALPs, were co-correlated to each other (Table 3).

So far, only NALPs 1–3 have been shown to form active inflammasome, while the functions of other NALPs remain obscure [9]. Some NALP proteins, for example, NALP12 and NALP6, have been previously shown to suppress inflammation through downregulation of NF-κB signaling subsequent to TLR activation [3]. Another revolving theme in the study of non-classic NALPs, for example, NALP5, NALP7, NALP8 and NALP9 is their involvement in oocyte maturation, implantation and other fertility-related processes [32],[33]. It is tempting to speculate that the decrease in adipocytic expression of these NALPs may contribute to decreased fertility in patients with NASH and metabolic syndrome, possibly through known association of NASH with polycystic ovary syndrome (PCOS) [34],[35]. Non-inflammasome related NALPs may also play as yet unknown roles in pathogenesis of obesity-associated disorders through modulation of adipocytic secretion or other pathways.

Interestingly, mRNAs encoding for NALP4 and its substrate IL1B were both downregulated in the cohort with hepatic pericellular fibrosis, while recently published study of Boaru et al. showed time and concentration-dependent increase in expression of NALP4 in cultured hepatic stellate cells upon lipopolysacharride (LPS) stimulation [29]. This would suggest a role for NALP4 in promoting fibrotic response by activated stellate cells. The contradiction between our data and observation of Boaru et al. [29] may be attributed to either yet unknown tissue specific differences in the function of NALP4, or to a negative feedback signal operating between adipose tissue and liver which may be, in fact, time dependent. It is possible that the inflammation and the fibrotic processes may be initiated in adipose tissue as described Reggio et al., [36], but later be shifted to distant organs such as liver. This may be accompanied with negative feedback signaling that originates in the liver and attempts to restore homeostasis.

The observed downregulation of adipose-specific production of IL1B mRNA in patients with pericellular fibrosis (Table 2 and Figure 1) as well as NASH (Table 2 and Figure 2) also supports this hypothesis. The downregulation at expression level is also supported by the lack of detectable IL1B protein levels in circulation (Assay Range: 0.125 - 8 pg/mL). Unlike the production of most inflammatory cytokines, the production of biologically active IL-1β is dependent on transcription, translation, maturation and secretion mechanisms, all of which are tightly regulated in tissue-specific manner. This may be attributed to tissue specific roles of IL1B. Animal studies showed that hepatic IL-1β protein and mRNA levels to be increased in various diet-induced NASH models in mice [28], while adipose specific IL1B deficiency in mice increase susceptibility to obesity [28],[37]. In another recent study, the authors demonstrate in animal models that IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity [38]. While the translation of animal studies to humans is difficult, the discrepancy in observed expression levels of IL1B in obesity associated NASH and pericellular fibrosis maybe attributed to the extremely high BMI of the cohort being examined (BMI ≥ 35). This along with the observed negative correlation of IL1B mRNA levels with BMI (r = −0.317; p = 0.04) indicates an ongoing negative feedback loop between adipose specific IL1B expression and an accumulation of VAT.

Further support to the hypothesis centering on ongoing negative feedback loop operating between adipose tissue and liver in morbidly obese individuals with severe NAFLD, is provided by result of the comparison in another cohort. In cohort of subjects with portal fibrosis compared to those without portal fibrosis, adipose specific NALP6 mRNA (Table 2 and Figure 3) and circulating IL18 (Table 2 and Figure 3) protein were upregulated with hepatic portal fibrosis, while adipose specific IL18 mRNA was not significantly different in the same cohort. This is interesting, since NALP6 has been shown to have an anti-inflammatory role by downregulating NF-κB signaling subsequent to TLR activation [3]. Conspicuously, whilst circulating IL-18 protein was upregulated in portal fibrosis (Table 2 and Figure 3), this was not accompanied with an upregulation of adipose specific IL18 mRNA. Additionally, circulating IL-18 was found to be positively correlated with BMI (r = 0.41; p = 0.012). This is in agreement with previous studies [39]-[43]. Notably, circulating IL-18 levels are also associated with increased liver injury markers [44]: AST (r = 0.33; p = 0.04) and ALT (r = 0.41; p = 0.01) levels respectively as seen in previous reports [43]. Since IL-18 is more widely expressed, this may indicate additional sources of circulating IL-18 protein such as from the gut [28] or the liver [45], playing a role in the inflammation and hepatic injury progression. Thus, IL-18 might contribute to the development of liver disease, albeit the origin of IL-18 may not be solely from adipose.

For the first time, we profiled gene expression profile of all the 14 members of NALPs in visceral adipose tissue. As can be seen from correlation analysis (Table 3), the expression of these genes is significantly correlated with each other. While most of the studies on obesity and insulin resistance have focused on NALP3 mediated inflammasome, our study showed that there is a need for exploring roles of other members of this family of proteins in systemic inflammation and chronic liver conditions.

Among the limitations of this study is that the gene expression analysis has been carried out in whole adipose tissue instead of studying isolated tissue components, i.e. adipocytes and stromal vascular cells. This study was limited to two markers of systemic inflammation, IL1B and IL18 that were selected as known targets released into circulation upon activation. The profiling of greater variety of inflammatory molecules may yield better mechanistic resolution of inflammatory responses in adipose. Another avenue to explore is the parallel study of NALP component expression in liver and other peripheral tissues, and the study of NALP components at protein level.

The results of our study indicate that non-inflammasome related NALPs may play as yet unknown roles in pathogenesis of obesity-associated disorders through modulation of adipocytic secretion or other pathways. Our observations also point at a possible shift in inflammation and fibrotic response from adipose tissue to liver in patients with severe obesity. While this hypothesis needs further experimental verification, it takes us a step closer in understanding the underlying reason for some obese individuals being metabolically healthy as compared to metabolically unhealthy obese. Additional study of matched liver and adipose tissues from same individuals will help delineate the cross talk mechanisms between inflammation and fibrosis in obesity and NAFLD.