To our knowledge, the present study included the first MR analysis to explore the association of IL family member levels with NAFLD and suggested that a potential the causal relationship between IL-1ra, IL-6, and IL-6RA on the risk of NALFD.
NAFLD is characterized by the excessive accumulation of lipids (triglycerides) in the liver and represents a spectrum of liver conditions with the possible outcomes of liver failure and hepatocellular carcinoma [1, 2]. Patients with NAFLD and NASH can present a variable combination of all of these different conditions, and NAFLD is thus considered a multifactorial disease with highly heterogeneous clinical manifestations . Inflammation participants in NAFLD development [6, 7], and insulin resistance has also been suggested to be an independent risk factor for NAFLD progression, since insulin resistance in both the adipose tissue and the liver leads to an accumulation of free fatty acids in hepatocytes, which results in lipotoxicity [22, 23]. To better understand the pathogenesis of metabolic diseases and to identify prevention and treatment strategies, it is required to identify factors causally related to disease developments. The genomics revolution offers new opportunities to perform MR studies to establish causal relationships between a wide range of biomarkers (exposure) and disease (outcomes).
IL-1ra is usually regarded as anti-inflammatory factor, since it is a naturally anti-inflammatory cytokine that competitively blocks the signaling of IL-1α and IL-1β . IL-1ra behaves as an acute-phase protein with its serum concentrations increasing in inflammatory conditions. It has been shown that mice with IL-1ra deficiency are associated with chronic inflammation and leaner phenotype which is the result of aberrant lipid metabolism . Even though being associated with fat, a close association between IL-1ra and CRP and fibrinogen was still found after adiposity adjustment [26, 27]. Accordingly, recombinant IL-1ra has been used to reduce the inflammatory markers in obese and non-obese diabetic patients  and treat several autoimmune and systemic inflammatory diseases .
However, one prior study analyzed liver biopsies from 119 morbidly obese subjects, reported that serum IL-1ra level was associated with NAFLD development, and suggested serum IL-1ra concentration could improve non-invasive diagnosis of NASH , also our result showed it was positively associated with NAFLD risk. As mentioned above, IL-1ra can prevent the binding of IL-1α and IL- 1β to IL-1R1 and attenuates inflammation. The dysregulation of this equilibrium has been reported in metabolic-related diseases . In our MR analysis, IL-1β was not increased along with NAFLD risk, so as the observation in the study among NASH , indicating imbalance between IL-1ra and IL-1β may play a role in NAFLD development. Moreover, IL-1ra was generated from adipose tissue and liver, and function both in systematic and organs, therefore, the systematic IL-1ra level tested in database may not reflect the roles in the liver.
Besides, IL-1ra are related to adipose tissue amount and insulin sensitivity [27, 33], that serum IL-1ra concentrations are increased in obesity and are further elevated in those with type 2 diabetes mellitus (T2DM), and IL-1ra is correlated with insulin levels and several surrogated markers of insulin resistance or sensitivity such as homeostasis model assessment (HOMA), quantitative insulin sensitivity check index (QUICKI) and triglyceride-glucose index (Tyg index), even after adjustment by the percentage of body fat. Thus IL-1ra upregulation is associated with IR, which can increase hepatic de novo lipogenesis and impairs insulin-mediated suppression of adipose tissue lipolysis with consequent increased fluxes of free fatty acids into the liver . All these factors may contribute to hepatic fat accumulation and increased lipids in circulation. IR is regarded as one of the key pathophysiological features in human NAFLD [23, 34], and is contributed by TNF-α, IL-6 and various IL-1 type cytokines . A rise in IL-1ra precedes the onset of T2DM  and discriminates which individuals will develop T2DM . One MR study revealed that genetically predicted IR was positively correlated with the higher risk of severe steatohepatitis and liver fibrosis and indicated IR would be a reason for steatohepatitis . The role of IL-1ra in IR is independent of binding to the IL-1R1, which suggests an alternative mechanism of IL-1Ra in metabolic regulation .
Our MR study provides another insight on the causal correlation of IL-1ra on the risk of NAFLD. IL-1ra has dual roles in regulating metabolism; its anti-inflammatory properties can prevent hepatic steatosis via blocking IL-1R1, and it can lead to IR via IL-1R1-independent mechanisms. For instance, IL-1ra can cause leptin resistance by inhibiting the anti-appetite signaling pathway of leptin . moreover, our study included patients diagnosed with NAFLD, but no further disease grading was carried out. IL-1ra may play different roles in different stages, which cannot be distinguished. Further comprehensive mechanistic research is warranted in investigate such effects on various organs and various cell compositions.
In this MR analysis, we also found IL-6 and IL-6RA are associated with risk of NAFLD. Many studies have demonstrated chronic exposure to IL-6 promotes hepatic gluconeogenesis, impaired lipid metabolism , and hepatic insulin resistance (IR) , indicating IL-6 blockade may offer a potential therapeutic target for NAFLD. Recently, researchers introduced the concepts of “pro-inflammatory diet” and “anti-inflammatory diet”. The former, including the consumption of a large amount of red meat, processed meat, refined carbohydrates and added sugar can promote chronic inflammation by the induction of inflammatory factors such as IL-6, TNF-α-R1, TNF-α-R2, CRP, etc., while the latter, including whole grains, fruit, and green leafy and dark yellow vegetables can inhibit the expression of inflammatory factors . Moreover, dietary supplements, such as n-3 poly-unsaturated fatty acids (PUFAs), vitamin C, and vitamin D, etc [43, 44], and physical exercise , can downregulate the production of a wide range of pro-inflammatory cytokines.
IL-6 exhibits a complex biology as it can signal via three different modes . In classic signaling, IL-6 binds to the membrane-bound IL-6RA, which is mainly expressed on hepatocytes and immune cells, and induces homodimerization of gp130 and downstream signaling in a cell-autonomous manner. In the trans-signaling mode, IL-6 uses the soluble IL-6RA (sIL-6RA). This mode of signaling is considered to cause proinflammatory properties, and enables cells lacking the membrane-bound IL-6RA to be stimulated by IL-6. In the third type of signaling, cluster signaling (also termed trans-presentation), IL-6 is presented bound to the IL-6RA on the surface of dendritic cells to cognate T cells, which are activated via gp130 homodimerization. The classical/ trans-signaling pathway of IL-6 is complicated, and the genetic variants in the IL-6RA region change how much soluble and membrane-bound IL-6RA is available and also their proportion, rather than changing the total level. The current study found both IL-6 and IL-6RA instrumental variables are associated with NAFLD risk, verifying the participation of IL-6 signaling in NAFLD development, but the SNP of IL-6RA cannot distinguish membrane-bound or soluble IL-6RA, or the balance between the two.
IL-6 family cytokines are now viewed as an appealing option in many metabolic diseases, including T2DM and NAFLD [46, 47]. Even though targeting IL-6 signaling through inhibition of IL-6 or IL-6R may elicit similar effects, classic IL-6 signaling is thought to represent a defense mechanism (eg. against pathogens), while trans-signaling has been suggested to play pathogenic roles in chronic inflammation . We speculated that neutralizing antibodies against IL classic pathway may influence the defense function of IL-6, implicated by infection cases due to impaired acute phase response after the treatment [48, 49]. The trans-signaling pathway might be a targeting point for NAFLD treatment. The newly developed target therapy by the blockade of IL-6 trans-signaling, Olamkicept (FE 999301; TJ 301), has been applied to inflammatory bowel disease (IBD) patients and induced clinical response in 44% and clinical remission in 19% of patients . Based on the result of our study, we hypothesized that a therapeutic approach specifically targeting IL-6 trans-signaling could specifically treat NAFLD without interfering with physiologic and host defense activities involving classic signaling.