Due to the potential association between TNF-α and NAFLD, there are experimental and clinical studies targeting to evaluate the effect of various therapeutic interventions on TNF-α and NAFLD. Furthermore, anti-TNF-α therapies in various diseases have opened a possible therapeutic window for the management of advanced NAFLD.
Pharmacologic Strategies Downregulating TNF-α in NAFLD
This section provides an overview of current and emerging pharmacologic strategies for NAFLD that reduce circulating or hepatic TNF-α based on prevailing experimental or clinical evidence. These data are also summarized in Tables
2 and
3.
Table 2
The effect of therapeutic interventions on TNF-α in experimental models of NASH.*,a
Obeticholic acid [ 54, 55] | FXR agonist | Male Ldlr − / − Leiden mice with HFD-induced NASH [ 54]; MC4R-KO mice with WD-induced NASH [ 55] | Hepatic TNF-α mRNA | ↓ |
Obeticholic acid + Losartan [ 56] | FXR agonist + ARB | Male Fischer 344 rats with CDAAD-induced NASH | Hepatic TNF-α mRNA | ↓ |
Obeticholic acid + Sitagliptin [ 57] | FXR agonist + DPP-4 inhibitor | Male Fischer 344 rats with CDAAD-induced NASH | Hepatic TNF-α protein | ↓ |
Obeticholic acid + Simvastatin [ 58] | FXR agonist + statin | C57BL/6 J mice with HFD-induced NASH | Hepatic TNF-α mRNA | ↓ |
Obeticholic acid + Elafibranor [ 59] | FXR agonist + PPAR-α/δ agonist | V-Lep ob/JRj (ob/ob) mice with HFD-induced NASH | Hepatic TNF-α mRNA | ↓ |
| PPAR-γ agonist | Male Sprague–Dawley rats with HFD diet-induced NASH [ 68]; Wistar rats with MCDD-induced NASH [ 69] | Serum TNF-α | ↓ |
| PPAR-α agonist | Male Sprague–Dawley rats with HFD-induced NASH [ 72]; male C57BL/6 mice with CDAHFD-induced NASH [ 73]; male Wistar rats with HFD-induced NASH [ 74] | Hepatic TNF-α mRNA [ 72, 74]; hepatic TNF-α protein [ 73] | ↓ |
| PPAR-α/γ agonist | Male C57BL/6 mice with CDAHFD-induced NASH [ 73]; male Wistar rats with HFD-induced NASH [ 74]; DIAMOND mice with WD-induced NASH [ 75] | Hepatic TNF-α protein[ 73]; hepatic TNF-α mRNA[ 74, 75] | ↓ |
| Pan-PPAR agonist | C57BL/6 mice with CDAHFD- and WD-induced NASH | Hepatic TNF-α mRNA | ↓ |
| SGLT2 inhibitor | C57BL/6 J mice with HFD-induced NASH | Hepatic TNF-α mRNA | ↓ |
| DPP-4 inhibitor | Male Fischer 344 rats with CDAAD-induced NASH | Hepatic TNF-α protein | ↔ |
| FGF-21 analog | C57BL/6 mice with MCDD-induced NASH | Hepatic TNF-α mRNA | ↓ |
| Statin | Male Sprague–Dawley rats with HFHCD-induced NASH | Hepatic TNF-α mRNA | ↓ |
| Statin | C57BL/6 J mice with HFD-induced NASH | Hepatic TNF-α mRNA | ↓ |
| ARB | Male Fischer 344 rats with CDAAD-induced NASH | Hepatic TNF-α mRNA | ↔ |
| ARB | Male Wistar rats with MCDD-induced NASH | Hepatic TNF-α mRNA | ↓ |
| ARB | Male Fischer 344 rats with CDAAD-induced NASH | Hepatic TNF-α protein and serum TNF-α | ↓ |
| ARB | Male Fischer 344 rats with CDAAD-induced NASH [ 106]; male Sprague–Dawley rats with MCDD-induced NASH [ 104] | Hepatic TNF-α protein [ 106] and serum TNF-α [ 104] | ↓ |
Table 3
The effect of therapeutic interventions on TNF-α in patients with NAFLD as derived from clinical trials.*,a
| PPAR-γ agonist | 30 mg od | Prospective cohort study | Biopsy-defined NASH (18) | - | 12 months | Circulating TNF-α | ↔ |
| GLP-1RA | 1.8 mg od | RCT | T2DM + US-defined NAFLD (30) | Metformin | 12 weeks | Circulating TNF-α | ↓ |
| Living microorganisms | Various | Meta-analysis of 21 RCTs | Biopsy- or MRS- or US-defined NAFLD (1037) | Placebo | NA | Circulating TNF-α | ↔ |
| Synbiotics (Probiotics + Prebiotics) | Various | Meta-analysis of 7 RCTs | NAFLD (419) | Placebo | NA | Circulating TNF-α | ↓ |
| Antibiotic | 550 mg bid | RCT | Biopsy-defined NASH (50) | Placebo | 6 months | Circulating TNF-α | ↓ |
| Antioxidant | 300 mg bid δ-tocotrienol | RCT | US-defined NAFLD (100) | α-tocopherol | 12 months | Circulating TNF-α | ↓ |
| Phosphodiesterase inhibitor | Various | Meta-analysis of 3 RCTs and 2 prospective cohorts | Biopsy- or US-defined NAFLD (147) | Placebo, UDCA | Various | Circulating TNF-α | ↓ |
| Phosphodiesterase inhibitor | Various | Meta-analysis of 5 RCTs | Biopsy- or US-defined NAFLD (157) | Placebo | 3–12 months | Circulating TNF-α | ↔ |
| Bile acid + Antioxidant | 12–15 mg/kg/day + 400 IU bid | RCT | Biopsy-defined NASH (41) | UDCA + placebo, placebo + placebo | 24 months | Circulating TNF-α | ↔ |
| Statin | 10 mg od | Prospective cohort study | Biopsy-defined NASH + dyslipidemia (42) | - | 12 months | Circulating TNF-α | ↓ |
| n-3 PUFA | 2700 mg od | Prospective cohort study | Biopsy-defined NASH (23) | - | 12 months | Circulating TNF-α R | ↓ |
ALA/ EPA/ DHA + Diet [ 101] | n-3 PUFA + Diet | 2 gr od + 30% caloric restriction | RCT | US-defined NAFLD (36) | Diet (30% caloric restriction) | 6 months | Circulating TNF-α | ↓ |
Obeticholic acid (OCA) is a potent farnesoid X receptor (FXR) agonist, an intriguing class of medication under investigation for the treatment of NAFLD. In addition to controlling a number of metabolic processes, including bile acid synthesis, glucose homeostasis, and lipid metabolism, OCA also exhibits anti-inflammatory and anti-fibrotic properties in the liver [
53]. OCA has been reported to reduce hepatic TNF-α expression, either as monotherapy [
54,
55] or more potently when combined with other agents (e.g., simvastatin, losartan, sitagliptin, elafibranor) [
56‐
59] in experimental animal models of NASH. In clinical trials, OCA improved histological features of NASH and, more importantly, fibrosis [
60,
61], thus being an important candidate for the treatment of NASH, as long as safety concerns, including unfavorable effects on lipid profile and pruritus are addressed.
Peroxisome proliferator-activated receptors (PPARs) are a superfamily of lipid-sensoring nuclear receptors, which are considered promising therapeutic targets for NAFLD, as they regulate glucose and lipid metabolism, inflammation and possibly fibrosis [
62]. Pioglitazone, a PPAR-γ agonist that belongs to thiazolidinediones, is a long-standing antidiabetic medication, usually preferred as a second- or third-line option [
63]. Although pioglitazone was shown to reduce TNF-α experimentally [
64], in human NASH, it improved hepatic steatosis, inflammation, and possibly marginally hepatic fibrosis [
65], by increasing the levels of circulating adiponectin but without affecting circulating TNF-α [
66]. Pioglitazone is currently recommended for
off-label treatment in selected patients with biopsy-proven NASH and fibrosis stage (F) ≥ 2 [
67]. Similarly, rosiglitazone, another PPAR-γ activator belonging to thiazolidinediones, also reduced circulating TNF-α in NASH rat models [
68,
69]; however, no data regarding its effect on TNF-α in human NASH are available. In addition, rosiglitazone did not improve NASH in the phase II trial (FLIRT) [
70]. It should be also noted that the use of rosiglitazone, initially approved as anti-diabetic medication, was restricted because of an increase in myocardial infarction risk [
62]. Fenofibrate, a commonly used drug against hypertriglyceridemia with an agonistic effect on PPAR-α, has been investigated against NAFLD, due to its pleiotropic properties, including lipid-lowering and anti-inflammatory action [
71]. In line, fenofibrate was shown to decrease hepatic TNF-α expression in mouse models of NASH [
72‐
74]. Similar to rosiglitazone, fenofibrate effect on TNF-α in human NASH has not been investigated to-date. Saroglitazar, which is a dual PPAR-α/γ agonist, i.e., exerting combined effects on PPAR-α and PPAR-γ, reduced hepatic TNF-α expression and improved histology in experimental NASH models [
73‐
75]. Saroglitazar is under evaluation in NASH patients with fibrosis, following encouraging results in a recent randomized controlled trial (RCT), in which saroglitazar successfully decreased liver function tests (LFTs) and liver fat content in NAFLD patients [
76]. Lanifibranor, a pan-PPAR agonist, ameliorated all histological features of NASH in mice, including fibrosis, and reduced activation of macrophages and TNF-α expression mainly via PPAR-δ agonism [
77]. Lanifibranor is another promising therapeutic candidate as it achieved both primary and secondary endpoints in human NAFLD (resolution of NASH and fibrosis), therefore is currently under investigation in a phase 3 RCT (NCT04849728). Of note, beyond pre-clinical studies, clinical evidence on the effect of dual-PPAR agonist (saroglitazar) and pan-PPAR agonist (lanifibranor) on TNF-α are scarce.
Following favorable effects on non-invasive biomarkers of hepatic steatosis and fibrosis by sodium-glucose co-transporter-2 (SGLT-2) inhibitors in patients with NAFLD [
78], a phase 3 RCT with dapagliflozin is ongoing in patients with biopsy-proven NASH (NCT03723252). SGLT-2 inhibitors act primarily by inducing glucosuria; however, data from T2DM preclinical and clinical studies have also revealed potential anti-inflammatory action and reduction of some circulating cytokines, including TNF-α [
79]. Clinical data on the effect of SGLT-2 on TNF-α in the setting of NAFLD are lacking. Experimentally, remogliflozin was found to reduce hepatic TNF-α mRNA in mice with diet-induced NASH [
80]; nevertheless, additional evidence is required to establish a potentially anti-TNF-α effect of SGLT2 in NAFLD. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) represent another class of anti-diabetic medication showing many favorable metabolic effects that make them an appealing therapeutic option for NASH. Semaglutide and liraglutide achieved higher rates of NASH resolution compared with placebo in phase 2 clinical trials, but without improving fibrosis [
2]. Besides metabolic properties, liraglutide was shown to exert anti-inflammatory action through downregulating TNF-α in a mouse model of NASH [
81], a finding that was further supported in an RCT of patients with concomitant T2DM and NAFLD, where liraglutide, as well as metformin, reduced circulating TNF-α [
82]. However, more studies are needed to establish any anti-TNF-α potential of liraglutide and other GLP-1RAs. On the other hand, sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, an approved anti-diabetic medication, failed to decrease hepatic TNF-α in an experimental NASH rat model [
57]; it is highlighted that DPP-4 inhibitors showed minimal or null effects on NAFLD [
2].
A number of RCTs have evaluated the efficacy and safety of probiotics in the treatment of NAFLD. The rationale lies on the proposed ability of probiotics to modulate the gut microbiome, thus beneficially affecting the gut-liver axis. At present, various strains, preparations, dosage schemes, and durations of treatment have been investigated on different NAFLD-related endpoints, which increases the heterogeneity between studies, thus rendering hard any secure conclusions. Probiotics improved LFTs, hepatic steatosis, plasma glucose and insulin levels, and lipid profile, but they did not affect body mass index (BMI) or circulating TNF-α, according to a meta-analysis of 21 RCTs involving 1037 patients with NAFLD [
83]. In contrast, synbiotic supplementation (i.e., nutritional supplements that combine probiotics and prebiotics), apart from improving LFTs, lipid profile, and glucose metabolism, had favorable effect on circulating TNF-α, in another meta-analysis of 7 RCTs including 419 NAFLD patients [
84].
Rifaximin is a broad-spectrum minimally absorbable antibiotic, which targets dysbiosis of intestinal microbiota and related endotoxemia. A 6-week course of 800 mg rifaximin daily in 15 histologically-proven NASH patients was not associated with robust changes in LFTs, hepatic steatosis, insulin sensitivity, and pro-inflammatory serum cytokines, including TNF-α [
85]. A previous 4-week trial using a higher dose of rifaximin (1200 mg) reduced lipopolysaccharides (LPS) and improved BMI and LFTs in 27 biopsy-proven NASH patients, but serum TNF-α concentrations remained unaffected [
86]. The authors suggested that the higher dose of rifaximin is most likely to reduce endotoxemia, but the relatively short treatment period may probably be insufficient to suppress cytokines, including TNF-α. In accordance with this hypothesis, a longer (6-month) RCT in histologically-confirmed NASH patients showed that daily administration of 1100 mg rifaximin reduced circulating TNF-α along with LFTs, IR, and presumable hepatic steatosis. [
87].
Vitamin E is a potent antioxidant, which may also exhibit anti-TNF-α action, as shown in rats with diet-induced NASH [
88]. Indeed, the anti-inflammatory properties of vitamin E were also evidenced in an RCT of patients with ultrasound-defined NAFLD, where vitamin E, mainly in the form of δ-tocotrienol reduced inflammatory mediators, including TNF-α [
89]. Of note, vitamin E at a daily dose of 800 IU resolves NASH, but not hepatic fibrosis, and may be prescribed
off-label for selected NASH patients with F ≥ 2 for maximum 2 years [
71].
Pentoxifylline is a xanthine derivative, which is a non-selective phosphodiesterase inhibitor, shown to histologically improve NASH in two meta-analyses [
90,
91]. These meta-analyses, however, do not agree on the effect of pentoxifylline on circulating TNF-α; one of them, including 3 placebo-controlled RCTs and 2 ursodeoxycholic acid (UDCA)-controlled prospective cohort studies, reported that pentoxifylline decreases circulating TNF-α [
90], while the latter, limited to RCTs (
n = 5), did not show a significant difference in TNF-α levels between pentoxifylline and placebo [
91]. Thus, more and probably more focused on TNF-α studies are needed to elucidate the potential effect of pentoxifylline on TNF-α.
UDCA, a secondary bile acid, was shown efficacy to reduce pro-inflammatory cytokines in animal studies [
92]. Clinical trials have shown some effects of UDCA on LFTs and possibly hepatic steatosis, but minimal, if any effect on hepatic inflammation and fibrosis [
71]. However, the combination of UDCA with vitamin E improved histology in patients with NASH, by increasing adiponectin levels and reducing hepatocellular apoptosis, but without affecting circulating TNF-α and other mediators of inflammation [
93]. Currently, existing evidence does not support the use of UDCA in patients with NASH.
Fibroblast growth factor-21 (FGF-21) analogs or mimetics act at the same receptors as the endogenous hepatokine FGF-21, which is regarded as a promising molecule against hepatic steatosis, inflammation and apoptosis [
94]. B1344, an FGF-21 analog, reduced the expression of TNF-α and other cytokines in the liver of mice with diet-induced NASH [
95], corroborating earlier evidence showing FGF-21 analogs to be effective in both animal models and humans with NASH, although their direct effect on TNF-α has not been investigated in human NASH to-date [
94]. As a result, numerous clinical trials with FGF-21 analogs are currently underway [
94].
Atorvastatin, a strong and widely used statin for the treatment of dyslipidemia [
96], was shown to improve lipid profile, LFTs, and NAFLD activity score (NAS) in an uncontrolled, interventional trial with 42 biopsy-confirmed NASH patients at a daily dose of 10 mg for 12 months; this effect was partly attributed to its lowering effect on circulating TNF-α [
97]. Similarly, rosuvastatin and simvastatin both decreased hepatic TNF-α mRNA in experimental mice models of NASH, implying their potential anti-inflammatory properties on the liver [
58,
98]. Overall, statin therapy appears to be safe in NAFLD patients and should be used at least to treat dyslipidemia and prevent cardiovascular events in patients with NAFLD, although their effects on hepatic histology are not well documented [
96].
Due to their triglyceride lowering effect, N-3 polyunsaturated fatty acids (n-3 PUFAs) were considered to be beneficial for NAFLD [
99]. Supplementation with eicosapentaenoic acid (EPA), one of the major components of n-3 PUFAs, or a mixture of EPA with docosahexaenoic (DHA) and alpha lipoic acid (ALA) plus caloric restriction, significantly reduced circulating TNF-α receptor or TNF-α, respectively, in NAFLD patients [
100,
101]. However, RCTs with histological endpoints did not provide favorable results in NAFLD [
102] and, therefore, n-3 PUFAs are not currently recommended for the treatment of NASH [
71]. Different compositions and formulas used in research and in clinical practice further complicate secure conclusions on their effects on NAFLD.
Angiotensin receptor blockers (ARBs) have been proposed as alternative therapeutic option for NAFLD, mainly owing to their potentially anti-inflammatory and anti-fibrotic effects in the liver [
103]. Both valsartan [
104] and olmesartan [
105] were reported to reduce hepatic TNF-α in experimental studies, and telmisartan was more effective than valsartan in reducing hepatic expression of TNF-α in rats with diet-induced NASH in a comparative study [
106]. In contrast, losartan had no effect on the hepatic TNF-α mRNA in another experimental NASH rat model [
56]. These variations may be attributed to structural differences between ARBs, as well as to telmisartan properties beyond angiotensin receptor, e.g., the activation of PPAR-γ [
103]. The experimentally observed anti-TNF action of some of ARBs has not been demonstrated to-date in human NASH. However, an open-label prospective study with short-term administration of telmisartan to hypertensive patients with metabolic syndrome (MetS) showed an increase in circulating adiponectin and improvement in IR but no effect on serum TNF-α levels [
107]; however, the results of this study could not directly be extrapolated to NAFLD patients.
Anti-TNF-α Therapies: a Promising Therapeutic Approach for NAFLD Treatment
Given the potential steatotic, inflammatory, and fibrogenic effects of TNF-α in the liver, targeting TNF-α may be promising for the treatment of advanced NAFLD. TNF inhibitors are a class of biologic agents, which include infliximab, adalimumab, etanercept, golimumab, and certolizumab pegol, and have been widely used to treat chronic immune-related diseases, such as rheumatoid arthritis (RA), psoriatic arthritis (PsA), and inflammatory bowel diseases (IBD) [
108]. Importantly, the extensive use of these agents in clinical practice over the last few decades has resulted in considerable experience on their efficacy and safety.
Experimental evidence has shown favorable effects of anti-TNF approaches on NAFLD outcomes; administration of infliximab in rats with diet-induced NASH resulted in histological regression of hepatic inflammation and fibrosis [
109]
. Hepatic steatosis, although improved, seems to be less affected by anti-TNF agents than other histological features of NAFLD [
110].
In the clinical setting, initial reports on the metabolic and hepatic effects of anti-TNF biologics derive mostly from observational studies in patients with RA and PsA. PsA patients receiving a 24-month course of etanercept and adalimumab showed an improvement in metabolic syndrome components, like waist circumference, triglycerides, high-density lipoprotein-cholesterol (HDL-C), and glucose, when compared with those treated with methotrexate [
111]. In line, other studies reported that anti-TNF reduced IR [
112] and cardiovascular risk [
113], both closely associated with NAFLD. Of note, administration of adalimumab to a young woman with rheumatoid arthritis and coexisting biopsy-proven NASH resulted in a remarkable biochemical response in terms of long-lasting improvements in LFTs [
114]. Importantly, liver stiffness, which is associated with hepatic fibrosis, was lower in PsA patients treated with anti-TNF agents compared with those not on anti-TNF treatment, suggesting a possible antifibrotic effect [
115]. On the contrary, some studies have raised the possibility that anti-TNF may increase body weight, which is a major risk factor for the development and progression of NAFLD [
116]. There are also studies showing adverse hepatic effect after treatment with anti-TNF biologics. Administration of anti-TNF agents for 12 months in patients with PsA and US-defined NAFLD resulted in higher rates of worsening of hepatic steatosis compared with controls (with NAFLD but without PsA); the worsening of hepatic steatosis was greater in PsA patients with more active disease [
117].
Concerning IBD populations, most studies showed higher prevalence of NAFLD in IBD patients than the general population [
118]. Most studies do not reveal an association between biologic therapy and NAFLD incidence or severity [
119]; however, existing studies are observational and were not designed towards this aim, i.e., the hepatic effect of biologics on NAFLD in IBD patients. It is highlighted that any possible benefit of anti-TNF inhibitors on NAFLD may be negated by a “rebound” weight gain observed following anti-TNF treatment, particularly in CD patients [
108]. Anti-TNF medications may, in fact, ameliorate intestinal inflammation and achieve disease remission, which may lead to increased BMI and visceral adiposity due to restored nutrient absorption and increased appetite [
108].
Clinical trials assessing anti-TNF medications specifically in NASH patients are not yet available. Before initiating such studies, observational studies with patients on anti-TNF agents for other conditions, who also have concomitant NASH, would be an excellent starting point [
108]. Also, post hoc evaluations of existing clinical trials in patients with other diseases and concomitant NASH at baseline receiving anti-TNF agents may provide indirect insights into the effectiveness of anti-TNF agents on NASH. Positive outcomes in such observational studies may possibly support clinical trials with anti-TNF agents specifically in NASH, ideally with histological endpoints or non-invasive biomarkers of hepatic steatosis and fibrosis as acceptable alternatives [
108]. Undoubtedly, we anticipate future studies designed to evaluate the role of anti-TNF-α agents in the treatment of NASH
.