Non-alcoholic fatty liver disease (NAFLD) is now considered to be one of the most widespread chronic liver diseases in the world and has a histologic spectrum between simple steatosis, non-alcoholic steatohepatitis (NASH), progressive fibrosis, cirrhosis and hepatocellular carcinoma. The idea of transition between uninspiring steatosis to inflammatory liver damage was traditionally outlined by Day and James, who suggested that steatosis predisposes the liver to secondary inflammatory and oxidative damage [1]. The later histopathological reports by Brunt et al. and Kleiner et al. developed grading and staging of NASH and NAFLD, which further supported the pivotal role of liver biopsy in assessing the severity of steatosis, ballooning, lobular inflammation and fibrosis [2,3].

The growing body of evidence indicates that inflammation is not only an epiphenomenal event in NAFLD, but a key contributor to hepatocellular injury, disease course, and fibrogenesis. Tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) have been of particular interest as some of the cytokines involved in this process due to their close association with insulin resistance, adipose tissue inflammation, Kupffer cell activation and hepatocyte injury. One of the first studies of human subjects showed that Crespo et al. found a greater mRNA expression of TNF-alpha in the liver of patients with NASH, indicating a direct relationship between intrahepatic inflammatory activation and severity of the disease [4]. Park et al. also demonstrated in a clinicopathological study of younger Asian patients with NAFLD that tissue-level inflammatory signaling is relevant to fatty liver disease, and that tissue-level inflammatory findings correlated with pathological severity [5].

Studies that correlate levels of cytokines with histological injury have also supported the relevance of TNF-a. Manco et al. found that the level of histologic liver injury in pediatric NAFLD was linked with serum TNF-a levels, which indicated that the activation of the pro-inflammatory cytokines reflects more aggressive disease biology [6]. In a similar study, Wieckowska et al. observed higher levels of hepatic and circulating IL-6 in NASH patients compared to simple steatosis, suggesting that IL-6 might somehow be involved in the crossroads between steatosis developed by metabolic factors and active steatohepatitis [7]. This idea was reinforced by Bertola et al. who showed that a variety of inflammatory and immune-response gene increases in liver tissue of patients with NASH, many of the most significant of which concerned pathways of cytokine-mediated damage [8].

These findings are a solid argument to analyze the expression of inflammatory markers in liver biopsy tissue. Even though the biochemical and radiological techniques have a role to play in the diagnosis of fatty liver, they fail to provide consistent information on the nature of the inflammatory microenvironment in the liver and its association with the histological grade and stage. The role of IL-6 and TNF- 2 as inflammatory mediators of NAFLD, and the potential contribution of immunohistochemical analysis of these cytokines in liver biopsies to the clinicopathological implications of cytokine expression in the disease progression could therefore be inferred. Here, the current research was conducted to assess the immunohistochemical expression of IL-6 and TNF-a in liver biopsies of NAFLD patients and to correlate the results with clinical, biochemical and histopathological variables.

AIM

To study the immunohistochemical expression of IL-6 and TNF-α in liver biopsies of NAFLD patients and its correlation with clinicopathological features.

OBJECTIVES

1. To evaluate the histopathological changes in liver biopsy specimens of NAFLD patients.
2. To assess the immunohistochemical expression of IL-6 and TNF-α in these biopsy specimens.
3. To correlate marker expression with histopathological grade and stage of NAFLD.
4. To correlate marker expression with relevant clinical and biochemical parameters.

Study design and setting This hospital-based prospective clinicopathological study was conducted in the Department of Pathology in collaboration with the Department of Medicine and Pathology at Malati multispeciality hospital and medical college, Khurd, Akola, Maharashtra, over a period of 10 Month from January 2025 to Oct 2025. The study was designed to evaluate the immunohistochemical expression of inflammatory markers IL-6 and TNF-α in liver biopsy specimens of patients with non-alcoholic fatty liver disease (NAFLD) and to correlate their expression with clinicobiochemical and histopathological parameters. Study population The study included 60 consecutive adult patients with biopsy-proven NAFLD who fulfilled the eligibility criteria during the study period. Patients were enrolled consecutively after clinical, biochemical, radiological, and histopathological evaluation. Inclusion criteria Patients were included if they fulfilled all of the following criteria: 1. Diagnosed clinically, biochemically, radiologically, and/or histologically with NAFLD. 2. Age 18 years or above. 3. Undergoing liver biopsy for evaluation of NAFLD during the study period. 4. Willing to participate and providing written informed consent. Exclusion criteria Patients were excluded if they had any of the following: 1. Significant alcohol intake. 2. Viral hepatitis, including hepatitis B or hepatitis C infection. 3. Other chronic liver diseases such as autoimmune hepatitis, Wilson’s disease, hemochromatosis, or drug-induced liver injury. 4. Primary liver malignancy or metastatic liver disease. 5. Inadequate or poorly preserved liver biopsy samples. 6. Refusal to provide written informed consent. Clinical, anthropometric, and biochemical assessment All patients underwent detailed clinical evaluation including history taking, physical examination, and assessment of associated metabolic risk factors. Demographic variables including age and sex were recorded. Clinical comorbidities including obesity, diabetes mellitus, hypertension, and dyslipidemia were documented. Relevant symptoms and examination findings, including fatigue, right upper quadrant discomfort, and hepatomegaly, were also noted. Anthropometric assessment included body mass index (BMI) and waist circumference. Biochemical investigations included serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), glycated haemoglobin (HbA1c), serum triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL). Viral markers were performed to exclude infective liver disease. Ultrasonography was performed in all patients, and ultrasound fatty liver grade was recorded for clinicopathological correlation. Liver biopsy and histopathological evaluation Liver biopsy specimens obtained from eligible patients were fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin, and stained with hematoxylin and eosin. Special stains were used wherever required for better assessment of fibrosis and associated histological changes. Histopathological examination was performed to evaluate the principal morphologic features of NAFLD, including steatosis grade, lobular inflammation, hepatocyte ballooning, and fibrosis stage. The NAFLD Activity Score (NAS) was assessed from the biopsy findings using the combined evaluation of steatosis, lobular inflammation, and hepatocyte ballooning. Based on the overall histomorphological features, cases were categorized into three histologic diagnostic groups: 1. Simple steatosis 2. Borderline steatohepatitis 3. Non-alcoholic steatohepatitis (NASH) The histopathological findings were subsequently correlated with immunohistochemical marker expression and clinicobiochemical parameters. Immunohistochemistry Immunohistochemical staining for interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) was performed on formalin-fixed, paraffin-embedded liver biopsy sections using standard immunohistochemical techniques. Appropriate positive and negative controls were included with each staining run. Cytoplasmic staining in hepatocytes and inflammatory cells was considered positive. The expression of IL-6 and TNF-α was assessed semiquantitatively using two parameters: staining intensity and extent of positive staining. Staining intensity was scored as follows: 0 = no staining 1 = weak staining 2 = moderate staining 3 = strong staining The extent of staining was scored according to the percentage of positively stained cells as follows: 0 = 0% positive cells 1 = 1-10% positive cells 2 = 11-33% positive cells 3 = 34-66% positive cells 4 = 67-100% positive cells A composite immunohistochemical score was obtained by adding the intensity score and extent score, yielding a total score ranging from 0 to 7 for each marker. Based on the final composite score, marker expression was categorized as: • Low expression: score 0-2 • Moderate expression: score 3-4 • High expression: score 5-7 Both the categorical expression pattern and the composite score were used for statistical analysis and correlation with histologic diagnosis, NAS score, fibrosis stage, biochemical parameters, anthropometric indices, and ultrasound fatty liver grade. Data collection Data were collected using a predesigned proforma. The following variables were documented: 1. Demographic details 2. Clinical history and metabolic risk factors 3. Anthropometric measurements 4. Biochemical parameters 5. Ultrasound fatty liver grade 6. Histopathological findings on liver biopsy 7. Histologic diagnosis category 8. NAFLD Activity Score 9. Immunohistochemical expression category and composite score for IL-6 and TNF-α Outcome measures The primary outcome measure was the pattern of immunohistochemical expression of IL-6 and TNF-α in liver biopsy specimens of patients with NAFLD. The secondary outcome measures were: 1. Histopathological spectrum of NAFLD in the study population 2. Association of IL-6 and TNF-α expression with histologic diagnosis 3. Correlation of IL-6 and TNF-α composite scores with NAFLD Activity Score and fibrosis stage 4. Correlation of marker expression with selected clinical, biochemical, anthropometric, and radiological parameters Statistical analysis Data were entered into Microsoft Excel and analyzed using appropriate statistical software. Continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed as number and percentage. Comparisons of continuous variables across the three histologic diagnosis groups were performed using the Kruskal-Wallis test. Categorical variables were compared using the chi-square test. Correlation between IL-6 and TNF-α composite scores and selected clinicopathological parameters was assessed using Spearman correlation analysis. A p-value of less than 0.05 was considered statistically significant. Ethical considerations The study was conducted after obtaining approval from the Institutional Ethics Committee. Written informed consent was obtained from all participants prior to inclusion in the study and before liver biopsy.

1. Study population and baseline clinicobiochemical profile

A total of 60 patients with biopsy-proven NAFLD were included in the analysis. The mean age was 44.9 ± 12.6 years, and 37 (61.7%) were men. Obesity was present in 45 (75.0%), diabetes mellitus in 20 (33.3%), and hypertension in 17 (28.3%). Across histologic categories, serum ALT and HbA1c differed significantly, with the highest values observed in patients with NASH (Table 1).

Table 1. Baseline clinical and biochemical characteristics overall and according to histologic diagnosis.

2. Histopathological spectrum of NAFLD

The most frequent histologic diagnosis was NASH in 36 patients (60.0%), followed by borderline steatohepatitis in 19 (31.7%) and simple steatosis in 5 (8.3%). Advanced fibrosis (stage 3–4) was present in 38 patients (63.3%). Grade 3 steatosis and grade 2 ballooning were the most common morphologic patterns (Table 2).

Table 2. Histopathological spectrum and morphologic features in liver biopsies of NAFLD.

3. Immunohistochemical expression of IL-6 and TNF-α

High IL-6 expression was observed in 35 patients (58.3%) and high TNF-α expression in 39 patients (65.0%). Both markers showed a clear shift toward higher expression categories in NASH compared with borderline steatohepatitis and simple steatosis. Composite IL-6 and TNF-α scores also increased significantly across worsening histologic diagnosis (both p < 0.001; Table 3).

Table 3. Semiquantitative immunohistochemical expression of IL-6 and TNF-α overall and according to histologic diagnosis.

4. Correlation of inflammatory marker expression with clinicopathological parameters

IL-6 and TNF-α composite scores showed very strong positive correlations with NAFLD activity score and fibrosis stage. For IL-6, the correlation with NAS was r = 0.938 and with fibrosis stage r = 0.858; for TNF-α, the corresponding correlations were r = 0.934 and r = 0.856 (all p < 0.001). Both markers also correlated positively with ALT levels and ultrasound fatty liver grade. Weaker but statistically significant correlations were observed with HbA1c, and for TNF-α with BMI as well (Table 4).

Table 4. Correlation of IL-6 and TNF-α composite scores with selected clinicopathological parameters (Spearman correlation).

The above Scatter plot shows the positive correlation between IL-6 composite IHC score and NAFLD activity score (Spearman r = 0.938, p < 0.001).

The above  Scatter plot shows the positive correlation between TNF-α composite IHC score and fibrosis stage (Spearman r = 0.856, p < 0.001).

 Continuous variables are presented as mean ± standard deviation and categorical variables as number (%). P values for Table 1 were derived using the Kruskal–Wallis test for continuous variables and the chi-square test for categorical variables.

The current paper confirms the opinion that inflammatory cytokine expression in NAFLD biopsy-selected is a better predictor of histologic severity than standard clinical indices. The high proportion of NASH in our cohort (60.0%), as well as the prevalence of advanced fibrosis (63.3%), is probably due to the enriched disease severity of biopsy-based series as compared to the general NAFLD population. The interpretation is in line with larger adult cohorts where biomarker profiles were compared with centrally read liver histology where higher centres of inflammatory and fibrotic phenotypes were overrepresented among biopsied patients and the most strongly correlated with inflammatory markers [14].

An important observation of our research was the gradual increase in the expression of IL-6 and TNF-alpha between simple steatosis and borderline steatohepatitis and NASH with mean composite scores falling within the low range in simple steatosis and with noteworthy high scores in NASH. This is consistent with previous human research that demonstrates that pro-inflammatory cytokine signaling increases with the progression of NAFLD to steatohepatitis. Abiru et al. also showed that circulating levels of TNF-alpha/sTNFR1 and IL-6/sIL-6R were significantly greater in NASH as compared to simple steatosis [9], and Kumar et al. also associated pro-inflammatory cytokines with the severity of disease and insulin resistance in NAFLD [11]. Combined with our tissue-based data, these studies lead to the conclusion that IL-6 and TNF-alpha are not simply epiphenomena of steatosis, but rather more likely associated with active hepatocellular injury and inflammatory remodeling.

There is a close correlation between the expression of cytokines and the histologic activity in our cohort which is of particular interest. Both indicators were very strongly correlated with NAS and fibrosis stage indicating that inflammatory signaling is still tied to necroinflammatory change as well as to the progression to structural liver damage. This is in agreement with Jamali et al., who discovered that biopsy-based liver histology was correlated with inflammatory/adipokine profiles [12], and with Ajmera et al., who detected inflammatory plasma biomarkers linked to the extent of steatosis, ballooning, lobular inflammation, and fibrosis in adults with NAFLD [14]. Goyale et al. also found that serum panels that included IL-6, TNF-a, and other similar mediators had significant correlations with the severity of fibrosis, further supporting the notion that cytokine-related mechanisms are more in line with disease activity and progression than with the mere presence of fat.

The other clinically important feature of our findings is that, cytokine expression correlated with ALT, ultrasound fatty liver grade, NAS and fibrosis as opposed to AST and triglycerides, and only weakly with HbA1c and BMI. This is a biologically realistic gradient. In most NAFLD cohorts, ALT is a better surrogate of continuing hepatocellular injury than AST, and BMI and glycemia are better indicators of systemic metabolic load that is not necessarily in tandem with the inflammatory phase of liver disease. Other related dissociations have been reported in other literature: Haukeland et al. pointed out that NAFLD is marked by systemic inflammation beyond standard liver tests alone [10], and Paredes-Turrubiarte et al. demonstrated that more severe NAFLD was linked to an increase in TNF- alpha levels even in the context of morbidly obese patients [13]. Our results thus lead to the conclusion that IL-6 and TNF-a can be more of an indicator of hepatic inflammatory process rather than merely an obesity or dyslipidemia correlate.

Pragmatically, these data indicate that the routine histology of IL-6 and TNF-a can be complemented with immunohistochemical evaluation to serve as an interpretative tool in NAFLD. Cytokine staining seems to map onto the biological shift between metabolically-driven steatosis and inflammatory and fibrogenic disease in a cohort in which simple steatosis had uniformly low expression but NASH was characterised by high-expression patterns. The above comparator studies support this interpretation by demonstrating a consistent pattern of TNF- and IL-6-related pathways to become more pronounced with increasing histology and fibrosis [9,11,14,15]. In this regard, the primary finding of our study is not their high levels only, but their time- and place-resolved tissue expression which reflects the disease activity in a manner that augments clinicopathological stratification in the biopsy-proven NAFLD.

Limitation

This was a single-center study with a relatively small sample size, which may limit the generalizability of the findings. In addition, the semiquantitative immunohistochemical assessment may be subject to observer-related variation.

The present study demonstrates that immunohistochemical expression of IL-6 and TNF-α increases with histological severity of NAFLD and is highest in patients with NASH. Their strong association with NAFLD activity score and fibrosis stage suggests that these inflammatory markers reflect ongoing hepatic injury and disease progression. Immunohistochemical assessment of IL-6 and TNF-α may therefore serve as a useful adjunct to routine histopathology in evaluating disease severity in NAFLD.

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