Twelve-week-old male leptin-deficient mice (Ob/Ob) and their littermates (n = 10 in each group) were obtained from Envigo (Harlan). To model MASH, two experimental mouse models were utilized. In the first model, Ob/Ob mice were fed a high-fat diet (HFD) for 4 weeks to induce hepatic and metabolic injury (Ob/Ob ^HFD). The diet used (Cat. # TD.06414) consisted of 60.3% kcal from fat, 21.4% from carbohydrates, and 18.3% from protein, replicating a lipotoxic nutritional environment. In the second model, immunodeficient NOD-SCID IL2rγ^null^ (NSG) mice, which lack functional T, B, and NK cells, were fed the same HFD for 16 weeks starting at 10 weeks of age. In week 12, these recipient NSG mice were i.v. injected with 10 × 10⁶ pancreatic tissue-resident (tr)NK isolated from Ob/Ob ^HFD, which were pre-treated with IL-6R antagonizing antibody (10 μg/10⁶ cells) or IgG-isotype control. The cell injection was administered twice a week for the remaining 4 weeks (n = 6 in each group). Ob/Ob ^HFD without adoptive transfer (untreated with trNK cells were used as a control. Mice received care in accordance with the ethical regulations of An-Najah National University (ANNU) and the NIH guidelines (Ref: Mas. May 2022/15). To minimize animal suffering, mice were monitored daily for signs of distress, weight loss, reduced activity, or abnormal grooming. Humane endpoint criteria were pre-defined, including a weight loss of more than 20%, significant behavioral changes, or signs of pain unresponsive to supportive care. All surgical procedures and sample collections were performed under anesthesia using 5% isoflurane inhalation followed by cervical dislocation to ensure rapid and humane euthanasia. Efforts were made to reduce the number of animals used by applying the principles of the 3Rs (Replacement, Reduction, and Refinement). Group sizes were based on minimal requirements to obtain statistically meaningful results while minimizing unnecessary animal use.

On the sacrifice day, whole blood was withdrawn through cardiac puncture and collected in heparinized tubes for isolating serum (blood was centrifuged at 3,500 rpm for 30 min at 4°C) and assessed by ELISA for liver injury enzymes and biochemical assessment as described below. The following laboratory measurements were obtained: serum ALT (Abcam; ab285263), AST (Abcam; ab263882), fasting blood sugar (Biocompare; MBS7200879), insulin (Abcam; ab277390), cholesterol (Abcam; ab285242), and triglycerides (Biocompare; MBS726589). In accordance with standard laboratory protocol, it was ensured that all reagents and samples were equilibrated to room temperature (18°C–25°C) before their use. A 100 µL volume of each standard and sample was added to the respective wells and incubated for 2.5 h at room temperature with moderate shaking. After incubation, the solution was discarded, and wells were washed four times with 1X Wash Solution. It is worth noting that the washing process involved filling each well with Wash Buffer (300 µL), using a multi-channel pipette or auto washer. Ensuring the complete liquid removal at each step is crucial for optimal performance. A 100 µL of 1x prepared detection antibody was added to each well for 1 h at room temperature with gentle shaking. Following this, 100 µL of the prepared streptavidin solution was added to each well and incubated for 45 min at room temperature with gentle shaking. A volume of 100 µL of TMB One-Step Substrate Reagent (Item H) was added to each well for 30 min at room temperature in the dark with gentle shaking. Finally, 50 µL of Stop Solution (Item I) was added to each well. Absorbance was immediately read at 450 nm using an ELISA reader (Tecan M100 Plate Reader).

Multiplexed sandwich enzyme-linked immunosorbent assay-based technology (Cat# MHSTCMAG-70K; R&D Systems) was used to simultaneously determine the concentrations of multiple cytokines (IL-6, IL-1β, and IL-8). Samples from each mouse were analyzed as described in the manufacturer’s instructions (Mateu-Borrás et al., 2025). Briefly, samples and standards were diluted and incubated overnight at 4°C with a premixed cocktail of fluorescent-coded magnetic beads, each conjugated to a specific capture antibody. The following day, plates were washed three times using a handheld magnetic separator and incubated sequentially with a biotinylated detection antibody cocktail for 1 h at room temperature, followed by streptavidin–phycoerythrin (PE) for 30 min. After a final series of washes, the beads were resuspended in assay buffer and analyzed using a Luminex MAGPIX system. Cytokine concentrations were calculated based on standard curves generated from known concentrations using xPONENT^® software. All samples were run in duplicate to ensure reproducibility, and intra-assay variation was monitored using quality control beads provided with the kit.

The liver and pancreas tissues were subjected to RNA isolation using 2 mL of TRI Reagent (Bio-Lab; Cat# 90102331) per cm³ of tissue. The samples were homogenized for 5 min at room temperature before adding 0.2 mL of chloroform (Bio-Lab; Cat# 03080521). Following a 15-min incubation period at an ambient temperature, the samples were centrifuged at 1,400 revolutions per minute for 15 min at 4°C. The supernatant from each sample was carefully transferred to a fresh micro-centrifuge tube during RNA precipitation. After adding 0.5 mL of isopropanol (Bio-Lab; Cat# 16260521), the mixture was incubated at 25°C for 10 min. The tubes were then centrifuged (12,000 rpm) for 10 min at 4°C, the supernatants were removed, and 1 mL of 75% ethanol was added to the pellet. Following centrifugation at 7,500 revolutions per minute (rpm) for 5 min, the resulting pellets were subjected to air drying at ambient temperature for 15 min. After adding 50 μL of DEPC, the samples were thermally treated at 55°C for 10 min. The R&D High-Capacity cDNA Isolation Kit (1406197) was used to produce cDNA. The αSMA and collagen I, Fibronectin, Reg1, and Reg3 gene expressions were quantified using real-time PCR with the TaqMan Fast Advanced Master Mix (Applied Biosystems; Cat# 4371130). The primers and probes used for RT-PCR targeting α-SMA, Collagen I, Fibronectin, Reg1, and Reg3 were commercially purchased as pre-validated TaqMan^® Gene Expression Assays from Rhenium (Israel), an authorized distributor of Thermo Fisher Scientific. These assays are proprietary and patented; therefore, the exact primer and probe sequences are not disclosed by the manufacturer. However, the use of TaqMan^® chemistry ensures high specificity and efficiency, as the assays are designed and optimized based on exon-exon junctions and genomic context using Thermo Fisher’s validated bioinformatics pipeline. Gene expression was normalized to that of the housekeeping gene GAPDH. In the first experimental model involving Ob/ObHFD mice, gene expression data were normalized and expressed as fold changes relative to the littermate control group, which was assigned a baseline value of 1. However, in the second adoptive transfer model, NSG mice fed an HFD without pancreatic trNK cell administration were used as the control group. The cycling conditions for the one-step RT-PCR involved 40 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The data were analyzed using a QuantStudio™ 5 Real-Time PCR System (Cat# A34322, Applied Biosystems).

Pancreatic β−islet cells isolation was performed using a standard collagenase digestion method followed by density gradient purification. Briefly, the pancreas was perfused via the common bile duct with a collagenase solution (typically Collagenase P or Liberase TL), excised, and incubated at 37°C for 15–20 min to enzymatically dissociate the tissue. The digested tissue was then washed and subjected to a Ficoll-based or Histopaque density gradient to separate intact β-islet cells from exocrine debris. Purified islets were hand-picked under a stereomicroscope and subsequently cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO₂ until further use.

The homogenized pancreas tissue was centrifuged at 9,000 g for 15 min. The separated supernatant was used to assess oxidative stress through the measurement of Malondialdehyde (MDA), which was determined using an ELISA kit according to the manufacturer’s instructions.

Under deep ether anesthesia, mice were euthanized using isoflurane, USP 100% (INH). The pancreases were then removed, and a portion of them was transferred to a Petri dish containing 5 mL of DMEM medium (Biological Industries; Cat# 01-055-1A). The pancreas tissues were thoroughly dissected using a stainless-steel mesh. The cells were then harvested in the medium and added to 50 mL tubes containing 10 mL DMEM. The cells were subsequently transferred to new tubes containing Ficoll (Abcam; Cat# AB18115269). Tubes were centrifuged for 20 min at 1,600 rpm at 20°C. The supernatant in each tube was transferred to a new tube for a second centrifugation, this time for 10 min at 1,600 rpm and 4°C. After the second centrifugation, the pellet in each tube was resuspended in 1 mL of DMEM for the NK isolation kit (Stem Cells; Cat# 19665). As mentioned below, pancreatic trNK cells were phenotyped as CD45⁺CD3⁻NK1.1⁺CD49a⁺DX5^- by flow cytometry. Before flow cytometry analysis, cells (10 (Tanaka et al., 2014)/100 μμΛ) were assessed for their viability, measured by propidium iodide (PI) (A35110, R&D Systems). PI-negative cells were considered viable, with a mean viability rate of 92.7% ± 1.5%.

All antibodies were incubated with the isolated trNK cells suspensions (1:100) at 4°C for 45 min. The cells were washed twice with PBS supplemented with 1% fetal calf serum (FCS), and a secondary antibody (1:100) was added for 45 min at 4°C. The primary mouse antibodies used were anti-CD3 (PE-CY7, BD Bioscience Cat# 560591), anti-NK1.1 (Percpcy-5; Abcam Cat# ab210337), anti-CD49a (PE; BD Bioscience Cat# 559596), anti-DX5 (APC; BD Bioscience Cat# 560591), anti-CD4 (FITC; Cat# ab269349, abcam), anti-CD8 (Percpcy-5; Cat# ab217334, abcam), anti-CD107a (lysosomal-associated membrane protein-1 (LAMP-1) (Pacific blue; BD Bioscience Cat# 564348), anti-NKp46 (FITC; BD Bioscience Cat# 560756), anti-IFN-γ (ab40465, abcam) and IL-6R (ab40465, abcam). The secondary antibodies used were goat anti-mouse IgG (AF488) (ab150113, Abcam) and goat anti-rabbit IgG (APC) (Thermo Fisher, Cat# A10931). Isotype IgG labeled with the relevant fluorochrome was used as a control for each antibody. Apoptosis was evaluated with annexin-V (A35110, R&D Systems) staining. Early apoptotic cells were defined as annexin-V⁺PI⁻ cells, and late apoptotic cells were defined as annexin-V + PI + cells. All stained cells were examined using a flow cytometer (BD LSR Fortessa, Becton Dickinson, Immunofluorometry Systems) and analyzed with FCS Express 7 by De Novo Software for flow cytometry.

β-Islet cells were harvested and fixed in 70% ethanol at −20°C for at least 2 h to ensure effective permeabilization. Following fixation, cells were washed with PBS and incubated with 0.1% Triton X-100 in PBS for 10 min at room temperature to permeabilize the nuclear membrane. After an additional wash, cells were incubated with an anti-Ki-67 monoclonal antibody (abcam; ab281847) directly conjugated to fluorescein isothiocyanate (FITC) at a 1:100 dilution in PBS with 1% BSA for 30–60 min in the dark at room temperature. Subsequently, samples were washed thoroughly and analyzed using flow cytometry, as mentioned above. A parallel sample stained with isotype-matched FITC-conjugated IgG served as a negative control.

Protein was extracted from pancreatic trNK cells using lysis buffer (50 mmol/L Tris-HCl (pH 7.6), 0.25% Triton X-100, 0.15 M NaCl, and 10 mM CaCl2) and complete Mini EDTA-Free Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany). Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States). The obtained proteins were then mixed with 5× loading buffer (protein: 5× loading buffer = 4:1) and boiled at 95°C for 10 min. Next, 20 μg of protein was separated per lane on a 10% (w/v) sodium dodecyl sulfate (SDS)-polyacrylamide gel (Bio-Rad) under reducing conditions. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane for immunoblotting. The membranes were then incubated for 1 h at room temperature in neutralizing buffer containing 5% skim milk (w/v) before being incubated with rabbit anti-human/mouse p-STAT3 (S727) (AF4934, R&D Systems) and anti-human/mouse STAT-3 (MAB1799, R&D Systems). All primary antibodies were diluted 1:1,000 and incubated with the membrane overnight at 4°C. The membranes were incubated with peroxidase-conjugated anti-mouse and anti-rabbit IgG (Abcam, 1:5000 dilution) for 1.5 h at room temperature. Immunoreactivity was detected using an enhanced chemiluminescence kit (Abcam). Imaging and data analysis were performed using a FUSION Solo S system.

The posterior third of the mice’s liver was dissected and fixed with 4% formalin for 24 h at room temperature, embedded in paraffin, and sliced (7 μm) in an automated tissue processor (microtome). Sections were deparaffinized by immersion in xylene, as mentioned in Amer et al. (Amer et al., 2022). The sections were then rehydrated using a graded series of ethanol concentrations, starting with absolute ethanol and ending with distilled water. Hematoxylin and eosin (H&E) staining was used to evaluate the level of steatosis, necro-inflammatory regions, and apoptotic bodies. A veterinary pathologist assessed all histopathological findings and reported assessment grades. Stained slides were scanned using a Zeiss microscope.

Statistical differences were analyzed using a two-tailed unpaired Student’s t-test for comparisons between two groups, or two-way analysis of variance (ANOVA) for comparisons among multiple groups, performed with GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA). When ANOVA indicated significant differences, Tukey’s post hoc multiple comparisons test was applied to assess pairwise group differences. A p-value ≤0.05 was considered statistically significant. Data are presented as averages ±standard deviation (SD).

In the current study, we adapted the Ob/Ob mice model to mimic MAFLD. Additionally, we introduced HFD [Ob/Ob ^HFD ] for 4 weeks to exacerbate systemic fat accumulation in the liver with a possible potential to cause liver fibrosis. To investigate the metabolic involvement in Ob/Ob ^HFD mice, we examined hyperglycemia, hyperlipidemia, and insulin resistance. Serum cholesterol and triglyceride levels were significantly elevated in the Ob/Ob ^HFD mice compared to their naive littermates. Serum cholesterol levels in naive mice were measured at 180 ± 20 mg/dL, while those in the Ob/Ob ^HFD mice were 800 ± 24 mg/dL, representing a four-fold increase (P = 0.001; Figure 1A). In addition, Ob/Ob ^HFD mice exhibited elevated serum triglyceride levels of 600 ± 31 mg/dL, compared to 120 ± 15 mg/dL in their littermate counterparts (P = 0.001), indicating a six-fold increase (Figure 1B). Furthermore, fasting blood sugar (FBS) levels in naive littermates were 100 ± 8 mg/dL, whereas in the Ob/Ob ^HFD mice, they increased to 350 ± 55 mg/dL, reflecting a 3.5-fold increase (Figure 1C). Insulin levels in naive mice were measured at 0.6 ± 0.02 ng/mL, while Ob/Ob ^HFD mice showed a significant 50% reduction, with insulin levels dropping to 0.3 ± 0.1 ng/mL (Figure 1D). This decline in insulin suggests impaired insulin production or function in the Ob/Ob ^HFD mice, further reinforcing their utility as a model for studying diabetes. In summary, this experiment demonstrated that Ob/Ob ^HFD mice developed significant metabolic dysfunction, mirroring the obesity and related metabolic disorders observed in humans. The severe increases in cholesterol, triglycerides, fasting blood glucose, and the reduction in insulin levels support the conclusion that this mouse model effectively represents key aspects of obesity, metabolic syndrome, and Type 2 diabetes.

Lab assessments of liver injury of serum levels of ALT levels were significantly elevated in Ob/Ob ^HFD mice compared to littermate controls (Ob/Ob ^HFD : 175 ± 35 IU/L vs. Littermate: 50 ± 12 IU/L, p < 0.001), indicating increased hepatocellular injury (Figure 1E). Similarly, AST levels were markedly increased in Ob/Ob ^HFD mice relative to littermate controls (Ob/Ob ^HFD : 200 ± 83 IU/L vs. Littermate: 90 ± 14 IU/L, p < 0.01), further supporting liver damage in the MASH model (Figure 1F). These findings confirm that the Ob/Ob ^HFD mouse model effectively recapitulates key features of MASH, including severe liver steatosis and elevated liver injury enzymes (ALT and AST). Moreover, Quantitative real-time PCR analysis revealed a significant upregulation of key fibrotic genes in the livers of Ob/Ob ^HFD mice compared to their littermate controls. The expression of α-smooth muscle actin (α-SMA) was markedly increased, demonstrating a threefold elevation in Ob/Ob ^HFD (3.0 vs. 1.0; Figure 1G). Similarly, collagen type I alpha 1 (Col1a1) expression was elevated to 2.3-fold relative to control mice (2.3 vs. 1.0, Figure 1H), indicating enhanced extracellular matrix deposition. These transcriptional changes reflect the onset of fibrotic remodeling and support the utility of the Ob/Ob ^HFD model in mimicking liver fibrosis associated with metabolic dysfunction. Histological evaluation using hematoxylin and eosin (H&E) staining revealed notable hepatic architectural disruption in Ob/Ob ^HFD mice compared to their littermate controls. The littermate panel represents a normal liver architecture, characterized by well-organized hepatic cords radiating from the central vein, with polygonal hepatocytes exhibiting round, centrally located nuclei and eosinophilic cytoplasm. There is no evidence of steatosis, inflammation, or fibrosis. In contrast, the Ob/Ob ^HFD panel exhibits marked macrovesicular steatosis, indicating significant lipid accumulation within hepatocytes. The hepatocyte cytoplasm is displaced by large lipid vacuoles, causing the nuclei to be pushed to the periphery. The presence of widespread fat droplets distorts the lobular architecture. No overt inflammatory infiltrate or fibrosis is visible at this magnification, but the steatotic changes are consistent with features of MASH in the appropriate clinical context (Figure 1I). The significant increase in these biomarkers validates the Ob/Ob ^HFD model as a robust tool for studying MASH pathophysiolog.

To assess the impact of liver disease on the extent of pancreatic injury in Ob/Ob ^HFD mice, we first measured the pancreas-to-mouse weight ratio and the expression of the regenerating islet-derived genes Reg1 and Reg3, which serve as sensitive markers of pancreatic injury and attempted repair (Girón-González and Salto, 2021). The pancreas-to-mouse weight ratio demonstrated a notable 1.7-fold increase in the obese mice model compared to the littermate control group (Supplementary Figure S1). This observation suggests a potential regenerative response in the pancreas, possibly due to injuries incurred within the organ. In addition, our experiments showed that Ob/Ob ^HFD mice exhibited a dramatic upregulation of both Reg1 (3.0 ± 0.2 vs. 0.9 ± 0.1 in controls) and Reg3 (2.7 ± 0.3 vs. 1.0 ± 0.1; p < 0.0001 for both; Figure 2A), confirming a heightened regenerative response in the Ob/Ob ^HFD pancreas. Nevertheless, additional tissue injury assessments of oxidative damage were performed via evaluating for MDA activity, a well-established measure of lipid peroxidation. Pancreatic tissue isolated from Ob/Ob ^HFD showed significantly higher MDA levels (2.6 ± 0.4 U/mg) as compared to their littermates’ controls that revealed levels of 1.4 ± 0.2 U/mg; p < 0.05; Figure 2B), emphasizing oxidative stress involvement in the regeneration process. To further investigate the behavioral phenotypic characteristics of β-islet cells, we assessed Ki67 and annexin V to evaluate proliferation and apoptosis, respectively. Figure 2C displayed a marked increase in Ki67 staining of isolated β-islet cells in the Ob/Ob ^HFD (60% ± 18%) as compared to their littermates’ controls that showed staining of 38 % ± 12% (p < 0.01), indicating a highly proliferative cell. In parallel, β-islet cells exhibited a substantial increase in apoptosis rate of 3-fold as compared to littermates’ controls (Figure 2D; p < 0.0001).

We next quantified mRNA levels of α-SMA, Collagen I, and Fibronectin in pancreatic tissue, as a key response to chronic pancreatic damage, which also plays a significant role in the progression of pancreatic diseases, including pancreatitis, pancreatic cancer, and metabolic disorders such as obesity and diabetes (Li X. et al., 2021). Figure 2E illustrates a 2.4 ± 0.6, 2.7 ± 0.6, and 2.6 ± 0.5-fold increase in α-SMA, Collagen I, and Fibronectin expression, respectively, in the Ob/Ob ^HFD mice as compared to littermates’ controls (p < 0.01), reflecting pro-fibrotic activation. Collectively, our results demonstrate pancreatic damage reflected by oxidative injury and compensatory regenerative process with highly proliferative cells, yet β-islet cells showed a low survival rate as the accumulation of ECM and fibrosis in the pancreas is thought to disrupt the microenvironment required for β-cell survival. Studies have shown that fibrotic tissue can limit the nutrient and oxygen supply to the remaining beta cells, leading to additional stress and further increasing the likelihood of apoptosis (Li J. et al., 2021). Furthermore, the altered extracellular environment can impair the regenerative capacity of the pancreas, making it more difficult for beta cells to survive and proliferate.

To elucidate the contribution of systemic inflammation to the deterioration of β-islet cells, phenotypic alterations, and it’s possible role in changes in pancreatic immunity, we aimed to assess circulating serum pro-inflammatory cytokines IL-1β, IL-6, and IL-8 by ELISA as a reflection of their involvement in mediating immune cell fluctuations. Figure 3A demonstrate Ob/Ob ^HFD mice exhibiting increase in serum IL-1β (240 ± 35 pg/mL vs. 18 ± 4 pg/mL in controls; p < 0.05), IL-6 (580 ± 110 pg/mL vs. 12 ± 3 pg/mL; p < 0.0001), and IL-8 (780 ± 80 pg/mL vs. 15 ± 5 pg/mL; p < 0.0001), indicating an association of obesity and pancreatic injury with systemic inflammation. Consequently, evaluating pancreatic immune cells in the context of obesity and pancreatic injury is crucial for understanding the mechanisms driving inflammation, fibrosis, and metabolic dysfunction, ultimately paving the way for targeted therapeutic strategies to prevent or reverse pancreatic damage and improve metabolic health. For this purpose, we next assessed tissue-resident (tr) pancreatic CD4 and CD8 T cells and NK cells via flow cytometry. Figure 3B showed Ob/Ob ^HFD mice having elevated percentages of CD4 (27% ± 5% vs. 11% ± 3% in controls; p < 0.01) and CD8 T cells (40% ± 4% vs. 22% ± 3% in controls; p < 0.01), with a dramatic reduction in trNK cells (5% ± 4% vs. 26% ± 4% in controls; p < 0.001), suggesting T cells playing critical roles in inflammation and tissue damage while reduction in trNK cells could in part potentially impair the body’s ability to control inflammation and tissue damage effectively. Therefore, to further focus on whether reductions in trNK cells are associated with their dysregulation, we assessed their activity as mentioned in the materials and methods.

Figure 3C shows the expression of degranulation marker CD107a, activating receptor NKp46, and intracellular IFN-γ in pancreatic trNK cells. In Ob/Ob ^HFD mice, CD107a⁺ trNK cells were markedly reduced (6% ± 2% vs. 25% ± 4% in control; p < 0.01), as were NKp46 (17% ± 3% vs. 45% ± 5% in control; p < 0.0001) and IFN-γ subsets (8% ± 2% vs. 42% ± 9% in control; p < 0.0001) with overall reductions of 2-fold. These findings indicate a severe impairment in the cytotoxic function of trNK cells, suggesting a compromised immune response in Ob/Ob ^HFD mice.

Given the pivotal role of cytokines in the pathogenesis of pancreatic injury and their impact on regulating the immune response, we have assessed IL-6R expression on pancreatic trNK cells. IL-6 promotes pancreatic injury, fibrosis, and β-islet cell dysfunction. Yet, IL-6’s role in impairing trNK cell cytotoxic potential and contributing to a dysregulated immune response is not well documented. Figure 4A illustrates a significant 2.2-fold elevation in trNK IL-6R (P > 0.05) in Ob/Ob ^HFD mice compared to littermate controls, a result positively associated with the increase in serum IL-6 levels in these mice, as presented in Figure 3A, and inversely correlated with trNK activity markers, as shown in Figure 3C.

To strengthen the translational relevance of our findings and emphasize the contribution of IL-6 signaling to the immune modulation of obesity-associated pancreatic injury, we employed an adoptive transfer model to validate our results. We aimed to clarify the immune impact of pancreatic trNK cells in modulating pancreatic injury outcomes. To achieve this, we employed the NSG mouse strain, which is widely used for modeling immunosuppression. The immunosuppressed NSG mouse strain lacks T, B, and NK cells (Aryee et al., 2023) and was fed with HFD as mentioned in Materials and Methods, mimicking the MASH model and serving as the recipient. On the other hand, isolated pancreatic trNK cells from Ob/Ob ^HFD mice served as donor-transplanted cells. Figure 4B shows regenerative markers of Reg1 and Reg3 to be comparable in the pancreatic tissue of mice of both received or did not receive the trNK treated with the IgG isotype control. In parallel, NSG mice fed HFD transplanted with pancreatic trNK cells obtained from Ob/ObHFD mice and pre-treated with IL-6R antagonizing antibody exhibited a 2-fold decrease in both Reg1 and Reg3 (P<0.01). Similar patterns were achieved in pancreatic tissue MDA levels (Figure 4C) and β-islet cells apoptosis rate (Figure 4D) and showed a 1.77-fold and 2.2-fold decrease, respectively was noticed following trNK treated with the IL-6R antagonizing antibody (Figure 4C; P<0.01). Additionally, to further confirm the role of trNK cells in attenuating pancreatic injury, isolated pancreatic trNK cells treated with an IL-6R antagonizing antibody from the recipient mice and demonstrated a significantly higher expression of the NK cell activation marker CD107a (2.3 -folds) compared to mice received trNK with no IL-6R antagonizing antibody treatments (Figure 4E; P < 0.05). These findings suggest that IL-6/IL-6R modulates trNK cell activity, contributing to the alleviation of obesity-related pancreatic injury mediated by NK cells. Moreover, further phenotypic characterization of the NSG mice fed an HFD and transplanted with an IL-6R antagonizing antibody showed an amelioration in the pancreatic fibrotic profile, as evidenced by a 1.6-fold increase in α-SMA mRNA expression compared to their counterparts (Figure 4F; P<0.01). To investigate whether IL-6R signaling directly modulates STAT3 activation in pancreatic trNK cells, we performed Western blot analysis for phosphorylated STAT3 (p-STAT3) and total STAT3 in trNK cells treated ex vivo with either an IL-6R neutralizing antibody or an IgG isotype control. As shown in Supplementary Figure S2, trNK cells treated with IL-6R-neutralizing antibodies exhibited a marked reduction in STAT3 phosphorylation levels compared to isotype-treated controls, while total STAT3 levels remained unchanged. Quantitative densitometric analysis confirmed a significant decrease in P-STAT3/STAT3 ratio in the IL-6R antibody-treated group (*p < 0.05). These findings indicate that IL-6R antagonism effectively inhibits STAT3 signaling in pancreatic trNK cells, supporting a direct role for IL-6R in modulating their activation state. Our data clearly showed the importance of immune modulation of IL-6R on trNK cells in remodeling pancreatic tissue affected by liver injury insults, highlighting the liver-pancreas axis as a target therapy to prevent pancreatic damage, particularly β-islet cells dysfunction or loss, pancreatic fibrosis, and therefore minimize the risk of diabetes mellitus and other associated metabolic syndromes.