The study was approved by the Ethical Committee of the Hospital Germans Trias i Pujol (Badalona, Spain) and followed the guidelines of the Declaration of Helsinki. Participants gave written informed consent prior to clinical data collection.

The cohort included 27 individuals: 13 with severe obesity and 14 with normal weight ( Table 1 ). All individuals were evaluated between October, 2015 and September, 2021 by the same endocrinologist (S.P.), who followed the institution’s protocol for bariatric surgery (BS), according to BS criteria (Spanish Position Statement between Obesity, Endocrinology, Diabetes, and Surgery Societies). vWAT and sWAT biopsies were taken from individuals with severe obesity during BS and from individuals with normal weight at the time of consultation/minor surgery, mainly cholecystectomy and biopsies that were finally negative for tumors. Exclusion criteria were having active infectious or inflammatory pathologies other than those related to obesity, treatment with immunosuppressant drugs, or suffering from other forms of immunosuppression.

Serum samples were collected after 12 h fasting on the day of surgery and 6 and 12 months afterward, and frozen at -20° C. An ELISA assay was performed at baseline, 6, and 12 months after surgery (R&D system Bio-Techne D1600). Glucose and insulin levels, and lipid profiles [total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein cholesterol, and triglycerides], were measured in our certified core clinical laboratory by enzymatic methods.

Homeostatic model assessment of insulin resistance (HOMA–IR) was calculated as:

Fresh WAT samples collected during surgeries were transferred to liquid nitrogen and then frozen at –80° C. Total RNA was extracted from whole adipose samples using a standard column-affinity-based methodology (NucleoSpin RNA II; Macherey-Nagel). Next, 500 ng of total RNA was retrotranscribed into cDNA using random hexamer primers and Multiscribe reverse transcriptase (TaqMan reverse transcription reagents, ThermoFisher Scientific) according to the manufacturer’s instructions. Platinum Quantitative PCR SuperMix-UDG with ROX reagent (ThemoFisher Scientific) was used as the master mix reagent and the expression levels of each gene of interest were assessed using specific TaqMan probes Hs00913644_m1 for IL-16 and Hs04194521_s1 for the housekeeping Ppia gene (ThermoFisher). Gene expression was calculated using the 2^-ΔΔCt method.

All procedures in the cell culture room were performed under a laminar flow hood. Cells were grown at 37°C with an atmosphere of 95% air, 5% CO2. All reagents used during cell culture procedures were heated to 37°C in a water bath before use.

The 3T3-L1 murine preadipocyte cell line (CL-173, ATCC) was used between passages 13 and 15.

To obtain mature adipocytes, 3T3L1 preadipocytes were plated into 12-well plates and grown to 100% confluency, with the growth medium being replaced every other day. Two days after 100% confluency was reached, an induction medium was added (day 0 of induction for the differentiation period), and cells were incubated for 48 h (day 2 of the differentiation period). The induction medium was then changed for a differentiation medium ( Supplementary Table S1 ), and cells were incubated for an additional 48 h (day 4 of the differentiation period). The differentiation medium was then changed to a maintenance medium ( Supplementary Table S1 ) until the cells acquired a mature adipocyte phenotype (day 9 of the differentiation period). IL-16 treatment was performed using recombinant mouse IL-16 protein (Invitrogen, RP-8610) at a concentration of 1 or 10 ng/mL during 3T3-L1 differentiation to evaluate the effect of this cytokine during in vitro adipogenesis. Differentiated adipocytes were treated for 24 h with the same concentrations of IL-16.

Fatty acid (FA) treatment: Sodium palmitate (Sigma-Aldrich, P9767) was conjugated with FA-free BSA at a 5:1 ratio (21). Mature adipocytes were incubated with 1 mM of this solution for 24 h. The control group was incubated with 0.1% BSA.

Free glycerol was measured using a Serum Triglyceride Determination Kit (Catalog Number TR0100, Sigma-Aldrich, USA).

Lipid accumulation was evaluated in 3T3-L1 cells by staining with Oil Red O (Sigma Aldrich, O0625). The stock solution was prepared by adding 300 mg of Oil Red O powder (Sigma Aldrich, O0625) to 100 ml of 2-propanol (Alfa Aesar, 36644). The solution was protected from light and stirred overnight. The final solution was filtered through Whatman paper to remove dye precipitates. Oil Red O working solution was prepared by mixing three parts of the Oil Red O stock solution with two parts of distilled water, incubated for 10 minutes at room temperature protected from light, and filtered through a syringe filter unit (Merck, SLHV033RB). Plated cells were carefully washed once with PBS, avoiding disruption of the cell monolayer. Next, cells were fixed with 500 μL of 4% paraformaldehyde (Sigma-Aldrich, 47608) diluted in PBS, and incubated for 30 min at room temperature. Once the cell monolayer was fixed, it was washed twice with distilled water and then incubated for 5 min with 500 μL of 60% isopropanol. Next, 500 μL of Oil Red O working solution was added and incubated for 15 min to stain intracellular lipids. The staining solution was removed and the cell monolayer was washed three times with distilled water. The cell monolayer was photographed using a microscope (Leica DM IL LED, Leica Biosystems) to assess the lipid vesicles in mature adipocytes. Staining was quantified by extracting the stain with 1 mL of 100% isopropanol, and 200 μL of this solution was placed in a 96-well plate (Greiner Bio-one, 655101) to measure the absorbance at 492 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Scientific).

RNA was extracted from cultured cells using Trizol™ reagent (Invitrogen, 15596026) according to the manufacturer’s instructions. RNA was precipitated using GlycoBlue® (ThermoFisher, AM9515) in the isopropanol step. Samples were then left overnight at -20°C and were processed the following day according to the manufacturer’s protocol. The RNA pellet was resuspended in 30 µL of nuclease-free water and quantified with Nanodrop. Next, 1 µg of total RNA was retrotranscribed using M-MLV reverse transcriptase (Invitrogen, 28025-0113) according to the manufacturer’s instructions. A LightCycler 480 SYBR Green I Master (Roche, 4887352001) was used as the master mix reagent, and expression levels of the genes of interest were assessed using specific primers ( Supplementary Table S2 ). Gene expression was calculated using the 2^-ΔΔCt method, with Ppia as the housekeeping gene, and expressed as arbitrary units.

A public database generated by our laboratory (GEO repository: GSE236145) containing RNAseq from human WAT-infiltrated T cells was used to decipher the potentially relevant role of IL-16 in pathways related to immunometabolism in obesity. Once this function was identified, additional statistical data analyses beyond these bioinformatic procedures were conducted using GraphPad Prism 7.01 (GraphPad Software, Inc., La Jolla, CA, USA) and IBM SPSS 25.0 (IBM, Armond, NY, USA). Data distribution within groups was analyzed using the Shapiro–Wilk test, while the presence of outliers was determined using Tukey’s rule. If data showed a normal distribution, a Student’s t-test was performed to assess comparisons between two groups, otherwise, a Mann–Whitney test was used. For comparison between more than two groups, a one-way ANOVA test was applied in the case of one independent variable, and two-way ANOVA when more than two independent variables were considered, followed by uncorrected Fisher’s least significant difference (LSD). Likewise, correlations between the expression levels of IL-16 and selected genes were conducted using Pearson’s (normally distributed data) or Spearman’s (non-normally distributed data) correlations. The statistical significance threshold for all analyses was established at the two-tailed 5% level (P< 0.05).

Individuals with severe obesity showed a higher weight, body mass index (BMI), % hemoglobin A1c, and elevated levels of triglycerides. On the other hand, patients with obesity had lower total cholesterol and HDL-cholesterol concentrations compared with the control group of normal-weight individuals. This suggests that they exhibited obesity-associated metabolic disarray beyond an increase in adiposity ( Table 1 ). IL-16 expression was evaluated in vWAT and sWAT from both groups, finding that IL-16 expression was increased in vWAT from individuals with obesity compared with sWAT and vWAT from normal-weight individuals (P< 0.0001) ( Figure 1 ). Higher serum levels of IL-16 were observed in patients with obesity compared with normal-weight individuals, with an increase 6 months after bariatric surgery that is reverted 12 months after the intervention ( Supplementary Figure S1 ).

Public database analysis from our laboratory (GEO repository: GSE236145) showed that IL-16 is associated with inflammatory processes, including regulation of cell activation, immune system process, adaptive immune response, and regulation of response to stimulus ( Figure 2 ). This analysis indicated an association between IL-16 and cell adhesion-positive processes and cell communication ( Figure 2 ).

In vitro experiments were performed using 3T3-L1 preadipocytes to evaluate the effect of IL-16 on adipocyte differentiation and the modulation of key markers involved in adipocyte biology. First, 3T3-L1 cell differentiation was evaluated by measuring the accumulation of intracellular lipid droplets and the mRNA expression of adipocyte differentiation markers ( Supplementary Figure S2 ). An increase in intracellular lipid droplets at day 7 and elevated levels of genes involved in fat storage, such as Perilipin 1 (Plin1) and the adipocyte fatty acid binding protein (Fabp4), was observed (P< 0.01). Moreover, markers of adipogenesis, such as adiponectin (AdipoQ) and peroxisome proliferator-activated receptor gamma (Pparg), also increased their expression at day 7 (P< 0.01) ( Supplementary Figure S2 ).

Then, 1 or 10 ng/mL of IL-16 was added to the cells to evaluate the effects on adipocyte differentiation in vitro. The results showed that IL-16 did not affect the lipid accumulation profile during preadipocyte differentiation ( Figure 3A ). While no changes were seen in AdipoQ mRNA expression, incubation with IL-16 induced an increase in Pref1 expression at day 12, independent of the concentration (1 or 10 ng/mL of recombinant IL-16, P< 0.0001) ( Figure 3B ).

The effect of IL-16 on inflammation, glucose and lipid metabolism, and remodeling, given their involvement in WAT during obesity, was analyzed.

In terms of inflammation, Il6 gene expression decreased in response to 10 ng/mL IL-16 (P< 0.05), while Tnfa and Ccl2 showed no response to any of the administered doses ( Figure 4A ).

The expression of genes associated with lipid and glucose metabolism (Plin1, Cd36, and Glut4) was studied, finding that incubation with 10 ng/mL of IL-16 decreased mRNA expression levels of Plin1 (P< 0.01), Cd36 (P< 0.001), and Glut4 (P< 0.01) ( Figure 4B ).

Finally, incubation with 10 ng/mL of IL-16 decreased the mRNA expression of Hif1a (P< 0.0001), Col4a (P< 0.01), and Col6a (P< 0.0001) ( Figure 4C ); additionally, both 1 and 10 ng/mL of IL-16 reduced the expression of Vegf (P< 0.0001) ( Figure 4C ). Furthermore, increased expression of the metalloproteinase Mmp9 and decreased expression of its inhibitor Timp1 (P< 0.0001) were found ( Figure 4C ; Supplementary Figure S3 ).

Individuals with obesity are reported to have high levels of circulating free fatty acids (FFAs), the molecules most associated with obesity and the development of co-morbidities (22). Palmitate (C16:0) is frequently used in in vitro models to enhance adipocyte differentiation and hypertrophy of the mature phenotype (23–25). On day 7, cells were treated with 1 mM palmitate conjugated to BSA for 24 h, according to previous protocols established in our laboratory (21). To corroborate the effect of this treatment, the mRNA expression of different inflammatory markers was measured ( Supplementary Figure S3 ). As expected, palmitate treatment promoted an inflammatory pattern, increasing the expression of Tgfb, Ccl2, and Il6 ( Supplementary Figure S4 ). Next, lipid accumulation and lipolysis were evaluated using Oil red O staining and measurement of secreted glycerol, respectively ( Figure 5 ). Palmitate treatment of mature adipocytes increased both lipid accumulation and glycerol release (P< 0.0001) ( Figure 5A ).

Mature adipocytes were co-treated with palmitate (1 mM) and IL-16 at concentrations of 1 or 10 ng/mL IL-16 for 24 h to study the impact of IL-16 in the context of obesity. Cells without IL-16 treatment (with and without palmitate) served as controls. The administration of 10 ng/mL IL-16 mitigated the palmitate-induced increase in both lipid accumulation and lipolysis (P< 0.05, P< 0.01, P< 0.0001) ( Figure 5B ).

Regarding inflammation, IL-16, together with palmitate, exerted a synergistic effect enhancing the increase in inflammatory markers Ccl2 and Il6 (P< 0.05, P< 0.01, P< 0.001, P< 0.0001) ( Figure 5C ). IL-16 treatment at both doses (1 and 10 ng/mL) did not prevent the reduction in Plin1, Cd36, and Glut4 caused by palmitate treatment ( Figure 5D ).