Overweight and obesity remain major global public health concerns, with prevalence and incidence rates continuing to rise worldwide (1,2). According to the World Health Organization (WHO), over 2.5 billion adults aged 18 years or older were overweight in 2022, of which over 890 million were classified as obese (www.who.int). Recently, the WHO updated BMI criteria for overweight and obesity class-I and class-II in adults in the Asia-Pacific region to 23.0-24.9, 25.0-29.9, and ≥30 kg/m², respectively (3,4). These revised cutoffs were proposed because Asians tend to be at greater risk of type 2 diabetes mellitus and cardiovascular diseases (CVDs) at lower BMIs than Caucasians (5).

Evidently, individuals who are overweight and obese are most at risk of dyslipidemia (6,7). Dyslipidemia and/or overweight/obesity are characterized not only by metabolic dysregulation but also by changes in innate and adaptive immune responses and, ultimately, higher chronic low-grade inflammation (8,9). NK cells are a key component of the innate immune system and may be altered under these aberrant conditions (10). As is well known, NK cells play vital roles in immunosurveillance and the elimination of virus-infected and malignant cells through signaling pathways that activate perforin-granzyme exocytosis, antibody-dependent cellular cytotoxicity and death receptor signaling mechanisms (11-13). Moreover, NK cells regulate immune homeostasis by secreting numerous cytokines and interacting with other immune cells to modulate inflammation (14). Typically, NK cells are divided into two subpopulations, classified by the intensity of anti-CD56 (NCAM1) staining: Weak (CD56^Dim) and strong (CD56^Bright). CD56^Dim NK cells mainly support cytotoxic activity by secreting perforin and granzymes, whereas CD56^Bright NK cells function mainly as specialized immunoregulators and cytokine producers rather than primary killers (11,15,16).

Accumulating evidence suggests that metabolic abnormalities associated with excessive adiposity and/or dyslipidemia may drive quantitative, phenotypic and functional changes in NK cells. These changes may contribute gradually to impaired immune surveillance, increased susceptibility to viral infections and the development of non-communicable diseases (NCDs) (10,17-19). However, the underlying mechanisms and consequences of dyslipidemia- and/or excessive adiposity-induced alterations in NK cells remain poorly understood, especially in Asian individuals. Moreover, the reported data have often been conflicting, and definitive conclusions remain unclear.

Therefore, the present study examined the frequency and functional changes linking metabolic dysregulation and NK cells. It examines the immunological profiles of NK cells, both overall and by CD56-based subpopulations, in 125 Thai individuals stratified by lipid profiles and BMI. The findings are expected to be beneficial for preventive medicine, particularly in the context of NCDs.

Overweight and obesity remain major health problems in the era of globalization, and their incidences are likely to increase in the future (1,2). Overweight and obesity are associated with a number of modifiable and non-modifiable risk factors, including lifestyle, low physical activity, diet, advanced age, genetics and the environment (21). Numerous studies on obesity/dyslipidemia have shown alterations in immune cell populations, phenotypes, and functions, predominantly T, NKT-like, and NK cells (10,17,22-24). However, research on NK cell changes remains limited, and the evidence remains inconsistent regarding NK cells' total and subpopulation percentages and functional capacities (10). Moreover, representative data on individuals with a BMI of 25.0-29.9 (obesity class-I) and their lipid profiles remain limited.

Therefore, the present study was the first, to the best of the authors' knowledge, to compare NK cell percentages, both overall and by subpopulations, and IFN-γ production status among 125 Thai (Southeast Asian) individuals without underlying diseases or anti-inflammatory using stratified by two-dimensional categories: Lipid profiles (normal lipid and dyslipidemia) and BMI (normal weight, overweight and obesity class-I). The participants were therefore distinguished into five subgroups (Table I). This classification differs from that used in prior studies, which mainly compared normal lipid vs. dyslipidemia or normal BMI vs. class-II obesity (BMI ≥30.0 kg/m²). Therefore, the present study provided a finer-grained analysis of NK cell changes associated with metabolic alterations and increasing adiposity, which may help reveal such changes. However, individuals with normal lipid plus obesity class-I were not included in the present study due to their rarity in the Thai population.

NK cells were examined, both overall and by subpopulations, by flow cytometry and compared across the five subgroups. Total NK cell percentages were higher in the DL groups and the group-wise comparisons exhibited a corresponding stepwise escalation. However, they were only markedly higher in the DL/OB-I group compared with the other groups. It was hypothesized that dyslipidemia may associate with the expansion of the total NK cell population, especially in obesity class-I. For instance, Wouters, et al (18) found that subjects with obesity had markedly higher total NK cell percentages than those who were lean. Obese individuals may associate greater low-grade inflammation. By contrast, Xu et al (22) found higher NK cell percentages in Chinese adults with dyslipidemia than in healthy controls, although the difference was not significant. Bähr et al (24) found no significant difference between subjects with obesity (BMI >30.0 kg/m²) and normal weight (BMI <25.0 kg/m²).

Regarding NK cell subpopulations, although CD56^Dim NK cell percentages were higher in the DL groups, only those in the DL/OB-I group were markedly higher than those in the other groups. This finding contrasted with a previous study on obesity (24). However, it was therefore hypothesized that under dyslipidemia, especially in higher BMI, individuals may experience greater low-grade inflammation. For CD56^Bright NK subpopulation, multiple group analysis showed no statistical significance.

Phan et al (25) previously showed that NK cell populations differ markedly by sex, both overall and by subpopulation. Thus, the present study further investigated whether sex affects NK cell populations using data from the NL/NW group, as it should best reflect sex differences without the confounding effects of dyslipidemia and high BMI. The proportions of total NK cells and CD56^Dim NK cells were significantly higher in males than in females (P<0.05; Fig. SI), consistent with Phan et al (25). However, the proportion of CD56^Bright NK cells did not differ markedly across groups. Nevertheless, the group-to-group comparison is unlikely to have been affected, as the sex ratio was adjusted to be comparable across subgroups.

The present study then observed the percentages of IFN-γ-producing total NK cells and their subpopulations. IFN-γ-producing CD56^Bright NK cell percentages were markedly higher in the DL/OB-I group than in the NL/NW and DL/NW groups. Accordingly, it is hypothesized that higher BMI may be associated with stronger immunological response driven by greater cytokine-induced low-grade inflammation. Consistent with a previous study (24), IFN-γ-producing CD56^Bright NK cell percentages were markedly higher in obese individuals who had BMI ≥30.0 than in normal weight groups (BMI <25.0), whereas IFN-γ-producing CD56^Dim NK cell percentages were markedly lower. However, the present study was limited to functional cytokine production capacity based on cellular responses stimulated by PMA (protein kinase C activator) and calcium ionophore (ionomycin), which activate the phosphoinositide 3-kinase (PI3k)/Akt signaling pathway (26). The present study was primarily an observational one that paves the way for further investigations into other functional aspects. In feasible cases of dyslipidemia combined with obesity condition, higher pro-inflammatory adipokines such as leptin and visfatin, as well as excessive long-chain fatty acid, were associated with signaling pathways involved in IFN-γ production. Possibly, leptin may activate Janus kinase 2/signal transducer and activator of transcription 3 (STAT3) prior to stimulating STAT4, T-box expressed in T cells (T-bet) transcription factor and ultimately IFN-γ production (27,28). As well as visfatin, it probably induces IFN-γ production via nuclear factor kappa-light-chain-enhancer of activated B cells and STAT3 signaling pathways (29). Additionally, long-chain fatty acids can be obtained from dietary fats and triglyceride components and also promote this cytokine production. However, the associated pathway remains inconclusive (30).

In other functional considerations of interest, previous studies have investigated the effects of obesity on NK cytotoxicity-related assay and degranulation function, consistently showing decreased cytotoxic activity against tumor cells (31-34) and reduced degranulation, as indicated by decreased lysosome-associated membrane protein 1/CD107a expression (31,33) or granzyme B production (33). However, Zhang et al (19) demonstrated that NK cell cytotoxicity, measured at an effector-to-target cell (human leukemic cell line K562) ratio of 12.5:1, was significantly higher in women with recurrent pregnancy loss and dyslipidemia than in those with normal lipid levels (P<0.05).

Since the clinical characteristics of various participants were found to be statistically significant following the Jonckheere-Terpstra test, MANCOVA was also examined the effects of variable factors on multiple outcomes of all NK cell percentages and cytokine production capacity while controlling for covariates. However, significant associations were not found. In the present study, age, PR, FBS, Cr, as well as variables including sex, smoking, and alcohol drinking, were tested simultaneously.

BMI correlated positively with the percentages of total NK cells, CD56^Dim NK cells, and IFN-γ-producing CD56^Bright NK cells. Thus, BMI may be possible physical indicator reflecting immunological status for NK cell and their function. Nevertheless, the observed significant positive correlations were also largely driven by dyslipidemia, especially in the DL/OB-I group, which made the effect appear more pronounced. Similarly, Viel et al (17) found that NK cell counts in peripheral blood correlated positively with BMI. While the findings of Bӓhr et al (24) are largely consistent, the authors reported negative correlations between BMI and both CD56^Dim NK percentage and cytokine production. Nevertheless, the existing evidence is based on only a few studies and further studies are required to advance and validate this concept.

Overall, it appeared that the findings of the present study leaned toward a higher low-grade inflammatory outcome. Larger NK cell populations and changes in their function might help drive occurring of the development of NCDs such as CVDs, kidney diseases and type 2 diabetes mellitus. In CVDs, NK cells were predominantly increased in human atherosclerotic lesions (35,36). Consistently, symptomatic patients showed increased percentages of IFN-γ-producing NK cells and CD56^Bright NK cells in atherosclerotic plaques (37), as well as higher levels of circulating NK cells (38). Moreover, elevated numbers of IFN-γ-producing NK cells increase cholesterol accumulation in macrophages, inducing foam cell formation and potentially activating macrophages, which, in turn, release matrix metalloproteinases to degrade endothelial cells, vascular smooth muscle cells and the extracellular matrix, ultimately leading to destabilization and rupture of atherosclerotic plaques (39,40). In kidney diseases, greater infiltration of activated NK cells might contribute to inflammation, stressed tubular and endothelial cells, interstitial fibrosis, and chronic damage to glomerular tissue through IFN-γ-mediated functions and cytolytic mediators (41-43). In non-diabetic individuals, a previous study showed that fasting glucose levels correlated positively with the number of total NK cells (44). An increase in NK cells may serve as a marker of immune activation and may also be associated with higher low-grade inflammation. However, the present study found no significant differences in FBS among the groups (all average values were within the normal range) and no correlation between FBS and NK cell total or subpopulation percentages, or between FBS and IFN-γ-producing NK cell total or subpopulation percentages. In type 2 diabetes mellitus, NK cells could contribute to insulin resistance primarily through obesity-induced low-grade inflammation by enhancing the release of proinflammatory mediators, such as the tumor necrosis factor-α and IFN-γ, which could disrupt the normal insulin signaling pathway and downregulate key insulin signaling components, including the insulin receptor, insulin receptor substrate 1 and the insulin-regulated glucose transporter type 4 (45,46). Therefore, greater numbers of NK cells and IFN-γ-producing NK cells might contribute to, and enhance, the microenvironment associated with the pathogenesis of these NCDs.

In conclusion, the present study was the first, to the best of the authors' knowledge, to report the immunological profiles of NK cells, both overall and by subpopulations, in Thai individuals stratified by lipid profiles and BMI, thereby likely revealing clearer patterns than would be possible by assessing each variable alone. The immunological status of individuals with dyslipidemia was shifted toward a more proinflammatory state. Notably, those with dyslipidemia and obesity class-I exhibited predominant changes in both NK cell percentage and function. Therefore, both BMI and lipid profiles must be controlled to prevent immunological changes and potentially reduce low-grade inflammation. However, even although the sample size achieved a test power (1-β) >80%, the small number of participants remains a limitation of the present study.