According to the WHO, nowadays there are more than a 1.9 billion overweight people worldwide and of these, over 600 million are obese (1). This situation elevates the prevalence of certain cancers and immunological disorders. The obesity induces hypertrophy of adipocytes and limits the access to oxygen and nutrients (2). The resulting stress leads to the activation of pro-inflammatory cells in adipose tissue (3). Some various immune cell subsets, including macrophages, eosinophils, natural killer cells (NKs), NKT cells, and T cells, are important in maintaining adipose tissue homeostasis (4–7).

Natural killer cells are able to eliminate malignant cells with a strong regulatory role, by modulating both innate and adaptive immunity via crosstalk with cells of the innate and the adaptive immune system (8). Conventional NKs originates from committing progenitors of the bone marrow. They go through a maturation process and in this way NKs are influenced by the transcriptional network that guides them for phenotypic changes into two functionally distinct subpopulations, the CD56^dim, and a minority subpopulation of CD56^bright cells (9). They are controlled by soluble factors such as chemokines, cytokines, and other secreted ligands of NKs receptors. Due to that, there is an extreme diversity of NKs repertoire over time (10). The receptors considered prevailing are those associated with receptor tyrosine-based activation motif (ITAM) bearing signaling molecules such as NKp₃₀, NKp₄₄, and NKp₄₆ natural cytotoxicity receptors (NCRs) (11). A difficulty in understanding the basis of the NKs unresponsiveness in many cases, especially against tumor evasion related to the inexpression of activating receptors or existence of the inhibitory signals that have not been identified yet. So, the microenvironmental niches could dramatically affect the generation and function of distinct NKs subsets. Illuminating the platforms governing NKs function is a wonder, as they usually contravene the principle of other lymphocytes status.

Some of the NKs receptors protect normal cells by recognizing major histocompatibility complex class I (MHC-I) molecules. Cells expressing low levels of the MHC-I (usually cancer cells) are attacked by the NKs (12), but, other activating receptors on NKs such as lectin-like NKG2D, a member of the NKG2s family can activate the NKs without MHC-I ligands (13). While most of the activating receptors are specific for non-MHC-I ligands that are expressed on both normal and malignant cells, most of the inhibitory receptors are specific for the MHC-I ligand (14). Killer cell immunoglobulin-like receptors (KIRs) family includes the major inhibitory receptors (KIR3DL1-3, KIR2DL1-3, and KIR2DL5) which recognize the MHC-I molecules (HLA-A, B, and C), specifically (14, 15), while those of the NKG2s family recognize MHC-I signal sequences bound to the non-classical MHC molecule HLA-E (15). Both the KIRs and NKG2s families are composed of activating/inhibitory molecules that regulate the NKs function; however, they are originated from distinct gene families. Even the differences in a family are evident as the cytolytic activity of the NKs is inhibited by the NKG2A and activated by the NKG2C molecules (14).

Natural killer cells could enter to the peripheral tissues and recognize the stressed cells. They could react to the adipose stress and cause inflammation and even insulin resistance (16). In this way, several mechanisms such as insulin-like growth factor, adipocytes derived hormones (i.e., leptin), steroid hormones, chemokines, and adipokines may alter the function of NKs in this niche (17). Also, mesenchymal stem cells (MSCs) that comprise about 1% of the adipose niche have an immunomodulatory role and could suppress immune reactions in vivo in a non-HLA-restricted manner. The MSCs inhibit several functions of T lymphocytes (18, 19) and dendritic cells (DCs) (20). Their immunomodulatory role directed scientists for using MSCs to treat graft vs host disease after allogeneic hematopoietic transplantation (21). Some studies show the activated phenotype of NKs in visceral adipose tissue (22) suggesting their role in adipose tissue inflammation in metabolic disease. They produce pro-inflammatory cytokines, notably tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ), and regulate macrophages to promote insulin resistance in obesity (16, 23). Although there are some data on visceral adipose tissue role on NK cell phenotypic alteration, we did start first to know about the subcutaneous adipose tissue (SAT) niche for NKs behavior and molecular changes. Here, we have confirmed the alteration of the CD56^dim NKs NCRs especially NKp30 and NKp44, with respect to their cytotoxicity potential against malignant cells. This finding can open a new window to our knowledge of the increased rate of cancers in obese persons (11) such as esophageal adenocarcinoma, breast and colon cancers (24), and solving the problems of how these NKs will affect human diseases.

In this study, we provide the phenotypic characteristics of NK cells reside in subcutaneous abdominal fat in obese persons. Also, it is important to mention that the visceral adipose tissue completely differs from the subcutaneous fat counterpart in behavior and gene expression profile (31). Epidemiological evidence indicate that obesity is responsible for more than 15% of all deaths for persons beyond 40 years of age in the United States (32). NK cells are a population of lymphocyte that enters in adipose tissue and reside there for their whole life. As NKs are the first line of innate defense vs tumor formation, the phenotypic changes in the surface markers, KIRs, and NCR expression may lead to the progress of transformed cells into a tumor. On the other hand, the impaired NKs function might cause autoimmune disease and persistence of autoimmunity. NK cell localization in the other tissues might be a prerequisite for maturation into the classical cytolytic phenotype, found predominantly in PB.

There are some reports that show the dysfunction of NKs caused by hormones and cytokines. In 2006, Kim et al. found that adiponectin is a negative regulator of NKs cytotoxicity (33), but there are no reports on the mechanism underlying this dysfunction. Our experiment was unable to detect the significant difference in the number of NKs between obese and lean cases (Figure 1A), but the shift of ADNKs to CD56^bright phenotype was obvious and our findings show a marked reduction in the function of NKs in obese cases. The expression of CD16 was reduced slightly in ADNKs of obese cases, and there was a significant difference between ADNKs and PBNKs groups (Figure 1B). In 2004, Dovio et al. investigated NKs activity in patients with uncomplicated obesity with an average BMI of 36. Our study confirms their findings as we found that obese subjects with no metabolic complications had similar NK cell levels to lean controls. They believe it was only metabolically unhealthy obese subjects which make up 80% of the obese population, have reduced NK cell levels, and altered phenotype (34). In our experiment that conducted on the healthy individuals, the cytotoxicity tests on the NB cell lines resulted in the poor cytolysis activity of ADNKs in comparison with PBNKs (Figure 4A). By increasing the body mass index, more and more NKs will reside in this niche and they would be non-functional for their entire life.

The responses to the question that why entrapped NKs in the adipose tissue lose their cytolytic effect on neoplastic cells may be found in the MSCs interaction with the NKs. Yet, so far 15 years that researchers found a large population of MSCs in the adipose tissue (ADSCs) (35). Probably these stromal cells could influence the niche of abdominal adipose tissue too, especially by exerting their immunomodulatory function (36) directly by cell–cell contact or via production of cytokines such as IL-10, IL-6, and TGF-β (37). Many piece of evidence are emphasizing on the stronger immunomodulation activity of ADSCs compared to bone marrow-MSCs (BMSCs). In 2009, Ivanova-Todorova et al. showed that ADSCs are more potent suppressors of DCs compared to BMSCs (38). Spaggiari and colleagues focused on the ADSCs secreted indoleamine 2,3-dioxygenase and prostaglandin E2 role in the suppression of proliferation, cytolysis, and cytokine production in the NKs (39). In this way, Selmani et al. (40) confirmed that human leukocyte antigen-G5 secretion by MSCs is required to suppress T and NK cell function and to induce CD25^highFOXP3⁺ regulatory T cells (40). An important change that detected in our flow cytometry analysis was related to the downregulation of Nkp30 and Nkp44 (Figure 2A). We know that NKp30 and NKp46 are constitutively expressed by all NK cells, while NKp44 is expressed only after activation. So, we detected the levels of Nkp44 in freshly isolated NKs and also after cytokine activation. In addition to the levels of NKp44, our results have been demonstrated the reduced levels of NKp30 receptors in ADNKs when compared with PB CD56^dim NKs (Figure 2B). The NKp44 suppression strongly contributes to the unresponsiveness of NKs against sensitive malignant cells that did not express MHC class I molecule (Figure 4A). The amount of Nkp46 expression was similar in ADNKs and PBNKs in both obese and lean persons (Figure 2B). As the gene coding for NKp46 is located in the leukocyte-receptor complex on human chromosome 19, while the genes for NKp44 and NKp30 are located on chromosome 6 (41) maybe this alteration happened only in one chromosome that belongs to Nkp30 and NKp44 NCRs. The NKp44 has unique features of several occasions including (1) its expression is closed to active NKs authoritative to initiate an immediate cytotoxic response (42), (2) its expression is responsible for killing of many tumor cells and also results in the release of cytotoxic granules, IFNγ, and TNFα (43), (3) it is also expressed on a subset of IFN-producing cells located in mucosal-associated lymphoid tissues (44). By downregulation of NKp44, tumor cells could induce NKs apoptosis via overexpression of Fas Ligands (45). NKp44 could be exploited and the tumor cells escape from the NKs recognition due to the changes in their niche. Our data show that the SAT in the abdominal region of obese persons provides this kind of microenvironment that induce these phenotypic changes in NKs (Figures 1B and 2A). However, we can see the similar expression of NKG2D levels in obese persons when compared with non-obese cases (Figure 2B). But, it has been reported by O’Rourke et al. that the adipose tissue NKs had an activated phenotype with increased levels of NKG2D (22). This increased expression of NKG2D may cause inflammation in obesity. Also, we have demonstrated the altered expression of granzyme B (Figure 1E) in stimulated NKs that might lead to NK-mediated death.

The cytokine secretion experiments showed decreased secretion levels of IFNγ and TNFα cytokines (Figure 4D). Recent studies show that MSCs cause a decrease in IFNγ secretion by NKs in coculture systems (46). We confirmed this situation for some HLA type I non-expressing neoplastic cells after cell to cell contact and/or coculture of PBNKs with ADMSCs (Data not shown). Some other studies have discussed the role of leptin that produced by adipocyte on the cytokine secretion by NKs.

Our findings provide preliminary data onto the relationship between abdominal adipose tissue and the NKs biology that undoubtedly influences the rate of malignant diseases or even metabolic disorders, especially in obese persons. The role of other cells and/or factors that affect NCRs under-expression is under investigation by our team and needs more evidence to be fully elucidated.

All methods were carried out in the experiments with human cells in accordance with the ethical committee of the Tehran University of Medical School. All the protocols were approved by the ethics committee of Tehran University of Medical Sciences and Ministry of Health and Medical education. Also, all of the volunteers provided the informed consent.

AS-H, MB, RM, and JA performed the experiments; SM-T performed statistical analysis; AH designed the study; SM-T and AH contributed in writing the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.