Nutritional status influences peripheral immune cell phenotypes in healthy men in rural Pakistan

The effects of malnutrition on immune functions are well established as many studies in the recent past have demonstrated that age-associated malnutrition contributes to immunodeficiency [1, 2]. Malnutrition is accompanied by a decrease in immunity and an increase in susceptibility to many infectious diseases. Particularly in the aged subjects, underweight or overweight and/or obesity confer increased risk of mortality [3]. Undernutrition affects many elderly not only because their nutrient intake, in general, may be inadequate [4], but also because older adults have altered requirements for several nutrients due to the effects of aging on absorption, utilization, and excretion of nutrients [5] as well as specialized nutrient needs associated with medication use, metabolic disorders, and chronic disease [6]. The effects of undernutrition on immune status may be judged from the fact that a substantial part of the lean mass of human body is comprised of lymphocytes [7]. Underweight may contribute to increased mortality caused by viral infections due to inability to meet the enhanced energy requirements associated with the immune response [8].

On the other hand, overweight and obesity are forms of malnutrition at epidemic proportions globally [1]. The comorbidities associated with obesity affect virtually every physiological system including the regulation of immunity and inflammation [2]. The relationship between obesity and immunity is logically to be expected mainly on the basis of three lines of evidence. First, obesity is linked to increased risks of virtually all types of cancers [2]. Second, obesity is closely associated with chronic, systemic inflammation, which may contribute to the development of obesity-related co-morbidity [9]. Third, a number of hormones including leptin, which is a satiety factor dysregulated in obesity, have been shown to play an important role in regulating immune functions [10]. In addition, some recent studies have shown a link between alterations in lymphocyte functions and the pathogenesis of a number of obesity-related comorbidities including atherosclerosis, steatohepatitis, and diabetes [11, 12], the presence of which may underlie a number of obesity effects on the immune system.

Obese and underweight individuals may present with a difference in their immune profile as compared to people of normal weight. Our objectives were to determine the effects of malnutrition in a sample of obese and underweight young and elderly individuals in order to determine whether malnutrition has any association with alterations in the frequency of peripheral blood lymphocyte subsets. Any such study of possible associations would provide further supporting evidence for the presence of an immune diathesis of obesity and underweight, which will help to identify specific lymphocyte subsets as those most important to monitor in further studies in nutritional immunology. Furthermore, this pilot study was conducted in a rural population in a developing country in order to ascertain the general applicability of the immunological findings worldwide.

The effects of nutrition on immune functions have been extensively investigated, but almost exclusively in so-called “WEIRD” subjects (Western, educated, industrialized, rich, and democratic) and rarely in concert. It is not established whether immune alterations found in these populations are representative of the majority of the world’s peoples. The possible detrimental effects of aging on the immune system are further aggravated by numerous physiological and physical conditions, which in many cases can be associated with malnutrition. In the present study, we investigated the effects of nutritional status on immune signatures in a group of healthy young and elderly individuals from a rural area in a developing country where nutritional issues tend to be even more extreme than in developed countries (both in terms of over-as well as under-weight).

The primary findings regarding relationships between nutritional status and immune parameters investigated in this study were that there were some significant differences in the subsets of CD4+ and CD8+ cells but only tendential differences in percentages of B cells and NK cells between young and elderly men based on their nutritional status. While the current study observed changes in both CD4+ and CD8+ compartments, the impact of nutritional status on CD4+ cells and their subsets was more profound than on CD8+ cells and subsets (Tables 234 and 5). Others have previously suggested that fat (one of the predictors of nutritional status) has a more direct effect on CD4+ T cell count, total lymphocyte count, and WBC count than on CD8+ T cells [17]. CD4+ subtypes are directly stimulated by various cytokines including tumor necrosis factor-α, which contributes to the differentiation of CD4+ T cells into the TH1 subset [18] and leptin. In contrast, cytokines produced by fat tissue are not central to CD8+ activation. Therefore, whereas fat directly influences CD4+ cell counts via the action of various adipokines, it may influence CD8+ counts only indirectly via its ability to activate CD4+ T cells.

In contrast to what we initially expected, the current study did not identify many differences among the three BMI categories versus normal. Nevertheless, our findings on the comparison between OB and NW subjects are in agreement with Lynch et al.[19], who reported significantly more CD8+ and NK cells in lean controls compared to obese individuals. They further reported no differences in CD4+ levels between obese and lean individuals. In addition, they observed that the phenotypes of immune cells were also different between obese and lean individuals with regard to expression of activation markers and that obese individuals expressed significantly less CD45RA on their T cells. However, when obese individuals were further divided into metabolically healthy (MH) and unhealthy (UH) groups, it was found that circulating NK cells and CD8+ T cell levels were significantly reduced only in the UH obese group. In our study, all the overweight and obese individuals seem more likely to be metabolically healthy as our inclusion criteria excluded all those who had a present or recent past history of diseases including diabetes, hypertension, CVD etc. We, therefore, need further studies in elderly Pakistani subjects including both metabolically healthy and unhealthy obese elderly individuals to investigate the differences as reported previously [20]. It has been suggested that the unique metabolically healthy subgroup of obese individuals appear to be protected or more resistant to the development of comorbidities associated with obesity [20]. Despite having excessive body fat, these individuals display a favorable metabolic profile characterized by high levels of insulin sensitivity, no hypertension, normal lipid, inflammation, and hormonal profiles and, importantly in the context of the present study, a favorable immune profile [19].

In the present study, young and elderly subjects were separated into high, medium and low risk categories on the basis of their WC and WHR values. The comparisons between the low risk (LR) and the other risk groups (high risk, HR; medium risk, MR) show that HR-WC young subjects had significantly (p = 0.0145) lower percentages of CD8+ cells (p = 0.014) and B cells (p = 0.0201) compared to the LR-WC category. In the elderly subjects, there were also differences between these categories but none of them was significant (p for all trends ≥0.05). In the present study, the high body fat (HF-% BF) category in young had significantly (p = 0.0201) lower percentages of CD4+ cells compared to the normal fat (NF) category. In general, it has been shown that a decrease in the lean body mass is related to the decrease in body cell mass [21] and that body cell mass depletion is out of proportion to loss of body weight for fat [22]. Furthermore, these results may suggest that compared to the elderly, in young people, WC, WHR and % BF are more sensitive anthropometric measurements influencing the percentages of circulating CD4+, CD8+ and B cells. We are not aware of concrete evidence from previous studies to support the present observations. Some conflicting results, however, demonstrated an increase in CD4+ cells and a decrease in CD8+ cells in obese people (based on BMI) [9]. Others have attempted similar studies of lymphocyte subset frequency in obesity, with conflicting results. Total circulating lymphocytes and monocytes were reported as increased [23] or the same [24] as in the lean controls. Lymphocyte subsets have also been studied, and some investigators have found no differences in numbers of circulating T-cells, B cells, and NK cells [25], while others have shown increased or decreased lymphocytes and T-cells in obese people, and correlated the magnitude of these differences with increasing BMI [26]. Still other investigators have demonstrated a relationship between morbid obesity and CD8+ count only, but not mere overweight and/or obesity when compared with normal weight [23]. Despite these conflicting results, a preponderance of evidence suggests alterations in the immune system of both underweight and obese individuals. For example, monocyte function has been shown to alter in obese humans resulting in increased oxidative burst and phagocytic activity [23]. Such alterations in monocyte function could contribute to the state of systemic inflammation associated with obesity. T-cell phenotypes are likewise altered in obesity as reported previously [23].

No significant differences were found in the four BMI categories of young and the elderly regarding the number of B cells (Tables 6 and 7). In the IgD+CD27-, IgD-CD27+ and NK cells (CD16/CD56) significant differences were noted, respectively, between UW vs. NW; UW vs. NW and OB vs. NW elderly (Table 7). Previous research concerning differences in these cellular phenotypes in the well- and malnourished elderly is scarce. Some studies on the relationship between weight and NK cell number in other age groups, however, have shown a close but conflicting link between body weight and NK cells. Kelley et al.[27] reported that individuals who reported losing weight had fewer and less effective NK cells than those who had never lost weight. Likewise, Scanga and co-workers [28] pointed out that obese women consuming a restricted diet had apparent decrements in NK cell cytotoxicity. However, another experimental trial by concerning the influence of obesity on immune response in an adult population indicates that obesity is related to lower T and B-cell mitogen-induced lymphocyte proliferation but normal numbers and function of NK cells [23], while Lynch et al., have shown decreased NK cell levels only in metabolically unhealthy obese compared to a similarly obese but metabolically healthy group [19]. This study had also shown that the levels and phenotypes of NK cells in metabolically healthy obese were similar to lean healthy controls. This decrease in number of NK cells in the unhealthy obese suggests that the immune system is altered in obese people who are at risk from obesity-related comorbidities. In children, a number of studies demonstrated lower proportions of B cells in malnourished compared to well-nourished individuals (nutritional status assessed by weight) [29]. Similarly, a study on overweight children found elevated counts in most types of circulating immune cells, including CD19+ B Lymphocytes, suggesting the presence of low-grade systemic inflammation [30]. In contrast, some other researchers have reported no differences between the percentages of B lymphocytes in malnourished versus well-nourished subjects [31]. If obesity is considered an inflammatory disease [32] then one might expect that NK cell number, or activity, should be increased. In contrast, results on adults suggest that NK cell number and activity may not be changed by obesity [23]. Thus, our results (Table 6) showing no significant difference in NK cells between OB and NW young are in agreement with these previous findings. Taken together, all these studies confirm that malnutrition (whether obesity or underweight) has an effect on the circulating B and NK cells, particularly in the elderly.

In the present study (Figure 1 C D), BMI and % BF were inversely correlated with percent CD4+ cells (p = 0.0223; p = 0.021, respectively), while none of the nutrients (including energy intake) correlated significantly with either CD8+ or CD4+ cells. Although energy intake has been shown to have some effects on the number of circulating lymphocytes, results are disparate. For example, some studies have shown that low energy intake is associated with a reduction in lymphocyte number and proliferation [33], while others have shown no effect of energy on the number of lymphocytes [34, 35]. Similarly, except for plasma CRP, none of the plasma factors measured (albumin, total protein, triglycerides, and ferritin) had a significant relationship with either percent CD8+ or CD4+ cells, although a positive correlation between serum albumin and CD4 count and between triglycerides, CD4 and CD8 counts has been reported in a previous study with a group of dialysis patients [36]. The same study also reported non-significant correlations between CD4, CD8 and BMI, as shown in another recent study in Korean elderly [34]. Yet another study [37] demonstrated serum albumin concentrations correlated positively with some lymphocyte sub-populations.

It is evident from the results of the current study that a state of malnutrition that is nonetheless not overtly associated with diseases such as diabetes, arthritis, and cardiovascular diseases, may still markedly affect cells of the immune system of otherwise healthy young and elderly men. Additionally, future studies will need to measure the activity of immune system cells as well cell counts, because increases or decreases in cell numbers may not necessarily mean a higher or lower associated activity. Even with its limitations, a striking finding of this study was that despite the great differences between the rural Pakistani population tested here and the commonly-reported studies on “WEIRD” subjects in the literature, the overall differences in immune profiles between younger and older people were generally very similar.