Gender Dictates the Relationship between Serum Lipids and Leukocyte Counts in the National Health and Nutrition Examination Survey 1999–2004

Leukocytosis, or elevation of leukocyte counts, serves as a marker of immune activation and inflammation [1]. While commonly observed in infection [2], injury [3], hematological malignancies [4], and allergy [5], elevated peripheral blood leukocyte counts have additionally been associated with increased risk of cardiovascular disease (CVD) [6,7,8], type 2 diabetes mellitus [9], metabolic syndrome [10,11,12], and nonalcoholic fatty liver disease [13,14]. Altered leukocyte profiles not only contribute to metabolic dysfunction and inflammation, but may impair immune responses to acute infection and injury [15,16]. Thus, it is essential to understand the complexity of factors that impact leukocyte activation, expansion, and inflammation in order to develop effective strategies to mitigate metabolic and immune disorders [17].

Evidence from clinical trials and mechanistic studies suggests that there is a direct relationship between lipid metabolism and leukocyte profiles, and that lipid-lowering interventions may provide therapeutic potential to markers of leukocytosis and autoimmunity [18,19]. Lipoproteins can modulate the activity of circulating blood leukocytes through interactions with immunomodulatory proteins [20,21,22,23], as well as through mediation of cellular lipid efflux and/or loading via mechanisms that have been elucidated within the context of lipid-laden arterial macrophages in atherosclerosis. Greater cholesterol loading of leukocytes has been shown to enhance lipid raft formation, cellular proliferation, and inflammatory potential, whereas promotion of cholesterol depletion, either through HDL-mediated efflux or statin-induced suppression of endogenous cholesterol synthesis, has been shown to suppress proliferation and pro-inflammatory immune responses [24,25,26,27,28]. Thus, dyslipidemias may directly exasperate leukocytosis and altered leukocyte profiles associated with chronic metabolic disease and immune disorders [23,29,30,31].

Despite promising findings from clinical and mechanistic studies, along with population studies from China [32], Taiwan [33], and Japan [34], the relationship between serum cholesterol markers and leukocyte profiles has not been extensively evaluated in the general U.S. adult population, or in comparison between genders. Given that serum lipid profiles [35,36], CVD risk [37], autoimmune disease incidence [38], and immune responses to infectious pathogens and viral vaccines vary between men and women [39,40], research is warranted to determine whether the relationship between clinical lipid and immune biomarkers is gender-specific to help inform diagnostic and therapeutic strategies. Thus, our objective was to investigate the association between serum lipids and clinical leukocyte counts in men and women from the National Health and Nutrition Examination Survey (NHANES) 1999–2004. We hypothesized that blood lipid markers can be used to predict leukocyte profiles, and that variations in the lipid-immune relationship would be observed between men and women.

Clinical and mechanistic studies have demonstrated that lipid metabolism plays an important role in leukocyte activity, which may have significant gender-specific implications for CVD, metabolic dysfunction, and immune disorders [17,19,26,29,38,40,55]. In this study of 5647 men and women from NHANES 1999–2004, we have demonstrated that there are significant relationships between clinical lipid markers and leukocyte counts in the general U.S. population, and that clinical lipid markers differentially predict specific leukocyte subsets to varying degrees in men and women. Our findings suggest that hypertriglyceridemia may impact immune profiles in both men and women, whereas hypercholesterolemia may only affect lymphocyte profiles in women. Further, increases in HDL-C may differentially affect immune profiles in men and women, leading to variable immune outcomes. To our knowledge, our study is the first to evaluate the lipid–leukocyte relationship in NHANES.

Differences in both lipid and leukocyte profiles between men and women have previously been reported [56,57], suggesting that the gender-specific lipid-immune relationships we observed may be attributable to innate variability in lipid metabolism and immune cell behavior. Clinical guidelines for optimal HDL-C levels are distinct for men (>40 mg/dL) and women (>50 mg/dL) [35,36], and men often present with higher total cholesterol, LDL-C, and triglycerides as compared to premenopausal women, although these trends are equalized or reversed in post-menopausal women [56,58,59]. Gender-specific patterns in lipid profiles are in line with observed differences in CVD risk for men vs. women at different life stages, and some studies suggest that there are gender differences in the efficacy of statin therapy [37,60,61]. Women also have greater risk for autoimmune disorders, viral infection, and adverse responses to vaccines, while exhibiting enhanced humoral and cell-mediated responses as compared to men [38,39,40,62]. Variability in lipid and immune profiles between men and women has not been solely attributed to hormonal differences, but may be further impacted by differences in body fat distribution and cholesterol, triglyceride, and fatty acid metabolism [58,63,64,65]. Thus, the complex dynamics of lipid metabolism and immune function between genders may explain the variability in relationships between specific clinical biomarkers.

Lipoproteins can directly impact leukocyte profiles and activity through modulation of cellular cholesterol flux, lipid raft organization, sequestration of pathogenic stimuli, and provision of immunomodulatory proteins [19,22]. Cellular cholesterol loading—either through enhanced LDL uptake or deletion of HDL-associated lipid efflux transporters such as ABCA1 or ABCG1—has been shown to enhance the proliferative and inflammatory potential of lymphocytes and macrophages [24,26,66]. However, mechanisms of lipid metabolism and expression profiles differ in monocytes [67], and hypercholesterolemia has been shown to impair neutrophil function [68]. Similar trends were observed on our study, as increased total cholesterol and LDL-C levels were associated with greater predicted levels of lymphocytes in women. Conversely, greater LDL-C was associated with lower predicted monocyte and neutrophil levels in women, whereas no associations between total cholesterol, LDL-C, or any leukocyte subset were observed in men. Thus, hypercholesterolemia—particularly due to elevated LDL levels—may promote selective, and gender-specific expansion lymphocytes and altered, and potentially impaired, monocyte and neutrophil profiles. These observations may be due to gender-specific variability in lipid metabolism and immune responsiveness, as described above [56,57,64].

In contrast to cholesterol loading, HDL-mediated efflux has been shown to promote cholesterol depletion of leukocytes, leading to reduced formation of membrane lipid rafts, suppressed toll-like receptor 4-mediated inflammatory signaling, and a lower proliferative capacity of lymphocytes [24,25,26,69]. Further, lipoproteins—particularly HDL—can sequester bacterial pathogens such as lipopolysaccharide and lipoteichoic acid, thereby preventing leukocyte activation and improving clinical outcomes of sepsis [19,70,71,72,73,74]. HDL and apolipoprotein A 1 (apoA1) have additionally been shown to inhibit neutrophil activation, recruitment, and adhesion [75]. Our study revealed similar associations, in that a 10% increase in HDL-C predicted lower lymphocyte, neutrophil, eosinophil, and basophil counts in men. In contrast, after adjusting for potential confounding factors such as age, smoking, BMI, central adiposity (waist circumference), race/ethnicity, and statin use [7,44,45,46,47,48,49], HDL-C levels were only able to predict lymphocyte and monocyte counts in women. Regardless of gender, each lipid marker was inversely associated with HDL-C levels, suggesting that cholesterol- and immune-modulating properties of HDL universally suppress indices of leukocytosis in monocytes and lymphocyte subsets, and that HDL appears to have limited impacts on granulocyte populations in women. Interestingly, differences in HDL particle size profiles and cholesterol efflux capacity have been observed between genders, with women exhibiting a greater degree of larger HDL₂- and scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux, whereas men exhibit greater preβ HDL- and ATP-binding cassette transporter A1 (ABCA1)-mediated efflux [76]. Thus, the variable relationships between HDL-C and differential cell subsets may represent differences in HDL particle size profiles and leukocyte cholesterol efflux patterns between men and women.

In addition to observing unique relationships between serum cholesterol and immune markers between men and women, we found significant linear trends between triglycerides vs. lymphocytes, neutrophils, and basophils in both men and women, whereas positive linear trends between triglycerides vs. monocytes were only observed in women. Accordingly, a 10% increase in triglycerides was associated with higher predicted lymphocyte, neutrophil, and basophil counts in both men and women, as well as greater predicted monocyte counts in women. Elevated serum triglycerides are indicative of elevated hepatic lipogenesis, very low-density lipoprotein (VLDL) production, and reduced systemic lipolysis [77,78]. Hypertriglyceridemia is often associated with physiological conditions that promote immune activation, including metabolic dysfunction, lipid deposition in metabolic and lymphoid tissues, and direct activation of leukocyte populations by fatty acids [17,79,80,81]. Given the universal positive associations between serum triglycerides and leukocyte subsets, with little variability between men and women, our findings suggest that hypertriglyceridemia may promote leukocytosis in a relatively gender-independent manner.

Our study is one of a limited number of reports that has evaluated the direct, predictive relationship between lipid markers and leukocyte profiles in large-scale populations—particularly in the general U.S. population. In hypertensive male and female patients from the China Stroke Primary Prevention Trial cohort, Liu et al. [32] found that serum total cholesterol, LDL-C, and triglyceride levels were positively associated with total leukocyte, lymphocyte, and neutrophil counts, whereas HDL-C was inversely associated with leukocyte counts. In 3282 Taiwanese subjects participating in a standard hospital health screening program, hyperlipidemia was positively associated with total leukocyte counts, as well as each differential cell counts aside from eosinophils [33]. In evaluating immune associations by lipid category, hypertriglyceridemia had a stronger positive association with levels of each differential leukocyte subset. Conversely, hypercholesterolemia was positively associated with lymphocyte, neutrophil, and basophil counts, yet inversely associated with monocyte counts [33].

Together, these population studies support findings from our study and mechanistic trials, in that lipid profiles indicative of lipid loading promote selective leukocytosis, whereas HDL-mediated pathways are associated with suppressed leukocytosis [19,24,25,69]. However, the majority of previous studies have not elucidated gender-specific relationships between markers of immunity and serum lipids [32,33], which we found to be significantly variable. In line with our study, Oda et al. [34] observed greater associations between hypertriglyceridemia, hypo-HDL cholesterolemia, and hyper-LDL cholesterolemia vs. differential leukocyte counts in Japanese men participating in general health screenings, as compared to women. While we observed stronger overall lipid-leukocyte associations in women—with the exception of HDL-C—this report supports our conclusions that complex, gender-specific associations between serum lipid markers and leukocyte counts exist at the population level.

The predictive, diagnostic, and therapeutic significance of our findings warrants further investigation. Global and/or selective elevation of leukocyte populations may be indicative of CVD [6], metabolic dysfunction [9,10], infection [2], injury [3], or allergy [5]. While a limitation of our study was that we were unable to account for confounding immune-modulating factors (e.g., infection and allergy status, steroid usage), we clearly demonstrate that serum lipid levels impact and predict leukocyte profiles, suggesting that lipid-modulating therapies—such as diet, weight loss, or statins—may regulate leukocyte expansion and behavior [17,19,55,82,83,84]. Importantly, the efficacy of targeting serum lipids to promote immune health may be gender specific, calling for greater examination of immune outcomes and gender effects in clinical lipid trials.

Conceptualization, C.J.A.; methodology and formal analysis, T.M.V.; data interpretation and writing/review of the manuscript, C.J.A and T.M.V.

The authors declare no conflict of interest.