The main findings of this study demonstrate that only the NL-ratio has a direct association with aging, which is demonstrated by a the statistically significant interaction between the birth-cohort effect and time effect. A gender dimorphism was found for the neutrophil absolute count and NL-ratio; for the same birth year, females had a lower number of neutrophils and a lower NL-ratio, compared to males.
Lymphocytes absolute number decreases inversely to birth-cohort, independently from sex. Sex modulates these two blood markers when categorized according to CHF and Creatinine Clearance. An inverse correlation between the birth-cohort and both neutrophils count, and NL-ratio was present only in females who developed CHF. In this same female group, higher values of all markers were measured in younger subjects, having similar CC values. Body mass index modulates the association between the blood markers and age differently according to sex, inversely in males and directly in females.
Aging
Circulating blood cells such as leukocytes, lymphocytes, and neutrophils, are widely utilized as markers of aging-related systemic inflammation. One of the most economical and widely available clinical markers of peripheral inflammation is the NL-ratio. Variation in the NL-ratio has previously been reported to be predictive of poorer prognosis [
17], longer stay after hospitalization for major illnesses, frailty [
18] and disability. Interestingly NL-ratio was also reported to be cross-sectionally associated with Alzheimer’s Dementia (AD) [
19]; but in the Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL cohort), longitudinal analysis of NL-ratio variation across time was limited for the problem of diagnosing AD, and unable to predict the transition from mild cognitive impairment to AD [
20]. Such correlations were weak, and disappeared when age and sex were considered, indicating that those covariates, rather than the underlying disease process, drove the changes observed in the ratio [
20]. In the Rotterdam study the NL-ratio levels were associated with an increased risk of all-cause mortality, independently from age; however the authors did not assess a potential association between age and NL-ratio [
16]. In the InCHIANTI study, the NL-ratio showed a departure from a linear trend, but maintained a direct correlation with age, and, more interestingly, with the interaction between birth-cohort and times of the study (aging effect). This would possibly suggest that the NL-ratio might be a worthwhile and stable marker of healthy aging.
Both the innate and the adaptive immune systems are dynamically remodeled with aging, in a process which is known as immunosenescence, characterized by great heterogeneity [
21]. Adaptive immunity changes can be tracked to a reduction of lymphocytes, mainly due to a decreased thymic output [
22], and with an imbalanced CD4:CD8 ratio, a decrease in the number of CD4 cells and a contextual increase in the number of CD8 cells [
23].
Peripheral T lymphocytes encompass a heterogeneous mix of cellular types, namely naive, effector, and memory cells. They have different lifespan and continuously recirculate through the lymphatic system. Investigations into the dynamics of T cell turnover have demonstrated that most peripheral T cells can remain in a quiescent state for prolonged periods, spanning months in rodents and even years in humans. Notably, typical naive T cells are known for their remarkable longevity as they persist as quiescent cells [
24]. Conversely, effector and memory T cells exhibit a more rapid turnover, indicating their active involvement in immune responses [
25]. Senescent-like features in highly differentiate T cells are characterized by a reduced proliferation rate, shorter telomere length and increased levels of p38 [
26]. These senescent-like T cells express NK cell receptors and high levels of cytotoxic molecules, that might be responsible for tissue damage [
27]. Also B cells show senescence signature mainly characterized by a pro-inflammatory senescence associated secretory phenotype [
28]. An expanded subset of B cells that accumulate with age (age-associated B cells) [
29] plays a significant role in various aspects of immunosenescence [
30] and they are found at higher levels in autoimmune and autoinflammatory diseases [
29]. The numbers of B lymphocytes are greatly reduced in aged individuals [
10] but their function is important for the aging process.
Neutrophils are part of the innate immune system and represent the main effector cells against bacterial infections. Neutrophils also play a critical role in disease control, as in cancer [
31]. The absolute number of neutrophils does not change significantly in immunosenescence [
9]; they show a short life [
32] (less than one day), coupled with a high production rate in the bone marrow (5–10×10
10 neutrophils-day) [
33]. During aging neutrophils show a decline in phagocytic functions that lead to an imbalance of tissue homeostasis [
34]. Their senescence features include shortened telomere length, reduced proliferation, and a pro-inflammatory senescence associate secretory phenotype [
34,
35].
In our study, the absolute number of circulating lymphocytes decreased with age independently from sex, whereas neutrophil count increased. This accounts for the declining NL ratio, however the reasons for the opposite trend are uncertain.
These results could be explained considering that immune system function undergoes a profound remodeling with age, even if it was extremely interindividual heterogeneous (immunobiography) [
36]. Elderly subjects show an increased risk for infective and degenerative disease manifestations, and that is thought to be a phenomenon associated with a less efficient adaptive immune response [
4]. On the contrary, some features of innate immunity seem to be preserved in immunosenescence; for example, inflammation is not dampened with age [
37]. The aging-related exhaustion of the adaptive immune system recognized large and complex interactions of factors as genomic instability (shortening of telomeres) [
38], epigenetic regulation (DNA methylation, histone modification, and noncoding RNAs) [
39], damage to mitochondrial function (reducing energy availability), as well as hormones imbalance, multimorbidity and environmental causes[
40,
41]. The prospective character of the study, with 20 years of follow-up (from 1998 to 2018), has enabled us to better capture this trend in the aging population.
Gender
Sex differences in the immune responses have been extensively demonstrated [
40,
42]. Generally, females have a more efficient innate and adaptive immune response than males, and this apparent beneficial effect, is counterbalanced by female increased susceptibility to inflammatory and autoimmune diseases [
43]. Reduced immune function in men might represent an example of pleiotropic antagonism, i.e. a side effect of positive selection for other traits, such as reproductive success or enhanced metabolism [
44].
Lymphocyte absolute numbers did not differ between sexes, but a gender dimorphism was found in neutrophils count, with females showing higher numbers and NL-ratio, compared to males. With aging this sex-biased gap tended to be tapering, as demonstrated by the significant inverse interaction between age and sex [
45]. These differences might plausibly be due to age-related changes in hormonal levels and immunological remodeling. For example, after a single i.m. injection of 17beta-oestradiol in male subjects, the number of neutrophils in blood doubled, although no significant changes in adhesion molecules were measured [
46]. Androgens suppress the pro-inflammatory responses via inhibition of leukotriene formation in neutrophils [
47]; progesterone enhance prostaglandin production of activated macrophages in female murine models inhibiting nitric-oxide production [
48], whereas in a male murine model, estrogen treatment improves cellular immunity through NF-kB activation and reduced IL-6 production [
49].
An alternative hypothesis explaining the gender differences considers the sexually dimorphic gene expression. The X chromosome is more complex than the Y chromosome, and contains many genes involved in immune function [
50], which are thought to be partly responsible for the hyperresponsiveness of the female immune system; for example, males experience more frequent severe infections, on the contrary females are more prone to autoimmune diseases [
51]. Those differences are attributable to X chromosome inactivation (XCI) during embryogenesis, female-specific mechanism to equalize gene expression between the sexes [
45], and the genes escape silencing, a possible mechanism that would account for the overexpression of X-linked immune genes [
52]. Approximately a quarter of X-linked genes are estimated to constitutively escape from XCI in humans and may contribute to the female autoimmune predisposition [
52], with higher serum IgM, higher number of B cells, and higher percentage of CD4 + T-helper cells in females compared to males [
52].
Diseases
Myocardial infarction is the leading cause of the development of CHF. Neutrophils are the first cells recruited to the site of myocardial injury, and they are of paramount importance in the clearance of necrotic tissue, for the local resolution of inflammation, and in tissue homeostasis [
53]. To explain the reason for the increase in the neutrophil count and the NL-ratio only in female subjects with CHF, we must consider reasons beyond the immunological gender dimorphism. Sex differences are reported in heart tissue from the early embryogenesis, and they are epigenetically perpetuated. Those differences may have sex-specific repercussions during organogenesis, persisting through adulthood and producing a different response of tissues to injury [
54].
Chronic kidney disease (CKD) progression has been associated with chronic inflammation and specifically with higher NL-ratio [
55]. A recent meta-analysis confirmed the NL-ratio predictive value for death in CKD patients, for all causes and for specific cardiovascular mortality [
55], suggesting also its use for monitoring inflammation, and renal insufficiency progression [
56]. In the InCHIANTI data, creatinine clearance was directly associated with neutrophil count and NL-ratio, although in female subjects only. Younger subjects with low creatinine clearance showed higher levels of the two blood markers, and this figure is in agreement with previous findings in CKD [
57].
Body composition
Lastly, in our study an increase in BMI across the follow-up times accompanies an increase of neutrophils count and NL-ratio in females, whereas in males for an increase in BMI a decrease in both parameters was found. Differences in body composition between sexes are well documented in aging; usually, women have a higher fat mass, and men have a higher muscle mass [
58], and excessive fat deposition can cause a proinflammatory state due to adipose tissue secretion of cytokines, and disturbances in metabolic homeostasis [
28]. Therefore, also sexual dimorphism in lipid metabolism must be considered and could explain the link among adipose tissue, aging, and chronic inflammation. Alternatively, starvation, malnutrition, as well as single nutrient deficiencies, may modify the balance between adaptive and innate immunity, probably as a consequence of bone marrow failure [
59]. Moreover, intermitting fasting and/or caloric restriction response is gender-specific [
60]. Even if, in females mice caloric restriction increases adipocytes infiltration, at least in the bone marrow [
61] and in the liver [
62], compared to males; those observations highlighted how different energy homeostasis is governed by sex hormones, and modulates chronic low grade inflammation.