Aging is associated with deregulation and general decline in immune responses contributing to increased susceptibility to infection. Human–based data looking at the association between aging and immune response is particularly sparse, with majority of the studies published so far based on rodent models. Furthermore, limited data is available on the impact of aging and menopause on human cytokine/chemokine expression in different tissues. Herein, we showed an association between both age and menopause and cytokine/chemokine expression in plasma and cervical lavage samples from female sex workers in Nairobi, Kenya.
We observed a number of age related changes in plasma cytokine/chemokine expression. Systemic expression of MCP-1 and IP-10 positively correlated with women’s age. Consistent with our data, Seidler et al. showed that in healthy adults, serum concentration of MCP-1, but not MIP-1α, MIP-1β and fractalkine increased with age [
30]. MCP-1, a chemoattractant for monocytes and lymphocytes mainly produced by endothelial cells and macrophages, is involved in the inflammation process [
31] and is reported to be involved in Th2 polarization [
32]. Increasing concentrations of MCP-1 in older individuals could partly be responsible for development of arthrosclerosis and an age dependant shift in Th1/Th2 responses [
33]. IP-10 is an interferon-gamma inducible protein predominantly produced by monocytes and plays an important role in leukocyte trafficking by promoting the migration of NK cells and activated and memory T cells but not naïve T cells [
33,
34]. Interestingly, in elderly individuals the number of naïve T cells is low and it is conceivable that a higher concentration of IP-10 is required due to the higher number of memory T cells observed with age. Additionally IP-10 is a strong chemoattractant for Th1 lymphocytes and is considered to be a reliable marker of strong Th1- mediated autoimmune disease [
35]. Our data agrees with the previous observations that an age-dependant increases in both MCP-1 and IP-10 chemokines in healthy women may affect both Th1 and Th2 phenotypes and contribute to the overall impairment of the immune system.
When we separated women into age groups (<30, 30–50 and >50) the expression of MCP-1 and IP-10 remained significant. Both MCP-1 and IP-10 were elevated in older women (>50 years old), suggesting that the expression of these 2 chemokines is predominantly associated with the higher end of the age distribution. Additionally, when we looked at the cytokine/chemokine expression in different age groups, MIG and MIP-3β were significantly higher in women aged >50. Interestingly, MIG and IP-10 belong to the same family of chemokines, suggesting that the IFN-gamma inducible pathway is influenced by age. To our knowledge this is the first study to show association of systemic MIP-3β and age. MIP-3β is produced by DCs and possibly other lymphocytes and has the ability to chemoattract T cells, B cells, macrophages and NK cells. MIP-3β could be one of the contributing factors in age dependant deregulation of the innate and adaptive immune responses.
Additionally we observed a negative association between age and IL-8 and sCD40L systemic expression. IL-8 is produced by macrophages and plays an important role in neutrophil recruitment. The percentage of T cells producing IL-8 has been shown to decrease with age in the acutely ill Malawian patients [
36] and spontaneous production of IL-8 was found to be significantly lower in elderly male and females [
37]. Our data agrees with the previous observations, suggesting that age is an important factor in IL-8 production. This age-dependant decrease in IL-8 production could contribute to the altered neutrophil function including reduced chemotaxis observed in older individuals [
38]. Major sources of soluble CD40L are activated T lymphocytes and platelets. CD40L plays an important role in activation of antigen presenting cells (APCs) and regulation of B cell proliferation, adhesion and differentiation by interacting with its receptor, CD40. Significantly reduced CD40L expression by the aged CD4 T cells was observed in mice [
39]. Lower CD40L expression was observed on lymphocytes from older subjects as well [
40,
41]. Aging has been previously associated with defects in platelet function [
42] and this might contribute to decreased sCD40L production observed here. Consequently, this decrease in sCD40L could contribute to age related B cell deregulation [
22].
To the best of our knowledge we are the first to examine the effect of age on the immune environment in cervical lavage samples. Interestingly, in this study no direct correlation was observed between the mucosal cytokine/chemokine expression and age overall. However, when we divided the women in three different age groups, we observed that the expression of sIL-2Rα and IL-15 was significantly higher in women above 50 years of age compared to younger women (<30). An increase in IL-15 mRNA was shown to be associated with aging in mouse model [
9] and higher IL-15 serum levels were reported in ultralongeval individuals (>95) [
43]. A relative increase in sIL-2Ra release by aged cells despite lower surface expression was reported previously [
1]. IL-15 is known to play a role in T cell homeostasis and could contribute to the age associated decrease in naïve to memory T cell ratio [
3‐
5] as well as increase in Treg cell numbers [
5]. Additionally, increased IL-15 mRNA expression was observed in aging muscles in mice studies. This is thought to be an age-related adaptation of skeletal muscle to counter muscle atrophy through IL-15 mediated increase in myosin heavy chain protein content [
9]. Aging and the associated muscle loss of the FGT could potentially contribute to increase in IL-15 observed here.
Menopause is associated with important physiological and immunological changes. The decline in production of estrogen associated with ageing in women has been linked with important changes in cell population and cytokine expression [
11]. Keeping this idea in mind, we looked at the cytokine/chemokine expression in women depending on their menopause status.
In the systemic compartment, expression of MIG and MCP-1 were higher in post-menopausal women. MIG did not directly correlate with age however it was significantly higher in women above 50 years of age. Multivariate analysis indicates that MCP-1 is primarily affected by age, while MIG expression was primarily affected by the menopause status. It has been previously shown that MCP-1 expression is inhibited by estrogen therapy leading to decrease in macrophage recruitment [
13‐
15]. Further studies are needed to determine weather increase in MIG and MCP-1 expression could in part be explained by menopause-associated decrease in estrogen levels.
Interestingly, when we looked at the impact of menopause status on the cytokine/chemokine expression in CVL we observed a higher impact when compared to plasma. Indeed, at the mucosal environment, it seems that the menopause associated estrogen deprivation has a bigger impact than age itself. Menopause status was significantly associated with lower mucosal expression of MIG, MIP-3α, IL-1b, IL-6, IL-8, IP-10 and MCP-1. Previous data showed an association of menopause with a decrease in T cell and B cell populations [
11]. This decrease in the lymphocyte population may be cause, or effect of the altered mucosal cytokine/chemokine expression observed in our study. Our data seems to indicate that post-menopausal women might have a lower capacity to generate a strong, inflammatory mucosal immune response, which matches the findings from the GI mucosa [
18,
19]. Vaginal infections, especially yeast infections, are one of the main symptoms of menopause. Menopause is associated with physiological changes in the urogenital epithelium including thinning out, loss of elasticity and increase in vaginal pH [
21]. These menopause-associated physiological changes in the FGT tissue result in increased ability of pathogens to establish infection. In addition to this, decreased pro-inflammatory cytokine/chemokine expression observed in our study could potentially contribute to increased susceptibility to microbial invasion and infection observed post-menopause. To our knowledge this is the first study to examine the effect of age and menopause status on cytokine/chemokine expression in CVL.
Some study limitations must be addressed. In this study we are looking at a specific population composed only of women age range 20–65 involved in sex worker cohort in Nairobi, Kenya, which might account for some of the difference between our study and others. Even if sex work has less or no impact on the systemic cytokine/chemokine expression, it is known to have an impact on the mucosal cytokine/chemokine expression dynamics (Lajoie et al., submitted). It is also important to note that p-values were discussed as observed without correction for multiple comparisons to avoid false negative results. Further investigations looking at changes in cell populations both systemically and mucosaly, are needed in order to fully understand the impact of age and menopause- associated hormonal changes on the immune environment.