Discussion
The immune parameters (leukocyte concentration and leukocyte profile) measured on the marmots varied with their age. At the within-individual level, although the leukocyte concentration remains stable over the course of an individual life, the relative number of lymphocytes decreased, while the relative numbers of neutrophils and eosinophils increased with age. Lymphocytes play a central role in acquired immunity, being involved in immunoglobulin and memory cell production (Jain, 1993; Roitt et al., 2001). Neutrophils and eosinophils are both involved in the innate immune response, and more specifically in the inflammatory process (Jain, 1993). While neutrophils are the primary phagocytic leukocytes and eosinophils are often associated with defences against internal parasites (Jain, 1993).
The decrease in the number of lymphocytes with age is often interpreted as a consequence of the gradual decline over age in the generation, in the thymus, of new naïve T lymphocytes, responsible for generating new input in the immune memory. Such gradual decline is often suggested to lead to a decrease in the efficiency of the acquired immune system (Dowling & Hodgkin, 2009; Hakim & Gress, 2007; Shanley et al., 2009). Similarly, the observed increase in the relative number of neutrophils does not necessarily mean a higher performance of the innate immune system with age. Indeed, the phagocytic ability of neutrophils could decrease with age (Gomez et al., 2008) and an adaptive compensatory mechanism for such decline in neutrophil performance could lead to an increase in their relative number.
However, a decrease in lymphocytes, together with an increase in neutrophils (Cheynel et al., 2017; Kirk et al., 2010; and in Roast et al., 2022, even if not significant), and more broadly, a decrease in the acquired immune system combined with an increase (or upkeep) in the innate immune system, with age, has been observed in various vertebrate species (Franceschi et al., 2000a; Franceschi et al., 2000b; reviewed in Peters et al., 2019). Such modification of the acquired/innate immune balance with age (McDade et al., 2016), called immune remodelling , could be interpreted as an increased resource allocation strategy towards the production of cheaper immune components, at the expense of the more costly immune components (Klasing, 2004). Indeed, the acquired immune components are generally thought to be more expensive to maintain (Lee, 2006), and therefore, more prone to decline with age than the innate immune components (Peters et al., 2019). This could reflect that immune parameters are traded-off with other life history traits, and/or that terminal investment is unlikely to be turned to these parameters since their costs of production would outweigh their future benefit when age-related mortality becomes imminent. In such a scenario, given the lower probability to encounter new pathogens at old ages, downregulating the acquired immune system would not necessarily be the sign of any malfunction, but could be adaptive (Fulop et al., 2018). Consequently, immune systems should not be considered to undergo unidirectional deterioration with age (i.e.senescence) but would probably be better described by taking into account remodelling and reshaping of the immune functions with age (Fulop et al., 2018).
Apart from the hypotheses exposed above, age-related changes in the level of environmental stress endorsed by an individual could also affect the relative numbers of lymphocytes and neutrophils. Indeed, stress hormones such as glucorticoïds stimulate an influx of neutrophils from tissues into the blood; concomitantly, it causes a migration of lymphocytes from the blood circulation to other compartments (Dhabhar, 2002). Thus, a rise of plasma glucocorticoïds caused by stress increases the neutrophils to lymphocytes ratio over a time span of hours (Davis et al., 2008; Lopez-Olvera et al., 2007). Nevertheless, to date, no link has been clearly established between environmental stress, levels of glucocorticoids and patterns of age variations of lymphocytes and neutrophils (see for instance Roast et al., 2022; Watson et al., 2016).
We observed fewer lymphocytes for marmot males than for females. Various hypotheses such as sex-differences in resource allocation strategy, intra-sexual competition (Metcalf & Graham, 2018; Sheldon & Verhulst, 1996) or inhibition of the immune system by some steroid hormones were often suggested to induce differences between males and females (Gubbels Bupp et al., 2018; Klein & Flanagan, 2016; Taneja, 2018). However, we did not observe sex-specific differences in the variation of the immune phenotype with age. So far, studies of sex-specific variation on immune parameters with age remain equivocal: some suggested sex differences (e.g. Bichet et al., 2022; Gubbels Bupp et al., 2018; Tidière et al., 2020; van Lieshout et al., 2020), while others did not (e.g. Brooks & Garratt, 2017; Cheynel et al., 2017; Kelly et al., 2018; Peters et al., 2019).  For instance, van Lieshout et al. (2020) found a decrease in the proportion of lymphocytes with age in male badgers (Meles meles ), but not in females. The authors argued that this result could be explained by the high testosterone levels observed in male badgers, due to their polygynandrous mating system (Buesching et al., 2009), contrary to monogamous species (Sugianto et al., 2019) such as the Alpine marmot (Allainé, 2000; Cohas et al., 2006).
In our study, individuals with fewer lymphocytes but more neutrophils were more likely to die (Table 3), as also indicated by a significant selective disappearance of individuals with this phenotype (Table 2). Innate cellular response (involving neutrophils) can be costly in terms of energy, as well as autoimmune (Lee, 2006) and inflammatory damages (Franceschi et al., 2018; Goto, 2008). Individuals with neutrophil-oriented response may be unable to mount an appropriate immune response against challenges encountered at old ages (Froy et al., 2019), and/or may pay an excessive cost to this response and die (Pawelec, 2018). Studies investigating the potential links between age variation in immune phenotype and individual fitness are still scarce and show contrasting results (see also Froy et al., 2019). For instance, in the greater sac-winged Bat (Saccopteryx bilineata ), the number of leukocytes decreased with age, both within- and among-individuals, while the immunoglobulin G concentration was higher in older individuals, but did not vary within individuals, and the bacterial killing capacity of the plasma did not vary with age, at both levels (Schneeberger et al., 2014). These variations with age also impacted the short-term survival probability (Schneeberger et al., 2014). However, in a study on purple-crowned fairy-wrens (Malarus coronatus ), Roast et al. (2020) found no evidence that high levels of innate immune functions impaired short-term survival, nor any other fitness traits (annual reproduction and dominance acquisition). In our marmot population, more investigations on the link between fitness and immune variations with age are needed to better understand the evolutionary consequences of the within-individual age variations and the selective disappearance we observed.
More generally, to understand the complexity of age-related changes in immune functions, as well as their evolutionary causes and consequences, we must not only supplement the existing longitudinal studies focused on age-related pattern of immune parameters (to the best of our knowledge, seven studies: Beirne et al., 2016; Bichet et al., 2022; Froy et al., 2019; Graham et al., 2010; Roast et al., 2022; Schneeberger et al., 2014; Vermeulen et al., 2017), but also relate the observed patterns to individual fitness. Such studies are crucial to disentangle whether remodelling of the immune system with age could or could not be adaptive.