Gabriele Sorci
email: gabriele.sorci@u-bourgogne.fr

Clothilde Léchenault-Bergerot

Bruno Faivre

Age reduces resistance and tolerance in malaria-infected mice

Keywords: anemia, immunity, Plasmodium yoelii, senescence, virulence cells, PBS = phosphate buffered saline, MCV = mean corpuscular volume

Introduction

Infection with parasites is usually associated with reduced host fitness. In response to this threat, hosts have evolved different strategies aiming at i) avoiding the encounter with infectious agents (i.e. avoidance), ii) limiting parasite proliferation (i.e., resistance), iii) reducing the damage induced by the infection (i.e., tolerance) [START_REF] Klemme | Vertebrate defense against parasites: Interactions between avoidance, resistance, and tolerance[END_REF].

Since resistance is defined as the host capacity to limit parasite multiplication, it usually involves the deployment of an immune response that can, ultimately, result in the clearance of the infection. On the contrary, tolerance aims at minimizing the damage, and therefore, the fitness cost induced by the infection [START_REF] Schneider | Two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases[END_REF][START_REF] Råberg | Decomposing health: tolerance and resistance to parasites in animals[END_REF][START_REF] Medzhitov | Disease tolerance as a defense strategy[END_REF]). As such, mechanisms of tolerance are manifold, possibly including the regulation of the inflammatory response [START_REF] Ayres | Tolerance of infections[END_REF][START_REF] Sonnenberg | Innate lymphoid cells in the initiation, regulation and resolution of inflammation[END_REF], the capacity to detoxify toxins produced during the infection [START_REF] Seixas | Heme oxygenase-1 affords protection against noncerebral forms of severe malaria[END_REF][START_REF] Larsen | A central role for free heme in the pathogenesis of severe sepsis[END_REF], the capacity to repair and/or regenerate tissues that have been damaged during the infection [START_REF] Chang | Modulation of the course and outcome of blood-stage malaria by erythropoietin-induced reticulocytosis[END_REF][START_REF] Soares | Tissue damage control in disease tolerance[END_REF], glucose and triglyceride metabolism [START_REF] Wang | Glucose metabolism mediates disease tolerance in cerebral malaria[END_REF][START_REF] Luan | GDF15 is an inflammation-induced central mediator of tissue tolerance[END_REF][START_REF] Wang | Opposing effects of fasting metabolism on tissue tolerance in bacterial and viral inflammation[END_REF], the microbiome (Palaferri [START_REF] Schieber | Disease tolerance mediated by microbiome E. coli involves inflammasome and IGF-1 signaling[END_REF], hormones [START_REF] Vandermosten | Adrenal hormones mediate disease tolerance in malaria[END_REF], or thermoregulation (Palaferri Schieber and Ayres 2016). While resistance has been the focus of much attention during the last decades, the study of tolerance in animal hosts has been boosted since the seminal paper by [START_REF] Råberg | Disentangling genetic variation for resistance and tolerance to infectious diseases in animals[END_REF], and it is now established that tolerance is widespread across different host-parasite systems [START_REF] Adelman | House finch populations differ in early inflammatory signaling and pathogen tolerance at the peak of Mycoplasma gallisepticum infection[END_REF][START_REF] Sternberg | Patterns of host-parasite adaptation in three populations of monarch butterflies infected with a naturally occurring protozoan disease: virulence, resistance, and tolerance[END_REF][START_REF] Sorci | Immunity, resistance and tolerance in bird-parasite interactions[END_REF][START_REF] Hayward | Natural selection on individual variation in tolerance of gastrointestinal nematode infection[END_REF][START_REF] Howick | Genotype and diet shape resistance and tolerance across distinct phases of bacterial infection[END_REF]Mazé-Guilmo et al. 2014;[START_REF] Athanasiadou | Tolerance and resistance to a nematode challenge are not always mutually exclusive[END_REF][START_REF] Kutzer | Maximising fitness in the face of parasites: a review of host tolerance[END_REF][START_REF] Decker | Elevated atmospheric concentration of carbon dioxide reduce monarch tolerance and increase parasite virulence by altering the medicinal properties of milkweeds[END_REF][START_REF] Grab | Host tolerance and resistance to parasitic nest flies differs between two wild bird species[END_REF][START_REF] Stehr | Resistance and tolerance to mixed nematode infections in chicken genotypes with extremely different growth rates[END_REF].

Age is a particularly relevant host trait that likely drives the capacity to resist or tolerate the infection. Aging is associated with profound changes in several physiological functions, including the immune system (i.e., immunosenescence) [START_REF] Albright | Aging, Immunity, and Infection[END_REF].

Immunosenescence is characterized by a gradual depletion of naïve T-cells, reduced antibody production, and a low-grade activation of the inflammatory response [START_REF] Desai | Leukocyte function in the aging immune system[END_REF][START_REF] Shaw | Age-dependent dysregulation of innate immunity[END_REF]. Immunosenescence is not restricted to humans and animal models, as age-related variation in immunity has also been reported for free-ranging species (e.g., [START_REF] Nussey | Age-related variation in immunity in a wild mammal population[END_REF][START_REF] Froy | Senescence in immunity against helminth parasites predicts adult mortality in a wild mammal[END_REF]. Due to altered immune functioning, senescence exposes hosts to increased susceptibility to infectious diseases [START_REF] Miller | Effect of age on outcome and epidemiology of infectious diseases[END_REF][START_REF] High | Infection as a cause of age-related morbidity and mortality[END_REF][START_REF] Nikolich-Zugich | The twilight of immunity: emerging concepts in aging of the immune system[END_REF], and reduced resistance to infection is one of the many declines in function that characterize the senescent phenotype (Giefing-Kröll et al. 2015, Glynn and[START_REF] Glynn | Systematic analysis of infectious disease outcomes by age shows lowest severity in school-age children[END_REF]. However, while there is evidence that age is associated with reduced resistance, we still have a very partial view of whether and how age affects tolerance. To the best of our knowledge, only a handful of studies have investigated whether tolerance to infection changes with age. Using a cohort of patients infected with HIV, [START_REF] Regoes | Disentangling human tolerance and resistance against HIV[END_REF] showed that age at infection was positively associated with disease progression, independently from viral load, suggesting a decreased tolerance with age. Similarly, fruit flies (Drosophila melanogaster) exposed to bacterial infections suffered from increased mortality when infected at old age, although the capacity to limit bacterial proliferation was not affected by age [START_REF] Ramsden | Functional analysis of the Drosophila immune response during aging[END_REF]. Again, this finding indirectly suggests that old flies were less tolerant to infection while maintaining similar resistance to young hosts. A positive effect of age on tolerance has also been reported. [START_REF] Jackson | An immunological marker of tolerance to infection in wild rodents[END_REF] found that tolerance to macroparasites improved in a natural population of voles (Microtus agrestis) between immature and mature individuals, through an increased expression of the transcription factor Gata3 (mediating the Th2 immune response).

Here, we aimed at investigating how resistance and tolerance change with age in house mice (Mus musculus domesticus) infected with the rodent malaria Plasmodium yoelii. Previous work conducted on models of rodent malaria has shown that resistance improves during the first months of life, which also mirrors the pattern found in malaria-infected humans [START_REF] Shan | Age-related susceptibility to nonlethal Plasmodium yoelii infection in C57BL/6 mice[END_REF][START_REF] Pinkevych | Decreased growth rate of P. falciparum blood stage parasitemia with age in a holoendemic population[END_REF]). However, whether individuals at the age when physiological functions start to decline have altered resistance and tolerance to malaria has not been investigated, yet. To fill this gap, we infected 2-and 12-month old house mice with a non-lethal strain of the rodent malaria Plasmodium yoelii, and we compared resistance and tolerance to infection between the two age classes. We decided to focus on 12-month old mice (not older ones), because very old individuals suffer from a general reduction in vigor [a condition called frailty, [START_REF] Clegg | Frailty in elderly people[END_REF]], and therefore reduced resistance or tolerance in very old individuals might reflect a general impairment of organismal functions rather than specific mechanisms related to malaria pathogenesis. Twelve-month old mice are considered to be at the onset of senescence of several (albeit not all) physiological functions. For instance, the expression of tissue-specific senescence markers, defining the senescence-associated secretory phenotype, is up-regulated from the age of 12 months onwards [START_REF] Hudgins | Age-and tissuespecific expression of senescence biomarkers in mice[END_REF]).

We measured resistance as the inverse of parasitemia (% infected red blood cells) during the course of the acute infection (the higher the parasitemia, the lower the resistance). To assess tolerance, we used a reaction norm approach [START_REF] Simms | Defining tolerance as a norm of reaction[END_REF], where the negative slope of the regression of health on parasitemia provides the estimate of the magnitude of the damage suffered per unit of increase in parasitemia (the steeper the slope, the lower the tolerance). This allowed us to assess i) the change in resistance and tolerance as a function of mouse age; ii) the pattern of phenotypic covariation between resistance and tolerance both between and within age classes.

Material and Methods

Animals and infections

Female C57BL/6 mice were housed in plastic cages (18.5 x 38 x 22.5 cm -enriched with shelters), maintained under a constant temperature (24°C) and a photoperiod of 12:12 (L:D), and given ad libitum filtered water and food (standard mouse pellets). At day 0, 2-month old (n = 20) and 12-month old (n = 20) mice received (by intraperitoneal injection) 100 µl of citrate saline diluted blood containing 10 5 red blood cells infected with Plasmodium yoelii 17XNL.

Parasitemia and hematology

Parasitemia was assessed as the percent of infected red blood cells (iRBCs). To this purpose, we collected a drop of blood from the tip of the tail every 2 or 3 days, from day 3 to day 33 post-infection (p.i.) (i.e., during the acute phase of the infection) to make blood smears.

Smears were fixed in methanol (3 min), stained with GIEMSA (20% during 45 min) and the number of iRBCs (out of 1,000 RBCs) counted under optical microscopy (at 1,000 magnification).

Starting from day 0 and up to day 33 p.i., we also collected every 2 or 3 days, two µl of blood from the tail tip and diluted them in 18 µl of Phosphate Buffered Saline (PBS). Within one hour of collection, these samples were used to count RBCs, lymphocytes and granulocytes.

We also assessed mean corpuscular volume (MCV) which refers to the mean size of red blood cells, as a proxy of reticulocyte production. RBC, lymphocyte, and granulocyte counts, and measurement of MCV were done using a SCIL Vet abc Plus+ hematology analyzer.

Body mass and mortality

Body mass (± 0.1 g) was measured daily (or every two days) from day 0 to day 33 p.i.

Mice were also checked daily for their health status. Animals with rapidly deteriorating health condition were humanely euthanized (three mice in the 12-month old group). Since 2-month old mice are still in a stage where they gain mass, we also monitored body mass of uninfected mice (2-month old, n = 10; 12-month old, n = 10), as to infer whether any possible agedependent variation in body mass was specifically due to the infection.

Statistical analyses

Parasitemia, counts of RBCs, lymphocytes, granulocytes, and values of MCV were all ln-transformed to normalize the distribution of model errors and to linearize the relationships among traits.

The temporal dynamics of traits over the course of the acute infection was modelled using mixed effects models. To take into account the non-linear relationship between traits and time p.i., we used a third-degree polynomial (time + time 2 + time 3 ). All models also included age and the interactions between age and time (including squared and cubic terms) as fixed effects and a random intercept for each individual (i.e., mouse identity was included as a random effect).

Initial differences (at day 0) between age classes in counts of RBCs, lymphocytes, granulocytes, MCV, and body mass were assessed using one-way ANOVAs.

Differences in survival between age classes were assessed using a log-rank test.

To investigate the relationship between RBC counts and parasitemia, we ran a mixed effects model where RBC count was the dependent variable and parasitemia and age class (plus the interaction) were included as fixed effects. This model also included random intercepts and slopes for each individual. The same model structure was adopted to explore the relationship between variation in body mass and parasitemia.

All models were run in SAS 9.4.

Results

Parasitemia

The dynamics of parasitemia differed between age classes, as shown by the statistically significant age x time p.i. interactions (table 1). Percent of iRBCs peaked earlier and at lower level in 2-compared to 12-month old mice (fig. 1A, table S1 in Supplementary Material). These results, therefore, show that 12-month old mice were less resistant to P. yoelii infection than 2month old mice.

Immune response

At day 0, 2-month old mice had similar numbers of circulating lymphocytes and lower numbers of granulocytes compared to 12-month old mice (table S2 RBC loss produces a physiological feedback to restore RBC number by erythropoiesis.

Reticulocytes (young RBCs) are larger than mature RBCs, and therefore it is possible to estimate the dynamics of RBC renewal by assessing the changes in RBC size [mean corpuscular volume (MCV)] over time. At day 0, 2-month old mice had higher MCV compared to 12-month old mice (one-way ANOVA, F1,38 = 245.96, p < 0.0001). However, MCV increased faster during the early phase of the infection for 2-compared to 12-month old mice, while during the late phase of the infection 12-month old mice had higher MCV than 2-month old mice (fig. 1C), resulting in statistically significant age x time interactions (table 2).

Body mass

Twelve-month old mice had initially much higher body mass values (table S6 andfig. S2 in Supplementary Material). To get rid of this initial difference, we computed the percent variation of initial body mass over the course of the study. Mice in the two age classes had very different trajectories of body mass variation during the infection. Two-month old mice gained, on average, up to 17% of the initial body mass, whereas 12-month old mice lost around 11% of their initial body mass at day 21 p.i., and subsequently tended to recover during the late phase of the infection (table S7 in Supplementary Material, fig. 1D). Including initial body mass as a covariate did not change the results of this model (table S8 in Supplementary Material).

Given that 2-month old mice are still gaining body mass (fig. S2 in Supplementary Material), we also expressed changes in body mass as variation with respect to non-infected controls. To this purpose, we computed the difference between body mass at day i and the mean body mass of non-infected mice at day i, and divided it by the body mass at day 0. Therefore, this variable describes the deviation in body mass of infected mice with respect to non-infected controls at each sampling time and for both age classes. Using this variable provided similar results to the previous model (table S9, fig. S3 in Supplementary Material), indicating that the difference in body mass trajectories between 2-and 12-old mice was actually due to a more severe effect of the infection in old mice.

Mortality

None of the 2-month old mice died during the course of the study vs. 20% (4/20) of 12-month old mice (one dead, three euthanized) (log-rank test, χ² = 4.33, p = 0.0374, fig. S3 in Supplementary Material).

Relationship between health status and parasitemia

We investigated how age affected changes in RBC counts per unit of change in parasitemia. The results revealed that the negative slopes relating RBC counts to parasitemia were significantly steeper in 12-compared to 2-month old mice, showing that 12-month old mice were less tolerant to the infection (table 3, fig. 2A).

Changes in body mass during the course of the infection can also be used as a general indicator of host health. We therefore ran the same mixed-effect model that included percent variation of initial body mass as the dependent variable. This model showed a difference in the relationship between changes in body mass and parasitemia (table 3). Twelve-month old mice showed a negative relationship between body mass and parasitemia, suggesting that mice with high parasitemia suffered from increasingly poor health (fig. 2B). On the contrary, the slope relating changes in body mass and parasitemia was flat in 2-month old mice (fig. 2B).

Relationship between resistance and tolerance

Over the whole sample, there was a negative correlation between maximum parasitemia (a measure of inverse resistance) and the slope of RBC counts on parasitemia (a measure of tolerance) (Pearson's r = -0.586, p = 0.0002, n = 36, fig. 3A). This finding suggests that mice that were tolerant to the infection were also the most resistant ones. However, this pattern was exclusively driven by an age effect. An ANCOVA model showed, indeed, that there was no correlation between tolerance and resistance within age classes (fig. 3B, table S10 in Supplementary Material).

Discussion

The aim of this study was to investigate whether resistance and tolerance to infection change with age in Plasmodium-infected mice, and to investigate the pattern of covariation between resistance and tolerance, both within and between age classes. We found that 12-month old mice harbored more parasites and suffered higher damage per unit of parasitemia compared to 2-month old mice. We also found that the two traits positively covaried, but only across ages.

These results show that age induces a reduction of both resistance and tolerance to infection with P. yoelii, while we did not find evidence for a trade-off between resistance and tolerance.

Age makes hosts more susceptible to infectious diseases and more likely to suffer from severe morbidities [START_REF] Glynn | Systematic analysis of infectious disease outcomes by age shows lowest severity in school-age children[END_REF]. However, increased susceptibility and/or morbidity might arise because of age-associated impairment of defense mechanisms aiming at controlling parasite proliferation (i.e., impaired resistance), age-associated impairment of mechanisms restoring homeostatic conditions during an infection (i.e., impaired tolerance), or both [START_REF] Ayres | The biology of physiological health[END_REF]. Previous work on the effect of age on infectious diseases has assumed that increased susceptibility in the elderly arises from impaired immune functioning, focusing on mechanisms of parasite control [START_REF] High | Infection as a cause of age-related morbidity and mortality[END_REF]). Only a handful of studies have explicitly addressed the possible role plaid by the impairment of mechanisms involved in the maintenance of organismal homeostasis as a cause of infection-induced damage in aged hosts (e.g., [START_REF] Regoes | Disentangling human tolerance and resistance against HIV[END_REF]).

Yet, understanding if aging impairs resistance, tolerance or both is key to have treatments that effectively promote health in the elderly [START_REF] Vale | Limiting damage during infection: lessons from infection tolerance for novel therapeutics[END_REF].

Although resistance and tolerance are fundamentally different host defense strategies, it has been suggested that they might be related through physiological and/or genetic trade-offs, finally producing an intertwined evolution of resistance and tolerance [START_REF] Fineblum | Tradeoff between resistance and tolerance to herbivory damage in a morning glory[END_REF][START_REF] Miller | The evolution of host resistance: tolerance and control as distinct strategies[END_REF][START_REF] Lefèvre | Genetic variation in resistance, but not tolerance, to a protozoan parasite in the monarch butterfly[END_REF]Mazé-Guilmo et al. 2014). Evidence for a genetic trade-off between resistance and tolerance has been provided for malaria-infected mice, with strains harboring the smallest number of parasite, also having the steepest slope between red blood cell count and parasitemia [START_REF] Råberg | Disentangling genetic variation for resistance and tolerance to infectious diseases in animals[END_REF]). Here, we could not assess the genetic correlation between resistance and tolerance because we only studied one mouse strain; however, we could assess whether the two traits were phenotypically correlated, possibly indicating a physiological trade-off. Physiological trade-offs might arise if investment into one trait diverts resources from competing traits. For instance, if investment into immune defenses diverts resources from mechanisms of tissue repair, this might produce a phenotypic trade-off between resistance and tolerance. Contrary to the hypothesis of a physiological trade-off between resistance and tolerance, we found that the two traits were positively correlated.

However, this pattern was exclusively due to the heterogeneity in the age structure of our sample of mice, since 2-month old mice were both more resistant and tolerant compared to 12month old mice. Looking at the correlation between resistance and tolerance within age classes provided no evidence for a phenotypic correlation between the two traits. This finding highlights the importance of taking into account the potential heterogeneity among individuals that, if ignored, might provide a biased view of the covariation pattern between resistance and tolerance. In agreement with this, [START_REF] Restif | Concurrent evolution of resistance and tolerance to pathogens[END_REF] modelled the evolution of resistance and tolerance under the specific assumption that the two traits were unrelated. Nevertheless, their analysis showed that across environments, resistance and tolerance can vary in similar or opposite directions [START_REF] Restif | Concurrent evolution of resistance and tolerance to pathogens[END_REF]. Field studies of the relationship between resistance and tolerance might, therefore, provide flawed results if potential confounding factors, such as host age or nutritional status, are not controlled for.

The lack of phenotypic correlation between resistance and tolerance within age classes suggests that i) the two strategies are based on different mechanisms; and ii) under the benign conditions experienced in the lab, the energetic requirements associated with the deployment of these mechanisms are not high enough to generate a physiological trade-off between resistance and tolerance.

There are numerous physiological mechanisms that might account for the observed ageassociated decline in resistance and tolerance, including impaired immunity, a highly regulated immune response (preventing infection-induced immunopathology), reduced regenerative capacity (impaired hematopoiesis), impaired detoxification of toxins produced during the infection, or impaired metabolic functions. Identifying all the possible mechanisms accounting for age-associated changes in resistance and tolerance was well beyond the scope of the current study. Nevertheless, we suggest that age-dependent changes in immunity and hematopoiesis might play a role to explain the observed results. Asexual multiplication of Plasmodium parasites causes the lysis of infected RBCs and hosts harboring high parasitemia might suffer from more severe anemia because of the direct destruction of RBCs during parasite multiplication. However, it is now well established that this direct mechanism of RBCs destruction only contributes to a relatively minor fraction of the total RBC loss, whereas the majority is due to the action of the immune system, through the removal of uninfected RBCs [START_REF] Evans | Severe malarial anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes[END_REF][START_REF] Fonseca | Quantifying the removal of red blood cells in Macaca mulatta during a Plasmodium coatneyi infection[END_REF]. We found that granulocytes had different dynamics during the course of the infection between age classes, with 12-month old mice having higher numbers. Granulocytes mostly refer to neutrophils that have phagocytic properties and contribute to the oxidative burst. It is, therefore, possible that an exacerbated innate response, mediated by neutrophils, might account for the accentuated loss of RBCs, as shown in other systems [START_REF] Evans | Severe malarial anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes[END_REF]). However, functional properties (i.e., phagocytosis) of neutrophils have also been shown to be compromised in senescing individuals [START_REF] Butcher | Ageing and the neutrophil: no appetite for killing?[END_REF]. Therefore, the causal involvement of neutrophils in the exacerbated anemic status of 12-month old P. yoelii-infected mice requires further investigation.

Anemia induces a physiological feedback on erythropoiesis, stimulating the renewal of RBCs that have been lost during the infection. Therefore, the capacity to rapidly and effectively restore a homeostatic RBC number should finally contribute to tolerance to malaria [START_REF] Chang | Modulation of the course and outcome of blood-stage malaria by erythropoietin-induced reticulocytosis[END_REF]. Age is often associated with a compromised regenerative capacity and impaired erythropoiesis is involved in the anemia of senescing humans and rodents [START_REF] Price | Aging and erythropoiesis: current state of knowledge[END_REF][START_REF] Stauder | Anemia at older age: etiologies, clinical implications, and management[END_REF]. Moreover, during a malaria infection, the interaction between several immune effectors and parasite-derived toxins might compromise the erythropoietic process [START_REF] Vigario | Inhibition of Plasmodium yoelii blood-stage malaria by interferon-α through the inhibition of the production of its target cell, the reticulocyte[END_REF], and age might further accentuate such impaired erythropoiesis. We assessed the agedependent capacity to renew RBCs by measuring mean corpuscular volume. Young RBCs (reticulocytes) have bigger size and therefore changes in MCV indicate a change in the age distribution of RBCs. As expected, malaria infection produced an increase in MCV in mice of both age classes; however, the dynamics of MCV was different between 2-and 12-month old mice. Two-month old mice had a faster increase in MCV during the first two weeks postinfection, whereas 12-month old mice had a slower increase in MCV but maintained higher values during the late phase of the acute infection. This finding, therefore, suggests that an impaired physiological feedback, compromising the efficacy of the erythropoietic process during the growing phase of the malaria infection, might account for the age-dependent weakened tolerance.

Aging is usually associated with a general decline in vigor [START_REF] Clegg | Frailty in elderly people[END_REF]. One might then wonder whether the observed results reflect a general increase in frailty rather than specific mechanisms related to malaria pathogenesis. With this respect, we decided not to use very old mice (lifespan of C57BL/6 mice can exceed 30 months), and instead to compare two age classes that correspond to mature adults (2-month old) and mice (12-month old) where senescent changes are found in some but not all physiological functions. On a more general note, it should be acknowledged that the relationship between age and physiological functions is rarely linear. For logistic reasons (housing capacity), we only studied two age classes, which obviously prevents us to assess the shape of the relationship between age, resistance and tolerance to malaria infection. Indirect evidence, nevertheless suggests that resistance to malaria increases during the first months of life, certainly reflecting the maturation of the immune system [START_REF] Shan | Age-related susceptibility to nonlethal Plasmodium yoelii infection in C57BL/6 mice[END_REF]. Whether tolerance also follows a curvilinear age-dependent variation is not known. Further work should therefore explore how resistance and tolerance to malaria (co)-vary over the entire individual lifespan.

To conclude, we showed that age is associated with both reduced resistance and tolerance to malaria. Further work using parasites with different life cycles and pathogenicity should confirm whether compromised tolerance is a hallmark of the senescent phenotype. In such a case, treatments for the elderly might, in addition to boost the immune response (i.e., improve the responsiveness to vaccines) [START_REF] Meyer | Improving immunity in the elderly: current and future lessons from nonhuman primate models[END_REF], also target specific mechanisms underlying a compromised tolerance [START_REF] Vale | Limiting damage during infection: lessons from infection tolerance for novel therapeutics[END_REF].

Table 1. Mixed-effect model exploring the effect of time p.i. and age (2-vs. 12-month old) on the dynamics of infection with P. yoelii (ln-transformed % iRBCs). The model included time 2 and time 3 to take into account the non-linear relationship with parasitemia. Mouse identity was included as a random effect. For the age effect (and the interactions between age and time), the estimate for the 2-month old group is included in the intercept. x 10 6 /mm 3 ) and MCV (ln-transformed µm 3 ). The model included time 2 and time 3 to take into account the non-linear relationship between the two traits and time. Mouse identity was included as a random effect. For the age effect (and the interactions between age and time), the estimate for the 2-month old group is included in the intercept. 3. Mixed-effect model exploring the effect of parasitemia (ln-transformed % iRBCs) on changes in RBC count (ln-transformed numbers x 10 6 /mm 3 ), and changes in body mass (g) in the two age classes (2-vs. 12-month old). Change in body mass is expressed as variation with respect to day 0. The model included random intercepts (mouse identity) and slopes (parasitemia) to allow for variation in the relationship between RBC count, body mass, and parasitemia at the individual level. For the age effect (and the interaction between age and parasitemia), the estimate for the 2-month old group is included in the intercept.

Fixed effects

RBC counts MCV

Fixed effects

RBC count

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. A) Changes in RBC count (ln-transformed numbers x 10 6 /mm 3 ) and B) changes in

Figure 3 .

 3 Figure 3. Covariance between tolerance and resistance. Tolerance refers to the value of the

  Figure 1.

  in Supplementary Material). Following the infection with P. yoelii, the number of circulating lymphocytes increased in both age classes, peaking at day 16 p.i. (table S2 in Supplementary Material). However, peak lymphocyte numbers were higher in 12-compared to 2-month old mice (table S2 in Supplementary Material). Therefore, lymphocyte dynamics differed between age classes (fig. S1A and table S3 in Supplementary Material). Granulocyte numbers peaked at an earlier stage of the infection and at lower numbers in 2-compared to 12-month old mice (table S2 in Supplementary Material). Therefore, granulocyte dynamics differed between age classes (fig. S1B and table S4 in Supplementary At day 0, 2-and 12-month old mice had similar RBC counts (table S5 in Supplementary Material). Infection with P. yoelii induced a decline in RBC counts, but the temporal dynamics of RBC counts differed between age classes (table 2). Minimum RBC count value was reached earlier on during the infection for 2-compared to 12-month old mice (fig. 1B, table S5 in Supplementary Material). Minimum RBC count was also lower for 12-compared to 2-month old mice (fig. 1B, table S5 in Supplementary Material).

	Material).
	3.3 Red blood cells

Table 2 .

 2 Mixed-effect model exploring the effect of time p.i. and age (2-vs. 12-month old) on the dynamics of RBC counts (ln-transformed numbers

		Estimate (se)	df	F	P
	Intercept	-2.792 (0.24)
	Time p.i.	1.050 (0.06)	1,450 524.06 <0.0001
	Time 2 p.i.	-0.064 (0.003) 1,450 373.65 <0.0001
	Time 3 p.i.	0.001 (0.0001) 1,449 230.48 <0.0001
	Age	0.802 (0.34)	1,480 5.64	0.0180
	Time p.i. x Age	-0.323 (0.08)	1,450 17.28 <0.0001
	Time 2 p.i. x Age	0.032 (0.01)	1,450 42.81 <0.0001
	Time 3 p.i. x Age -0.0007 (0.0001) 1,449 62.30 <0.0001
	Random effect	Estimate (se)		Z	P
	Mouse ID	0.024 (0.01)		1.8	0.0356

Acknowledgements -We are grateful to Valerie Saint-Giorgio and the staff of the mouse house of the Université de Bourgogne. We are grateful to Carmen Garrido who allowed us to use the SCIL Vet abc Plus+ hematology analyzer located at the "Lipides, nutrition, cancer" department.

Author contributions -GS and BF conceived the study. All authors collected the data and analyzed the samples. GS performed the statistical analyses and wrote the first draft of the manuscript. All authors revised and approved the final version of the manuscript.

Conflicts of interest -None to declare.

Ethics statement -The experiment was approved by the Comité d'Ethique de l'Expérimentation Animale Grand Campus Dijon, France (CNREEA n C2EA -105) and by the ''Ministère de la Recherche et de l'Enseignement Supérieur" (project #8675).

Funding -This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.