Aged mice display altered numbers and phenotype of basophils, and bone marrow-derived basophil activation, with a limited role for aging-associated microbiota

The human adult gut contains about 10¹³–10¹⁴ bacteria [1, 2], which is comparable to the number of human cells in the total body of a 30-year-old adult [3]. These commensal gut microbiota modulate the immune system [4] and contribute to immune homeostasis in the mucosal immune system [5]. Gut microbiota play an important modulatory role beyond mucosal immunity, for instance by changing the stem cell niche in the bone marrow (BM) [6]. Furthermore, absence of microbe-derived peptidoglycan in the circulation impairs the killing by BM neutrophils of Salmonella pneumoniae and Staphylococcus aureus [7]. In addition, in the absence of microbiota, CD123 (IL-3Rα) expression on basophil precursors was upregulated, thereby enhancing their responsiveness to interleukin (IL) 3 [8].

During aging the immune system develops several defects and undergoes various changes in differentiation, distribution, and activation [9]. Anti-parasitic immune responses in aged mice are impaired [10], which may indicate age-related changes in basophil function [11]. With aging, gut microbiota composition changes [12]. Basophil hematopoiesis and function are regulated by gut microbiota. Absence of gut microbiota lead to increased basophil frequencies and enhanced T helper (Th) 2 immune responses [8]. In addition, basophils express Toll-like receptor (TLR) 2 and TLR4, and respond to microbial ligands like peptidoglycan [13] and lipopolysaccharide (LPS) [14]. Histamine release and sensitivity of basophils from elderly were reported to be increased upon anti-immunoglobulin (Ig) E stimulation [15], but in a different study, no age-related difference was found in histamine release of human blood basophils upon anti-IgE or anti-IgG4 stimulation [16]. Basophil counts were not associated with frailty or mortality in elderly women [17, 18]. Basophil frequencies and absolute numbers decreased in blood from healthy elderly volunteers and patients suffering from Alzheimer’s disease [19, 20]. It is, however, largely unknown what effect age has on basophil differentiation and function.

Basophils are granulocytes which are involved in mounting and perpetuating Th2-mediated responses [21]. Basophils are an important source of IL-4 and IL-13, which direct the immune response towards Th2 type responses [22]. After IgD crosslinking, basophils produced IL-1, IL-4 and B cell activating factor (BAFF), supporting B cell functions [23]. Basophils are the major source of IL-4 after Streptococcus pneumoniae infection, contributing to humoral memory immune responses [24]. In addition, the basophil is crucial in the pathophysiology of systemic lupus erythematosus [25, 26], and its counts are a marker for disease activity [27]. Recently, basophil infiltration into tumors after depletion of regulatory T cells was implicated in tumor rejection via C-C motif chemokine ligand (CCL) 3- and CCL4-mediated recruitment of CD8⁺ T cells to tumors [28], indicating a role beyond classical Th2 responses.

Basophil differentiation and functions are dependent on IL-3 or thymic stromal lymphopoietin (TSLP) [29]. Basophils can be activated in an IgE-dependent and IgE-independent manner. Regarding IgE-dependent activation, FcεRIα crosslinking by complexes of IgE and antigen activates basophils, resulting in IL-4 and IL-13 production [30]. Basophils express IL-18R and IL-33R (ST2), and upon stimulation with IL-18 and IL-33, basophils produce IL-4, IL-6, IL-13, granulocyte-macrophage colony stimulating factor (GM-CSF), and several chemokines [31]. This effect is further enhanced in the presence of IL-3 [32]. CD200R3-mediated activation of basophils leads to IL-4 production in vitro, and to anaphylaxis in vivo [33].

Here we studied the influence of the aging-associated microbiota on basophil frequency and phenotype, and differentiation from precursors of basophils. We compared basophils from young germ-free recipients of microbiota of 4-month-old to young germ-free recipients of microbiota of 18-month-old mice. In addition, we studied changes in frequency and phenotype of basophils in BM and spleen, correlation between microbial genera and basophils, and changes in differentiation from precursors of basophils during aging by comparing 4-month-old and 18-month-old mice.

In this study, we found that basophil frequencies, numbers, and phenotype in the spleen change in mice during aging. Less effects on phenotype were found in the BM, although absolute numbers of basophils increased. This however should not be interpreted as a suggestion that no aging effects in the BM exist, as significant effects of age were found on the in vitro activation of basophils differentiated from precursors in the BM. Partly these in vitro effects were caused by the aging microbiota, as age-dependent changes in the activation of BM-derived basophil were also observed in young germ-free recipients of microbiota of 18-month-old mice. Fecal microbiota analysis showed that the microbiota composition significantly changed with age, and after microbiota transfers. Several microbial genera were correlated with basophil frequencies and phenotype.

Our report confirms age-related effects on basophils, showing for the first time that basophil phenotype changes. Intriguingly, CD123 expression by basophils from old mice consistently tended to increase. CD123 is crucial for IL-3 signaling and basophil hematopoiesis [29], and might explain the increased basophil numbers. IL-3 is, in higher amounts, also able to induce IL-4 production in basophils via the IL-3 receptor [35]. Aged basophils showed a tendency to lower expression of CD200R3, which inhibits FcεRIα-mediated activation of basophils [36]. CD200R3 also activates basophils to produce IL-4 and to degranulate [33]. Lower CD200R3 expression by basophils from aged mice (versus basophils from young mice) might indicate that aged basophils are less readily activated [33]. Together, these age-related changes might indicate an increased sensitivity to IL-3, and at the same time an altered threshold for activation. Thus, we were able to show differences in BM and spleen basophils with age.

Recently, we have shown a correlation between B cell precursors and abundance of specific microbial genera [37]. Similarly, in this study, we found an association between specific microbial genera and basophil frequencies and phenotype (both in BM and spleen). Most strikingly, the abundance of Alistipes, Oscillibacter, Bacteroidetes RC9 gut group, and S24–7 family positively correlated with CD123 expression in BM and spleen. As indicated above, CD123 is crucial for basophil hematopoiesis and function. Transfer of specific microbiota into germ-free recipient mice would further support the association of microbial genera with basophil frequencies and phenotype we found in this study. Because basophils express TLR2 and TLR4 [13], it would be of high interest to determine expression of these receptors as well as responsiveness to their ligands in the context of aging.

To gain insight into the effect of aging on the precursors of basophils, we used IL-3-dependent BM cultures as a proxy (Fig. 5). First, we improved the method to generate basophils by at least 70-fold compared with a recent, detailed protocol [31]. Yoshimoto et al (2012) reported using femurs and tibias of ten 9- to 12-month-old Balb/c male mice. A conservative estimation of the starting number of BM cells in their cultures is 4 × 10⁸, which resulted in 20-40 × 10⁶ cultured cells (culture efficiency ≤10%). After purification, 1-4 × 10⁶ basophils were collected (purification efficiency ≤10%). Under the best conditions, the mentioned protocol ends with a 1% yield. In our hands, the culture efficiency of the improved BMB generation protocol was higher than previously reported, with each 10⁶ BM cells generating on average 2 × 10⁶ cultured cells. Taking into account the withdrawal of cells for direct assessment three times during the culture, our culture efficiency was a bit higher than 200%. Our purification method, which includes dendritic cell removal, resulted in higher numbers of pure basophils: we isolated on average 6.9 × 10⁶ pure basophils per 20 × 10⁶ cultured cells (35% purification efficiency). Regardless different origins of BM (Table 1), our protocol ends with an average yield of 70%. The vast difference between the yields are most likely explained by the cell density at the start of the culture. Other differences that might cause improved yield are mouse strain, fresh versus frozen BM, and the purification method. Thus, using our robust method, we were able to assess basophil function by using a few million BM cells as input. It is important to underline the importance of excluding the adherent cells during the culture and the targeted depletion of CD11c⁺ dendritic cells during the isolation of BMB. This enables to specifically look at BMB responses, without bystander effects of stromal cells or dendritic cells.

We identified additional differences between young and aged BMB (Fig. 6). CD11b expression was decreased, whereas IL-4⁺ (but not IL-13⁺) frequencies were increased upon activation in BMB from aged mice. IL-4⁺ basophil frequencies were particularly increased after CD200R3 stimulation, in line with previous studies [33]. BMB derived from germ-free recipients receiving microbiota of aged mice (versus microbiota of young mice) also showed increased IL-4⁺ basophil frequencies. Thus, we found that microbiota from aged mice influence basophil precursors and subsequent in vitro activation.

The functional implications of these findings remain to be elucidated. It is conceivable that basophils may differ in their functional response in vivo, because Hill et al (2012) showed that antibiotics under steady state conditions in vivo did not alter basophil frequencies in lymph nodes. Basophil frequencies, however, were increased after papain treatment in antibiotic-treated mice (compared with control mice) [8]. Allergic challenges or helminth infections in young versus aged mice would give insight in the functional consequences in vivo of the observed changes between young and aged basophils, and after microbiota transfers of young and aged mice.

Our study has a number of limitations: 1) Due to the relatively small populations of basophils and the required numbers of aged and germ-free mice, we were not able to sort basophils directly from spleen or bone marrow to evaluate in vitro basophil function. 2) We could not study alterations in in vivo production of e.g. IL-4 by basophils with aging, as could be done by using aged or germ-free IL-4-eGFP reporter mice. 3) We used total aging-associated microbiota, rather than selected microbial strains that were altered upon aging and correlated with basophil phenotype or numbers.

Our study shows that aged mice display increased basophil numbers and altered phenotype, which seems independent of aging-associated microbiota. In vitro activation of BM-derived basophils is impaired with aging, which in part is explained by aging-associated microbiota. Further functional in vivo studies are warranted to investigate the consequences of our findings for Th2-mediated immune responses in aging.