Whether obesity may be considered a disease is still under debate (24). Despite this, it is now evident that increased body weight is linked to a broad spectrum of diseases, which overall reduce healthspan in the elderly. Several studies showed that immune cell types such as macrophages, innate lymphoid cells, T cells and B cells are contained within the adipose tissue (25-28). In particular, lymphoid cells such as NK cells, NKT, and B and T cells may play a fundamental role in the inflammatory process associated with obesity. Among the different types of adipose tissues present in the body, MAT has recently gained considerable importance in the field of immunological memory (18). In our lab, we recently reported that MAT can produce survival factors for adaptive immune cells, therefore playing an important role in establishing niches for memory T cells and long-lived plasma cells in the BM. Despite this, as MAT additionally produces pro-inflammatory molecules such as IL-6 and TNF, its accumulation may be detrimental for the maintenance of immunological memory in the BM. Indeed, reduced plasma cell function with increased numbers of adipocytes has been documented (18).
The first aim of the current study was to assess whether the BM niche environment supporting the maintenance of adaptive immune cells may change with increased BMI (Figure 1). Although the focus of our work was not on MAT but on BMMCs, a close interaction between BM adipocytes and other BM cells is expected. Increased production of IL-15, mainly important for the survival of highly differentiated T cells, and IL-6, a pro-inflammatory molecule additionally involved in plasma cell and T cell maintenance, was found in overweight compared to lean persons. In addition, a positive correlation was found between ROS in BMMCs and BMI, in line with the observations that obese persons are characterized by high levels of oxidative stress (3). Increased oxygen radicals found in obese persons were linked to a decreased production of pro-inflammatory molecules by CD8+ T cells. Indeed, we saw a negative correlation between IFNγ+ and TNF+CD8+ T cells and BMI in the BM, suggesting that CD8+ T cell effector functions in the BM may be impaired in overweight persons. This situation could be observed in the BM but not in the PB. This aspect slightly differs from the situation observed in the BM during aging (14). Although, also in this case, an increase in the levels of IL-15, IL-6 and ROS was documented, the expression of IFNγ and TNF was increased. We can therefore speculate that, in the BM of obese persons, pro-inflammatory stimuli coming from the BM environment may activate signaling pathways within T cells, which counteract the production of IFNγ and TNF.
Another important aspect to investigate is whether the frequency of immune cell subsets in the BM change with increased BMI. For this part, the influence of CMV also needs to be considered, and therefore the samples were divided into CMV- and CMV+ groups. CMV is a persisting herpes virus, which, depending on the cohort, is present in 60-100% of individuals (29,30). By itself, CMV has been linked to diseases and/or pathologies such as cardiovascular diseases and cancer (31-33). More recently, we described how CMV affects the phenotype of subsets of highly differentiated CD8+ T cells in the BM (15). After a first general definition of immune cell populations in the BM, we observed that both CD4+ T cells and B cells significantly increased in persons with high BMI, but only in the CMV- group. Higher frequency of both subsets in the PB of obese persons has been documented (8). Activated CD4+ T cells accumulate in WAT of mice on high fat diet, supporting the recruitment of M1 macrophages with a pro-inflammatory phenotype, therefore leading to WAT inflammation (34). Although it is still unknown whether CD4+ T cells may additionally infiltrate into MAT, we can speculate that this subset may contribute to the onset of a pro-inflammatory environment within the marrow. As B cell frequency was described to correlate with BMI, further studies must be performed in order to assess whether specific B cell subsets may change with increased body weight.
As a next step, we investigated whether the phenotype of “classical” CD8+ and CD4+ T cell subsets in the BM may be influenced by BMI. This and the following parts were performed in both BM and PB in paired samples. Interestingly, most of the significant correlations within CD8+ T cell subpopulations in the BM were found in CMV- persons. This suggests that the CMV+ group may be more homogeneous regarding CD8+ T cell parameters, and the effects of obesity may be more evident in CMV- persons. In alternative, in CMV+ donors, the impact of CMV on the T cell compartment may be so profound that other more subtle changes such as BMI may be missed. CD8+ TCM and CD8+ TEM subsets are known to include bona fide memory cells, which are classically characterized by expression of costimulatory receptor CD28 and lack the marker of terminal differentiation CD57. Overall, our results indicate that BM CD8+ TCM and CD8+ TEM decreased in overweight compared to lean persons, in either the CMV- (CD8+ TCM) or in the CMV+ (CD8+ TEM) groups. In addition, the frequency of IL7Rα+KLRG-1- MPEC, known to differentiate into memory T cells (21,22), was negatively associated with BMI in the BM of both CMV- and CMV+ persons. Similar trends were described for the PB, although no differences were found in the CMV- group. Altogether, these data indicate that the maintenance of memory CD8+ T cells in the BM may be negatively affected by body weight. In addition, it is still unknown whether MAT may be involved in the competition for space between immune cell populations in the BM, supporting the preferential accumulation of certain subsets (35,36).
We recently reported that, using the markers CD28 and CD57, populations of non-activated/early-activated/memory CD28+CD57-, activated/immune regulatory-like CD28+CD57+, activated/early-senescent CD28-CD57- and terminally differentiated/senescent-like CD28-CD57+ cells could be identified within CD8+ T cells (37). Although the overall amount of CD8+CD57+ T cells decreased while the frequency of CD28-CD8+ T cells did not change in relationship to BMI, differences were observed when the four subsets were considered. Interestingly, both the CD28+CD57- population, which includes memory CD8+ T cells, and the CD28+CD57+ subset, which may play an important role in regulating immune responses (37), decreased in the BM of overweight compared to lean persons in the CMV- group. Despite this, although a link between obesity and T cell senescence has been suggested in murine adipose tissue (38), only few senescent-like populations were influenced by body weight in our cohort, in both BM and PB. In particular, while CD28-CD57+, IL7Rα-KLRG-1+ SLEC and CD8+TEMRA T cells did not change, only KLRG-1 expression positively correlated with BMI in some subsets. Despite this, it is important to consider that the expression of KLRG-1 alone may not represent an optimal marker for T cell senescence (39). We can clearly observe that the frequency of CD28-CD57- CD8+ T cells, which accumulates in the BM compared to PB, significantly increased with body weight. Thus, we can speculate that, although the “early steps” of CD8+ T cell senescence may be triggered by obesity, the “late steps”, which involve the upregulation of CD57, may be somehow inhibited. The reduced expression of IFNγ and TNF by CD8+ T cells may partially be explained by the expansion of CD28-CD57- CD8+ T cells in obese individuals, as low production of both cytokines was described in this subset (37).
Furthermore, in CMV- persons, PD-1 expression on BM CD8+ T cell subsets is negatively associated to BMI. As this co-inhibitory molecule is expressed by activated T cells and inhibits their further activation (therefore making T cells “exhausted”) (40), CD8+ T cells may be less activated in obese persons.
We next investigated whether some relationships could be found between phenotype of CD4+ T cell subsets and BMI, in the BM and in the PB. In this case, significant differences were obtained only in the BM, indicating that the BM environment may specifically influence CD4+ T cell parameters. Interestingly, only in CMV+ persons, CD4+ TN and CD4+ TCM increased while CD4+ TEM decreased in overweight persons. While TN and TCM are stable, TEM cells are known to display rapid turnover (41). Thus, our data suggest that, in obese CMV+ individuals, the maintenance of naïve and long-lived memory CD4+ T cells in the BM may be improved. Whether this aspect may be linked with the expansion of MAT observed with increased body weight must be investigated in future studies. As observed for CD8+ T cells, the effector functions of BM CD4+ T cells may be impaired with obesity, as reduced expression of activation/exhaustion marker PD-1 could be observed. Although the levels of BM CD4+ TEMRA positively correlated with BMI in the whole cohort, other senescent-like CD4+ T cell subsets were not associated with body weight. Indeed, no correlations were observed when the markers CD28 and CD57 were considered.