Ageing and obesity similarly impair antibody responses

Summary

Ageing is characterized by increased low‐grade chronic inflammation, which is a significant risk factor for morbidity and mortality of elderly individuals. Similar to ageing, obesity is considered to be an inflammatory predisposition associated with chronic activation of immune cells and consequent local and systemic inflammation. Both ageing and obesity are characterized by reduced innate and adaptive immune responses. This review focuses on B cells, how they may contribute, at least locally, to low‐grade chronic inflammation in ageing and obesity and on the mechanisms involved.

OTHER ARTICLES PUBLISHED IN THIS REVIEW SERIES

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The convergence of senescence and nutrient sensing during lymphocyte ageing. Clinical and Experimental Immunology 2017, 187: 4–5.

Immune senescence: significance of the stromal microenvironment. Clinical and Experimental Immunology 2017, 187: 6–15.

Innate immune responses in the ageing lung. Clinical and Experimental Immunology 2017, 187: 16–25.

Age‐related alterations in immune responses to West Nile virus infection. Clinical and Experimental Immunology 2017, 187: 26–34.

Intracellular signalling pathways: targets to reverse immunosenescence. Clinical and Experimental Immunology 2017, 187: 35–43.

Ageing and inflammation in patients with HIV infection. Clinical and Experimental Immunology 2017, 187: 44–52.

Considerations for successful cancer immunotherapy in aged hosts. Clinical and Experimental Immunology 2017, 187: 53–63.

The life cycle of a T cell after vaccination – where does immune ageing strike? Clinical and Experimental Immunology 2017, 187: 71–81.

Herpes zoster and the search for an effective vaccine. Clinical and Experimental Immunology 2017, 187: 82–92.

Adult vaccination against tetanus and diphtheria: the European perspective. Clinical and Experimental Immunology 2017, 187: 93–99.

Ageing and related inflammation decrease B cell responses

With ageing, the innate and adaptive immune responses deteriorate, leading to greater susceptibility to infectious diseases and reduced responses to vaccination 1. The decreased ability of aged individuals to respond effectively against infectious agents and vaccines includes defects in T cell signalling to B cells 2, 3, 4, reduced somatic hypermutation (SHM) 5, 6 and class‐switch recombination (CSR) in germinal centre B cells 7, and intrinsic shifts in the V_H repertoire 8. Functional alterations in T cells have been considered for a long time to be sufficient per se to explain the age‐related decrease in antibody responses to exogenous antigens and vaccines in elderly people. However, a large amount of work has been conducted more recently showing that defects in other components of the innate and adaptive immune systems also occur with age and contribute to the increased frequency and severity of infectious diseases in elderly people.

Our laboratory has characterized age‐related intrinsic B cell defects, which are responsible for suboptimal antibody responses of elderly individuals to infections and vaccines 9, 10, 11, 12, 13. A reduction in activation‐induced cytidine deaminase (AID), the enzyme necessary for CSR, SHM and immunoglobulin (Ig)G production, as well as in E47, a key transcription factor regulating AID 14, has been identified by our group. It has also been established that AID correlates with optimal B cell function and therefore AID can be used a predictive marker of optimal B cell response. The decrease in AID and E47 leads to a reduced ability to generate higher‐affinity vaccine‐specific antibodies 5. The antibody response to the seasonal and pandemic influenza vaccines measured in serum is associated with the B cell response after vaccination to the vaccine in vitro. In‐vivo and in‐vitro B cell responses have been measured, respectively, by the haemagglutination inhibition assay and by AID mRNA expression by quantitative polymerase chain reaction (qPCR) after B cell restimulation with the vaccine. It has been shown that the specific response of B cells to vaccination in vivo and in vitro are both decreased by ageing and are correlated significantly 5, 11, 13. Moreover, the percentages of switched‐memory B cells and cytosine–phosphate–guanine (CpG)‐induced AID before vaccination are both good B cell biomarkers that are reduced in elderly people and are correlated significantly with the in‐vivo antibody response to the vaccine 11, 13, indicating that they can be used as predictive biomarkers of optimal vaccine‐induced antibody responses.

It has been shown that elderly individuals have a significant reduction in B cell repertoire diversity and that this correlates with their health status, and that B cell clonal expansions with age had been reported previously 15. Using high‐throughput long read sequencing of human antibody repertoires in the context of Epstein–Barr virus (EBV) or cytomegalovirus (CMV) serum positivity 16 and influenza vaccination 17, it has been shown that elderly individuals have decreased numbers of lineages but increased prevaccination mutation load in their repertoire and the diversity of the lineages is reduced greatly compared to young individuals, consistent with earlier reports on contraction of B cell repertoires in elderly people 18. These findings could help to explain the impaired vaccine responses observed in elderly people.

The inflammatory status of an individual may impact upon the function of cells of the immune system. B cells from elderly individuals spontaneously make higher amounts of tumor necrosis factor (TNF)‐α than those from young subjects and B cell intrinsic TNF‐α levels are correlated positively with serum TNF‐α. Importantly, these B cell levels of TNF‐α before stimulation are correlated negatively with the response of the B cells from the same individual after in‐vitro stimulation which is measured by AID 9. Additionally, high prevaccine serum and B cell TNF‐α levels are also correlated negatively with the in‐vivo serum response to the influenza vaccine 9. In line with these results, an anti‐TNF‐α antibody was found to increase significantly the response in cultured B cells from elderly individuals, providing a proof‐of‐principle that it is possible to improve antibody production in elderly individuals by counteracting autocrine TNF‐α 9. These findings identify serum and cytoplasmic B cell TNF‐α as other B cell‐specific biomarkers, which can help to predict the quality of in‐vivo and in‐vitro B cell responses. Although our studies have shown that serum and B cell TNF‐α are correlated positively in the majority of individuals, some people show lower levels of B cell TNF‐α and lower AID. These results suggest that not only TNF‐α but also other markers of intrinsic B cell inflammation may contribute to the down‐regulation of AID in B cells from aged individuals; for example, microRNAs, which have been correlated negatively with AID 19.

Ageing is characterized not only by increased circulating levels of proinflammatory cytokines [TNF‐α, interleukin (IL)−6, C‐reactive protein (CRP)], but also by latent infections with viruses such as CMV. Stimuli triggering inflammation can also be generated by the age‐related increase in the amount of self‐debris due to the continuous turnover of cells and tissues, such as circulating mitochondrial DNA (mtDNA) and post‐translationally modified macromolecules (DNA or proteins modified by oxidation, acylation, glycosylation), which are recognized by immune sensors as exogenous and represent a potent inflammatory stimulus 20.

CMV‐seropositivity has been shown to have a negative effect on influenza vaccine‐specific antibody responses. Our group has demonstrated recently for 21 the first time a negative association between CMV seropositivity and the B cell predictive biomarkers of optimal vaccine responses characterized previously in our laboratory, and found CMV seropositivity associated with increased levels of serum and B cell‐intrinsic TNF‐α; this increase in inflammation, contributed in part by CMV, may be one of the mechanisms to down‐regulate the B cell antibody response. One proposed mechanism by which CMV decreases B cell function may be an increase in systemic/serum TNF‐α which induces B cell‐derived TNF‐α which, in turn, activates the promoter/enhancer of CMV and proinflammatory cytokine production. In addition to this mechanism acting directly on B cells, CMV may also down‐regulate the antibody response to the influenza vaccine indirectly through the induction of terminally differentiated T cells and accumulation of senescent T cells 22, 23, which could lead to reduced generation of memory T cells 24, 25.

Obesity is associated with decreased B cell responses

Obesity and obesity‐related diseases are a significant risk to public health, and the numbers of obese individuals in the United States have increased dramatically in the last few years (Obesity Data Prevalence Map, http://www.cdc.gov/obesity/data/prevalence-maps.html). More than one‐third of US adults are obese, with more in the African American population, and these numbers are predicted to increase alarmingly in the next few years. Increased weight and abnormal accumulation of fat tissue lead to detrimental health consequences, mainly because increased adipose tissue is associated with increased inflammation, insulin resistance (IR), p53 activation and telomere shortening 26, 27. The growing interest in the field of obesity research is aiming to discover and unravel the cellular and molecular mechanisms leading to the increase in body weight and how this affects health outcomes. While prevention is of great importance, it is medically relevant to identify biological pathways with the potential to treat obesity and related disorders, particularly in adults with fully established obesity and associated conditions.

Obesity is associated with chronic activation of cells of the innate immune system and consequent local and systemic inflammation, which contributes to pathological conditions such as type 2 diabetes (T2D) 28, 29, 30, cancer 31, psoriasis 32, atherosclerosis 33 and inflammatory bowel disease 34. Obesity is linked to increased susceptibility to bacterial, viral and fungal infections 35, 36, and obese individuals develop more postsurgical infections than do lean individuals 37, 38.

The adipose tissue is a major immunologically active organ that contributes to systemic inflammation. Adipose tissue inflammation is characterized by infiltration and activation of immune cells that produce cytokines and chemokines that contribute to the ongoing chronic inflammation that promotes the degradation of metabolic pathways in obesity. Most of the studies conducted so far support a crucial role for proinflammatory T cells and macrophages in promoting local inflammation in the visceral adipose tissue (VAT) leading to IR. It has been shown that, in obesity, IFN‐γ‐producing CD8⁺ and T helper type 1 (Th1) CD4⁺ T cells infiltrate VAT 39 and promote secretion of proinflammatory cytokines from M1 macrophages which contribute to both local and systemic IR 40. Conversely, in lean individuals, IL‐4/5/13‐producing Th2 CD4⁺ T cells, CD4⁺ regulatory T (T_regs) and invariant natural killer (iNKT) cells are predominant in the VAT and promote secretion of IL‐10 and other anti‐inflammatory cytokines from M2 macrophages which maintain insulin sensitivity (IS). There is increasing evidence that subcutaneous adipose tissue (SAT) in the belly may also be dangerous in promoting inflammation 41.

Studies elucidating B cell function in obesity are limited, although B cells have emerged recently as crucial players in regulating inflammation in murine VAT by presenting antigens to T cells, secreting proinflammatory cytokines and secreting pathogenic antibodies 42. B cells infiltrate the expanding adipose tissue in response to hypernutrition 43. B cells can be activated by products of altered lipolysis in the expanding adipose tissue to release proinflammatory cytokines (TNF‐α/IL‐6) or chemokines (IL‐8), thus contributing to local and systemic inflammation 44, 45. Antibodies can also regulate obesity at the level of lipid absorption from the gut and B^null mice show reduction in lipid absorption 46. Consistently, B^null mice fed with a high‐fat diet show reduced visceral fat pad weights compared to wild‐type controls, suggesting that B cells play a role in changing nutrient absorption, perhaps through local inflammatory responses which could also shape mucosal immunity and change gut microbiota 42, 47. Moreover, murine and human B cells support T cell inflammation in obesity 48.

Our group has shown recently 49 that obesity is associated with attenuated in‐vivo and in‐vitro antibody responses in both young and elderly individuals and that the peripheral B cell pool of individuals with obesity is characterized by decreased percentages of anti‐inflammatory B cell subsets (transitional B cells) and increased percentages of proinflammatory late/exhausted memory B cells. Moreover, total B cells from both young and elderly individuals with obesity, compared to lean individuals, have impaired function, as measured by AID in response to CpG stimulation, and they secrete more proinflammatory (IL‐6) and fewer anti‐inflammatory (IL‐10) cytokines in culture supernatants. Before stimulation, total B cells from obese individuals show higher immune activation (IA), as measured by increased levels of intracellular TNF‐α (icTNF‐α), TLRs and inflammatory micro‐RNAs (miRs), all of which associate negatively with AID in stimulated B cells. B cells from young and elderly individuals with obesity also support the production of the proinflammatory cytokines IL‐17 and IFN‐γ in T cells. These results are summarized in Table 1. In conclusion, the effects of ageing seem to be more pronounced than those of obesity on in‐vivo responses to the influenza vaccine and on circulating B cell subsets. Conversely, obesity, more than ageing, seems to affect the induction of IA markers in unstimulated B cells (higher icTNF‐α and IL‐6 in young obese versus lean individuals and lower IL‐10 in elderly obese versus lean individuals). Also, CSR seems to be affected more by obesity in elderly individuals, although values are already very low in the lean ones.

Table 1.

Effect of obesity and ageing on B cell function in humans

	Individuals
	Young Lean	Young Obese	Elderly lean	Elderly Obese
In vivo influenza vaccine response a	196 ± 33	80 ± 16**	56 ± 8 ∧∧∧	28 ± 4** ∧∧
B cell subsets in blood b
– Switched Memory	15 ± 0.9	6 ± 0.6****	4 ± 0.5 ∧∧∧∧	2 ± 0.3** ∧∧∧∧
– IgM Memory	29 ± 1	27 ± 2	22 ± 2	21 ± 3
– Naïve	50 ± 0.8	57 ± 2**	61 ± 2 ∧∧∧	64 ± 0.9 ∧∧
– Late/Exhausted Memory	4 ± 0.4	12±i ****	12 ± 0.6 ∧∧∧∧	14 ± 2
– Transitional B cells	8 ± 0.9	4 ± 0.5**	3 ± 0.6 ∧∧∧	1.7 ± 0.4* ∧∧
Cytokine production
– icTNF‐a in unstimulated B cells c	5 ± 3	18 ± 2*	14 ± 2 ∧	26 ± 1*** ∧∧
– IL‐6 in stimulated B cells d	68 ± 6	244 ± 24****	181 ± 20 ∧∧∧	323 ± 2o** ∧
– IL‐10 in stimulated B cells d	58 ± 3	37 ± 2**	26 ± 4 ∧∧∧∧	6 ± 0 6*** ∧∧∧∧
CSR
– AID expression in stimulated B cells e	0.2 ± 0.03	0.09 ± 0.01*	0.08 ± 0.01 ∧∧	0.03 ± 0.003* ∧∧∧
– E47 expression in stimulated B cells e	0.16 ± 0.008	0.08 ± 0.008***	0.06 ± 0.007 ∧∧∧∧	0.03 ± 0.004* ∧∧∧
– IgG in sups of stimulated B cells d	41 ± 4	2o±3***	21 ± 2 ∧∧	7 ± 1*** ∧∧

Results are from reference 48 (Frasca et al., Obesity 2016).

^aResults are reciprocal of the titers after vaccination.

^bResults are percentages of CD19+ B cells.

^cResults are percentages of icTNF‐a‐positive B cells, as evaluated by flow cytometry and intracellular (ic)staining.

^dMeasured by ELISA.

^eMeasured by qPCR.

*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicate significant differences between lean and obese within the same age group.

^∧p<0.05, ^∧∧p<0.01, ^∧∧∧p<0.001, ^∧∧∧∧p<0.0001 indicate significant differences between young and elderly within the same weight group.

Mechanisms for the down‐regulation of B cell responses in ageing and obesity

As summarized above, ageing and obesity are associated with metabolic, physiological and functional changes. Both ageing and the increase in fat mass lead to higher production of proinflammatory cytokines and chemokines, which increase the risk of developing chronic diseases and decrease life expectancy. Moreover, glucose tolerance decreases with age 50 and percentages of abdominal (visceral and subcutaneous) fat also seem to be the major determinants of IR in elderly individuals. Because of these similarities, it is likely that these conditions share similar cellular pathways.

In humans, adiposity increases with age. Computational tomography scans have shown that as age increases SAT decreases, whereas VAT increases 51. Aged mice also develop increased fat mass, with an increase in VAT, similar to elderly humans 52. In humans and mice, VAT and SAT are biologically distinct in terms of secretion of proinflammatory mediators, including leptin, the adipocyte‐derived factor linking nutritional status with neuroendocrine and immune functions, with VAT being more inflammatory. Furthermore, expression of adipokines from adipose tissue is regulated by nutrients, and these responses are increased with ageing 53.

One of the mechanisms responsible for reduced B cell function in individuals with advanced age or obesity may be leptin‐induced systemic and B cell intrinsic inflammation. Plasma levels of leptin, produced by fat cells, correlate with the amount of body fat and body mass index (BMI) and increase with age 54, 55, 56. High serum levels of leptin contribute to the inflammatory state of the adipose tissue associated with obesity 57, 58. Leptin can modulate both innate and adaptive immune responses. For example, it regulates the macrophages’ acute inflammatory response by inducing the secretion of proinflammatory cytokines 59, 60, activates B cells to produce cytokines 61, promotes B cell survival by inhibiting apoptosis and inducing cell cycle entry 62, controls the activation of CD4⁺ effector T cells 63 and regulates the balance between proliferation and anergy in T_regs 64. In‐vitro incubation of B cells from lean individuals with leptin increases phospho‐signal tranducer and activation of transcription factor (STAT)‐3, crucial for TNF‐α production, and decreased phospho‐AMP‐activated protein kinase (AMPK), the energy‐sensing enzyme upstream of AID activation 49.

Another mechanism by which the adipose tissue may down‐regulate B cell function and induce the phenotypical and functional changes in B cell subsets observed in individuals with obesity is the secretion by the adipocytes of proinflammatory mediators contributing to systemic chronic inflammation, and of chemokines promoting the migration of B cells to the VAT and regulating B cell function in the VAT. Experiments in progress in our laboratory are investigating these hypotheses.

Telomeres are key markers of biological ageing 65, 66 and are predicitve biomarkers of effective B cell responses to the influenza vaccine 67. In particular, B cells from individuals with protective titres to the influenza vaccine have significantly longer telomeres than those with poor antibody responses 67. The age‐related increase in inflammation decreases telomere length. Shorter telomeres have also been associated with increased BMI and increased waist : hip ratio and VAT accumulation 68, suggesting that obesity may accelerate the ageing process.

Therapeutic interventions to reduce obesity and delay ageing

Although recent studies have shown that overweight and mildly obese elderly individuals may live longer than their normal weight controls, obese elderly individuals may live a greater portion of their life with some disability 69, 70. With the increasing obese population worldwide and with the increasing elderly obese population 71, the development of safe and effective therapeutic interventions is needed urgently. These will not only enhance immune responses to infectious agents and vaccines, but will also improve the biological quality of life in these individuals.

Several epidemiological studies have evaluated the effects of different types of diet in protecting subjects from diseases associated with chronic low‐grade inflammation. Traditional diets (Mediterranean) and other less‐known diets (Okinawan, DASH, Portfolio) share similar dietary patterns, all of which are associated with reduced risk for inflammation‐associated diseases. The primary goal is to treat obese patients to reduce their weight, and this would also correct their other co‐morbidities, as not all obese individuals will have and/or take the option of bariatric surgery. Calorie or food restriction without malnutrition has been shown to delay ageing in animal models 72. This intervention prolongs the lifespan of different animal species, from single‐cell organisms to mammals, and prevents or delays age‐related diseases such as cardiovascular disease 73, cancer 74 and renal failure 75. In humans, short‐term calorie restriction decreases the expression of biomarkers of reduced longevity, such as fasting insulin, glucose levels and body temperature 76, similar to what has been observed in mice 72.

Obesity is associated with low circulating levels of 25‐hydroxyvitamin D [25(OH)D], VitD 77, which decrease the development of obesity‐related pathophysiological disorders such as adipose tissue inflammation and subsequent IR. Using high‐throughput methodology (transcriptomics), it has been shown that VitD reduces chemokine expression by adipocytes and macrophage migration in vitro and limits NF‐κB activation 78. By limiting inflammation, VitD supplementation has been proposed to also benefit frail elderly people by enhancing long‐term health 79.

Pharmacological approaches are also being considered, and these include inhibitors of glycolysis, growth hormone/insulin‐like growth factor 1 (GH/IGF‐1) and inflammatory pathways, as well as activators of the AMPK pathway and treatments with metformin, statins and β‐blockers 80. Some of these interventions have already been shown to improve immune responses in elderly individuals. As an example, a recent paper has shown that pharmacological inhibition of mTOR with the mTOR inhibitor RAD0 increased significantly the in‐vivo response to the influenza vaccine in elderly healthy individuals, and reduced the percentage of CD4 and CD8 T lymphocytes expressing the programmed death‐1 (PD‐1) receptor, which inhibits T cell signalling and is expressed more highly with age 81.

In conclusion, autonomous B cell biomarkers of ageing and inflammation, which affect the production of protective antibodies, have been identified and characterized. Investigation of the mechanisms whereby inflammation and immune activation disrupt a functional immune response adds a novel dimension to the current focus on the relationship of inflammation with long‐term metabolic disease outcome. These studies will allow targets for design of possible adjuvants, new drugs and/or non‐invasive lifestyle changes to improve the immune and effective vaccine responses.

Disclosure

No potential disclosures relevant to this paper are reported.