Response In Old Mice Research Articles

PD1 blockade improves survival and CD8+ cytotoxic capacity, without increasing inflammation, during normal microbial experience in old mice.

By 2030, individuals 65 years of age or older will make up approximately 20% of the world's population1. Older individuals are at the highest risk for mortality from infections, largely due to the pro-inflammatory, dysfunctional immune response, which is collectively known as immunosenescence2. During aging, CD8+ T cells acquire an exhausted phenotype, including increased expression of inhibitory receptors, such as programmed cell death 1 (PD1), a decline in effector function and elevated expression of inflammatory factors3-7. PD1 reduces T cell receptor activity via SHP2-dependent dephosphorylation of multiple pathways; accordingly, inhibiting PD1 activity through monoclonal antibodies increases CD8+ T cell effector response in young mice8-11. Attempts to improve CD8+ T cell responses by blocking inhibitory receptors are attractive; however, they can lead to adverse immune events due to overamplification of T cell receptor signaling and T cell activation12,13. Here we investigated the effect of monoclonal anti-PD1 immunotherapy during normal microbial experience, otherwise known as exposure to dirty mice, to determine whether it either improves exhausted CD8+ T cell responses in old mice or leads to a heightened inflammatory response and increased mortality.

SUPPRESSOR CELLS (MDSC) REGULATE THE T-CELL RESPONSE IN OLD MICE IMMUNIZED WITH THE WHOLE-VIRION SARS-CoV-2 VACCINE

Myeloid-derived suppressor cells (MDSCs) are an immunosuppressive population involved in pathological processes associated with chronic inflammation. In recent years, the role of these cells in the regulation of a number of non-pathological processes, in particular, in the development of vaccine-induced immune response, has been actively discussed. This work was aimed at investigating the role of MDSC in the development of an immune response in old mice immunized with an inactivated whole virion SARS-CoV-2. Immunization of old animals induced an increase in the relative content of B cells and the production of specific antibodies to S1 and N proteins of SARS-CoV-2 in titers comparable to those of young animals. At the same time, elevated MDSC levels in aged animals negatively correlated with the level of CD8+ T cells. Splenic MDSCs in old animals were characterized by increased production of reactive oxygen species compared to young animals. In vitro experiments showed that MDSC depletion increased antigen-specific production of IFNγ by CD4+ T cells. The data obtained suggest a negative role for MDSC in regulating the T-cell vaccine-induced response to inactivated SARS-CoV-2 and encourage further studies in this direction, which may be a key to developing approaches to increase the efficacy of vaccines against SARS-CoV-2 in at-risk groups.

Depletion of preexisting B-cell lymphoma 2-expressing senescent cells before vaccination impacts antigen-specific antitumor immune responses in old mice.

The age-related decline in immunity reduces the effectiveness of vaccines in older adults. Immunosenescence is associated with chronic, low-grade inflammation, and the accumulation of senescent cells. The latter express Bcl-2 family members (providing resistance to cell death) and exhibit a pro-inflammatory, senescence-associated secretory phenotype (SASP). Preexisting senescent cells cause many aging-related disorders and therapeutic means of eliminating these cells have recently gained attention. The potential consequences of senescent cell removal on vaccine efficacy in older individuals are still ignored. We used the Bcl-2 family inhibitor ABT-263 to investigate the effects of pre-vaccination senolysis on immune responses in old mice. Two different ovalbumin (OVA)-containing vaccines (containing a saponin-based or a CpG oligodeoxynucleotide adjuvant) were tested. ABT-263 depleted senescent cells (apoptosis) and ablated the basal and lipopolysaccharide-induced production of SASP-related factors in old mice. Depletion of senescent cells prior to vaccination (prime/boost) had little effect on OVA-specific antibody and T-cell responses (slightly reduced and augmented, respectively). We then used a preclinical melanoma model to test the antitumor potential of senolysis before vaccination (prime with the vaccine and OVA boost by tumor cells). Surprisingly, ABT-263 treatment abrogated the vaccine's ability to protect against B16 melanoma growth in old animals, an effect associated with reduced antigen-specific T-cell responses. Some, but not all, of the effects were age-specific, which suggests that preexisting senescent cells were partly involved. Hence, depletion of senescent cells modifies immune responses to vaccines in some settings and caution should be taken when incorporating senolytics into vaccine-based cancer therapies.

Heterologous DNA-prime/protein-boost immunization with a monomeric SARS-CoV-2 spike antigen redundantizes the trimeric receptor-binding domain structure to induce neutralizing antibodies in old mice.

A multitude of alterations in the old immune system impair its functional integrity. Closely related, older individuals show, for example, a reduced responsiveness to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines. However, systematic strategies to specifically improve the efficacy of vaccines in the old are missing or limited to simple approaches like increasing the antigen concentration or injection frequencies. We here asked whether the intrinsic, trimeric structure of the SARS-CoV-2 spike (S) antigen and/or a DNA- or protein-based antigen delivery platform affects priming of functional antibody responses particularly in old mice. The used S-antigens were primarily defined by the presence/absence of the membrane-anchoring TM domain and the closely interlinked formation/non-formation of a trimeric structure of the receptor binding domain (S-RBD). Among others, we generated vectors expressing prefusion-stabilized, cell-associated (TM+) trimeric "S2-P" or secreted (TM-) monomeric "S6-PΔTM" antigens. These proteins were produced from vector-transfected HEK-293T cells under mild conditions by Strep-tag purification, revealing that cell-associated but not secreted S proteins tightly bound Hsp73 and Grp78 chaperones. We showed that both, TM-deficient S6-PΔTM and full-length S2-P antigens elicited very similar S-RBD-specific antibody titers and pseudovirus neutralization activities in young (2-3 months) mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. The trimeric S2-P antigen induced high S-RBD-specific antibody responses in old (23-24 months) mice through DNA-prime/DNA-boost vaccination. Unexpectedly, the monomeric S6-PΔTM antigen induced very low S-RBD-specific antibody titers in old mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. However, old mice efficiently elicited an S-RBD-specific antibody response after heterologous DNA-prime/protein-boost immunization with the S6-PΔTM antigen, and antibody titers even reached similar levels and neutralizing activities as in young mice and also cross-reacted with different S-variants of concern. The old immune system thus distinguished between trimeric and monomeric S protein conformations: it remained antigen responsive to the trimeric S2-P antigen, and a simple change in the vaccine delivery regimen was sufficient to unleash its reactivity to the monomeric S6-PΔTM antigen. This clearly shows that both the antigen structure and the delivery platform are crucial to efficiently prime humoral immune responses in old mice and might be relevant for designing "age-adapted" vaccine strategies.

Delineating the relationship between immune system aging and myogenesis in muscle repair.

Recruited immune cells play a critical role in muscle repair, in part by interacting with local stem cell populations to regulate muscle regeneration. How aging affects their communication during myogenesis is unclear. Here, we investigate how aging impacts the cellular function of these two cell types after muscle injury during normal aging or after immune rejuvenation using a young to old (Y‐O) or old to old (O‐O) bone marrow (BM) transplant model. We found that skeletal muscle from old mice (20 months) exhibited elevated basal inflammation and possessed fewer satellite cells compared with young mice (3 months). After cardiotoxin muscle injury (CTX), old mice exhibited a blunted inflammatory response compared with young mice and enhanced M2 macrophage recruitment and IL‐10 expression. Temporal immune and cytokine responses of old mice were partially restored to a young phenotype following reconstitution with young cells (Y‐O chimeras). Improved immune responses in Y‐O chimeras were associated with greater satellite cell proliferation compared with O‐O chimeras. To identify how immune cell aging affects myoblast function, conditioned media (CM) from activated young or old macrophages was applied to cultured C2C12 myoblasts. CM from young macrophages inhibited myogenesis while CM from old macrophages reduced proliferation. These functional differences coincided with age‐related differences in macrophage cytokine expression. Together, this study examines the infiltration and proliferation of immune cells and satellite cells after injury in the context of aging and, using BM chimeras, demonstrates that young immune cells retain cell autonomy in an old host to increase satellite cell proliferation.

Detrusor Underactivity as an HCN-Mediated Failure of Resilience in Aging

Sympathetic relaxation of the bladder wall permits low pressure urine storage and allows central regulation of afferent sensitivity to volume. Impaired regulation of volume sensitivity has been linked to symptoms of underactive bladder and cystometric detrusor underactivity. Hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels) are mediators of sympathomimetic-induced detrusor relaxation in young mouse bladder tissue, however in bladder strips from old female mice, HCN blockade enhanced age-diminished isoproterenol-induced relaxation. We therefore hypothesized that loss of HCN would compromise cystometric function and enhance sympathomimetic responses in old mice. Male HCN1 KO mice (20-22 mo) and their WT littermates underwent pressure-flow cystometry under urethane anesthesia to assess urinary performance at the level of the autonomic nervous system in the absence of cortical control. Following cystometry, bladders were harvested and pharmacomyography was performed on bladder strips to determine tissue-level changes in the absence of CNS input. All mice responded to continuous-fill cystometry by establishing regular filling/voiding cycles. HCN KO mice function showed discrete changes in volume sensitivity vs. WT. Bladder strip studies showed minimal response to isoproterenol regardless of HCN status, and no significant differences in response to carbachol based on HCN status. We conclude that HCN status impacts the brainstem-bladder reflexic control over urine storage/voiding, but not by regulating bladder wall tensions during filling. The absence of HCN influence on the loss of end-organ responsiveness to sympathetic control in old mice points to an increasing dependency on central control mechanisms with aging.

Dangerous γδ T cells in aged mice.

Progressive susceptibility to tumors and infectious diseases in the elderly are a serious threat to public health in aging societies. For this reason, there is growing interest in mechanisms and predictive biomarkers that accompany and potentially cause this process. In this issue of EMBO Reports, Chen etal [1] report the surprising finding that a specific subset of γδ T cells with very limited clonal diversity strongly expands in lymph nodes of aging mice. These T cells uniformly express a T-cell receptor (TCR) composed of a Vγ6 and a Vδ1 chain and show an effector T-cell phenotype characterized by the swift production of the pro-inflammatory cytokine interleukin-17 (IL-17) upon exvivo stimulation (γδT17 cells). Since γδT17 cells are suspected to be pro-tumorigenic [2], the authors next compared how mice of different age coped with an experimental lung cancer challenge and found impaired anti-tumor responses in old mice. Based on these observations, they propose a link between changes of the composition of γδ T cells in the aging lymph nodes and increased risk of cancer development in aged mice.

A plant-derived VLP influenza vaccine elicits a balanced immune response even in very old mice with co-morbidities

BackgroundThe elderly are at high risk from influenza, in part because immunity wanes with age and through the accumulation of comorbidities. A novel plant-derived virus-like-particle (VLP) vaccine bearing influenza hemagglutinin can induce a balanced humoral and cellular response in old mice (16–18 months) while split virion vaccines elicit mostly antibodies. Because mice also collect comorbidities and lose immune competence as they age, we wished to determine how the plant-derived VLP vaccine would perform in animals approaching the end of their life-span.Materials and methodsOld (24–26 months) female BALB/c mice received two intramuscular doses of H1-VLP vaccine, an inactivated H1N1 vaccine (IIV) (both based on A/H1N1/California/07/09) (3μg each) or PBS. Serum was collected on day 42 and humoral responses were measured by enzyme-linked immunosorbent assay (ELISA), microneutralization (MN) and hemagglutination inhibition (HI) assays. Influenza-specific splenocyte CD4+ & CD8+ T cell responses were measured by flow cytometry. Full body computed tomography (CT) and structured necropsies were performed on day 42. Comorbidities including reduced lung volume (kyphosis), masses, abscesses, etc. were assessed using a standard scoring system (1–21) and mice with scores ≥5 were considered to have important comorbidities.ResultsOverall, 53.3% of the animals had significant comorbidities. Three weeks post-boost, HI and MN titres were mostly undetectable but ELISA titres were significantly higher in the H1-VLP animals compared to the IIV group (GMT (95% CI): 961 (427, 2163) vs 425 (200, 903): p = 0.03). Both CD4+(TNFα, IFNγ) and CD8+ (IFNγ) T cell responses were also greater in the H1-VLP group than the IIV.ConclusionsEven in very old mice with comorbidities, the plant-made H1-VLP vaccine elicited a stronger and more balanced immune response than IIV. Animals with fewer comorbidities tended to have the better composite (humoral and cellular) responses. These novel vaccines have the potential to address some of the limitations of current vaccines in the elderly.

Lifelong CMV infection improves immune defense in old mice by broadening the mobilized TCR repertoire against third-party infection

Lifelong interactions between host and the ubiquitous and persistent cytomegalovirus (CMV) have been proposed to contribute to the age-related decline in immunity. Prior work from us and others found some support for that idea, yet evidence that this led to increased vulnerability to other infections was not obtained. Moreover, evidence has accumulated that CMV infection can be beneficial to immune defense in young/adult mice and humans, dominantly via enhanced innate immunity. Here, we describe an unexpected impact of murine CMV (MCMV) upon the T cell response of old mice to Listeria monocytogenes expressing the model antigen, OVA (Lm-OVA). Single-cell sequencing of the OVA-specific CD8 T cell receptor β (TCRβ) repertoire of old mice demonstrated that old MCMV-infected mice recruited many diverse clonotypes that afforded broad and often more efficient recognition of antigenic peptide variants. This stood in contrast to old control mice, which exhibited strong narrowing and homogenization of the elicited repertoire. High-throughput sequencing of the total naïve CD8 TCRβ repertoire showed that many of these diverse OVA-specific clonotypes were present in the naïve CD8 repertoire of mice in all groups (adult, old control, and old MCMV+) yet were only recruited into the Lm-OVA response in MCMV+ old mice. These results have profound implications for our understanding of T cell immunity over a life span and suggest that our coevolution with CMV may include surprising, potentially positive impacts on adaptive heterologous immunity in late life.

Increased Inflammatory Response in Old Mice is Associated with More Severe Neuronal Injury at the Acute Stage of Ischemic Stroke.

Stroke occurs mostly in patients with advanced age. Elderly patients have a less favorable prognosis compared with young adult patients. To understand the underlying mechanisms, we tested our hypothesis that an increased inflammatory response to acute ischemic injury in old stroke mice leads to more severe brain damage and behavioral dysfunction. An ischemic stroke model was created in 2- and 12-month-old C57BL/6 mice through permanent occlusion of the left distal middle cerebral artery (dMCAO). Infarct/atrophy volumes were quantified by staining the brain sections with Cresyl Violet. Sensorimotor function was assessed using the corner test and adhesive removal test. Quantification of CD68+ cells in the peri-infarct region was performed at 1, 3 and 14 days after dMCAO. Interleukin-6 (IL-6), interleukin-1 β (IL-1β) and vascular endothelial growth factor (VEGF) levels in the ischemic brain tissue were measured using ELISA. Western blot was used to determine the expression levels of tight junction proteins, claudin-5 and zonula occludens (ZO)-1. Blood-brain barrier permeability was measured by Evans blue (EB) extravasation. Gelatinase B (MMP-9, type IV collagenase) was measured by gel zymography. Compared to 2-month-old mice, 12-month-old mice had more severe behavioral deficits at both the acute and chronic stages of stroke. Compared with the 2-month-old mice, 12-month-old mice had larger infarct/atrophy volumes at 1 and 14 days after dMCAO, higher levels of IL-6 and IL-1β, higher MMP9 activity, and lower levels of claudin-5 and ZO-1 at 1 and 3 days after dMCAO. 12-month-old mice also had more CD68+ cells in the peri-infarct region at 1, 3 and 14 days after dMCAO and more EB leakage at 3 days after dMCAO. A higher inflammatory response at the acute stage of ischemic stroke in old mice is associated with more severe neuronal injury and long-term behavioral dysfunction.

Restoration of Retarded Influenza Virus-specific Immunoglobulin Class Switch in Aged Mice.

ObjectiveThe declined immune response to infection causes significant higher morbidity and mortality in aging in spite of the coexisted hyperimmunoglobulinemia (HIG). This study is to reveal the cellular basis of HIG and mechanism of weakened HA-specific IgG response in aged mice and to test cell therapy in the treatment of age-related IgG antibody production deficiency with immunocyte adoptive transfer.MethodsBALB/c mice was immunized with Influenza A/Taiwan vaccine and challenged with the same strain of virus. ELISA was used to assess the levels of total immunoglobulins and antigen specific antibody response. The flow cytometry and ELISPOT were used to evaluate the frequencies of total immunoglobulin- and specific antibody-producing and secreting B lymphocytes. In vitro expanded mononuclear cells, CD4+ T lymphocytes and CD20+ B lymphocytes from old and young mice were adoptively transferred into influenza virus-challenged aged mice, and HA-specific IgG responses were observed.ResultsIt is found that old mice exhibited higher levels of total serum IgG, IgM and IgA, higher frequencies of IgG+, IgM+ and IgA+ cells, and greater antigen-specific IgM and IgA responses to influenza infection, in comparison to young mice. However, influenza antigen- specific IgG and its subclass responses in old mice were significantly lower.ConclusionThe retarded specific IgG response could be attributed to an insufficiency of immunoglobulin class switch in aging. Correlation analysis indicated that HIG and deficient specific IgG production in aged mice could be independent to each other in their pathogenesis. Correction of deficient specific IgG production by adoptive transfer of in vitro expanded and unexpanded CD4+ cells from immunized young mice suggests the CD4+ cell dysfunction contributes to the insufficiency of immunoglobulin class switch in aged mice. The transfusion of in vitro expanded lymphocytes could be a potential effective therapy for the age-related immunodeficiency and could play a role in the infection prevention in aging.

Dysregulation of Toll-Like Receptor 7 Compromises Innate and Adaptive T Cell Responses and Host Resistance to an Attenuated West Nile Virus Infection in Old Mice.

The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), a mosquito-borne flavivirus, has induced severe neurological symptoms, mostly in the elderly population. No vaccines are available for human use. Recent work showed that an attenuated WNV, a nonstructural (NS) 4B-P38G mutant, induced no lethality but strong immune responses in young (6- to 10-week-old) mice. While studying protective efficacy, we found unexpectedly that old (21- to 22-month) mice were susceptible to WNV NS4B-P38G mutant infection but were protected from subsequent lethal wild-type WNV challenge. Compared to responses in young mice, the NS4B-P38G mutant triggered higher inflammatory cytokine and interleukin-10 (IL-10) production, a delayed γδ T cell expansion, and lower antibody and WNV-specific T cell responses in old mice. Toll-like receptor 7 (TLR7) is expressed on multiple types of cells. Impaired TLR7 signaling in old mice led to dendritic cell (DC) antigen-presenting function compromise and a reduced γδ T cell and regulatory T cell (Treg) expansion during NS4B-P38G mutant infection. R848, a TLR7 agonist, decreased host vulnerability in NS4B-P38G-infected old mice by enhancing γδ T cell and Treg expansion and the antigen-presenting capacity of DCs, thereby promoting T cell responses. In summary, our results suggest that dysregulation of TLR7 partially contributes to impaired innate and adaptive T cell responses and an enhanced vulnerability in old mice during WNV NS4B-P38G mutant infection. R848 increases the safety and efficacy during immunization of old mice with the WNV NS4B-P38G mutant. The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), an emerging mosquito-borne flavivirus, has induced severe neurological symptoms more frequently in the elderly population. No vaccines are available for human use. Here, we used an aged mouse model to investigate the protective efficacy of an attenuated WNV, the nonstructural 4B-P38G mutant, which was previously shown to induce no lethality but strong immune responses in young adult mice. Studies that contribute to a mechanistic understanding of immune defects in the elderly will allow the development of strategies to improve responses to infectious diseases and to increase vaccine efficacy and safety in aging individuals.

Differential proteomics reveals age-dependent liver oxidative costs of innate immune activation in mice

Individual response to an immune challenge results from the optimization of a trade-off between benefits and costs of immune cell activation. Age-related immune disorders may have several mechanistic bases, from immune cell defects to chronic pro-inflammatory status and oxidative imbalance, but we are still lacking experimental data showing the relative importance of each of these mechanisms. Using a proteomic approach and subsequent biochemical validations of proteomics-derived hypotheses, we found age-dependent regulations in the liver of 3-months and 1-year old-mice in response to an acute innate immune activation. Old mice presented a chronic up-regulation of several proteins involved in pathways related to oxidative stress control. Interestingly, these pathways were weakly affected by the innate immune activation in old compared to young individuals. In addition, old mice suffered from lower glutathione-S-transferase activity and from higher oxidative damage at the end of the experiment, thus suggesting that they paid a higher immune-related cost than young individuals. On the whole, our data showed that a substantial fraction of the liver costs elicited by an activation of the innate immune response is effectively related to oxidative stress, and that ageing impairs the capacity of old individuals to control it. SignificanceOur paper tackles the open question of the cost of mounting an innate immune response. Evolutionary biologists are familiar since a long time with the concept of trade-offs among key traits of an organism, trade-offs that shape life history trajectories of species and individuals, ultimately in terms of reproduction and survival. On the other hand, medicine and molecular biologists study the intimate mechanisms of immune senescence and underline that oxidative imbalance is probably playing a key role in the progressive loss of immune function with age. This paper merges the two fields by exploring the nature of the cellular pathways that are mainly affected by age when the innate immunity is triggered. To this purpose, a proteomic approach was used to explore liver protein profiles and provide for the first time convincing data supporting the idea that oxidative stress constitutes a cost of innate immune response in old mice, possibly contributing to senescence. Proteomics-derived hypotheses were furthermore validated using biochemical assays. This paper therefore illustrates the added value of using proteomics to answer evolutionary biology questions, and opens a promising way to study the inter-specific variability in the rates of immune-senescence.

Impaired Immune Response in Old Mice Suffering from Obesity and Premature Immunosenescence in Adulthood.

Obesity and aging share an impaired immune system and oxidative and inflammatory stress. Therefore, the hypothesis of obesity as a possible model of premature immunosenescence has been proposed. In this study, we investigated whether adult obese mice, as a consequence of being fed with a fat-rich diet during their adolescence, showed premature immunosenescence and if this was aggravated with aging. Peritoneal cell suspensions were obtained when ICR/CD1 obese female mice were adults (28 weeks) and old (72 weeks), and several functions and antioxidant defenses were evaluated. The results showed that the chemotaxis of both macrophages and lymphocytes, phagocytosis of macrophages, activity of natural killer cells, proliferative response of lymphocytes, interleukin-1β, tumor necrosis factor-alpha, interleukin-6, interleukin-2, and interleukin-10 released in leukocyte cultures, as well as antioxidant and oxidant capacity were significantly impaired in adult obese mice with respect to adult nonobese mice, with values similar to those in chronologically old mice. When these obese animals grew older, although having been fed with a standard diet, they showed a higher deterioration of their immune functions in comparison with the old control group. In conclusion, these results demonstrate that a high fat intake during adolescence can produce an obesity state in adult age associated with a premature immunosenescence, which is aggravated through aging.

Age Related Phenotype of Diabetic and Obese Mouse Models

Our purpose was to examine age-related phenotypic changes in db/db and ob/ob mice, compared to their genetic background control C57bl/6 mice. The population of mice includes animals from many studies, some presented in smaller group sizes elsewhere. Here we compiled data from N=228 C57, N=63 db/db and N=30 ob/ob mice. Mouse age was grouped as juvenile (<70 d), young adult (70-110 d), mid-age (111-220 d) and old (> 220 d). Weight, blood glucose, hematocrit and baseline arteriolar diameter and vasoactive responses were compared. In juveniles, both weight and blood glucose was significantly higher in db/db or ob/ob compared to C57. In young or mid-aged adults, the weight ranking is C57<db/db<ob/ob, while the blood glucose ranking is C57<ob/ob<db/db. Despite increased variability in the genetically modified groups, these comparisons show statistical differences from C57. Old C57 show increased weight and blood glucose, while db/db decreased weight and blood glucose to an equivalent level as C57. Baseline arteriolar diameter increased with age for all strains, and was smaller for db/db and larger for ob/ob mice; this was found for both the cremaster and mesenteric arterioles. Vasoactive responses in old mice show a similar tendency in all strains for diminished dilation to adenosine or acetylcholine and constriction to phenylephrine that is enhanced in db/db or ob/ob mice. Thus, although weight and blood glucose are not different between old control and diabetic mice, loss of vasoactivity is greater at all ages in the genetically modified mice. NIH DK68401 AHA 0655908T

Effects of aging on osteogenic response and heterotopic ossification following burn injury in mice.

Heterotopic ossification (HO) is a common and debilitating complication of burns, traumatic brain injuries, and musculoskeletal trauma and surgery. Although the exact mechanism of ectopic bone formation is unknown, mesenchymal stem cells (MSCs) capable of osteogenic differentiation are known to play an essential role. Interestingly, the prevalence of HO in the elderly population is low despite the high overall occurrence of musculoskeletal injury and orthopedic procedures. We hypothesized that a lower osteogenicity of MSCs would be associated with blunted HO formation in old compared with young mice. In vitro osteogenic differentiation of adipose-derived MSCs from old (18-20 months) and young (6-8 weeks) C57/BL6 mice was assessed, with or without preceding burn injury. In vivo studies were then performed using an Achilles tenotomy with concurrent burn injury HO model. HO formation was quantified using μCT scans, Raman spectroscopy, and histology. MSCs from young mice had more in vitro bone formation, upregulation of bone formation pathways, and higher activation of Smad and nuclear factor kappa B (NF-κB) signaling following burn injury. This effect was absent or blunted in cells from old mice. In young mice, burn injury significantly increased HO formation, NF-κB activation, and osteoclast activity at the tenotomy site. This blunted, reactive osteogenic response in old mice follows trends seen clinically and may be related to differences in the ability to mount acute inflammatory responses. This unique characterization of HO and MSC osteogenic differentiation following inflammatory insult establishes differences between age populations and suggests potential pathways that could be targeted in the future with therapeutics.

Blunted hypertrophic response in old mouse muscle is associated with a lower satellite cell density and is not alleviated by resveratrol

BackgroundSarcopenia contributes to the decreased quality of life in the older person. While resistance exercise is an effective measure to increase muscle mass and strength, the hypertrophic response may be blunted in old age. ObjectivesTo determine 1) whether hypertrophy in the m. plantaris of old mice was blunted compared to adult and 2) whether this was related to a reduced satellite cell (SC) density and 3) how resveratrol affects hypertrophy in old mice. MethodsIn adult (7.5months, n=11), old (23.5months, n=10) and old-resveratrol-treated (n=10) male C57BL/6J mice, hypertrophy of the left m. plantaris was induced by denervation of its synergists. The contralateral leg served as control. ResultsAfter six weeks, overload-induced myofiber hypertrophy and IIB–IIA shift in myofiber type composition were less pronounced in old than adult mice (P=0.03), irrespective of resveratrol treatment. Muscles from old mice had a lower SC density than adult muscles (P=0.002). Overload-induced SC proliferation (P<0.05) resulted in an increased SC density in old, but not adult muscles (P=0.02), while a decrease occurred after resveratrol supplementation (P=0.044). Id2 and myogenin protein expression levels were higher in old than adult muscles (P<0.05). Caspase-3 was expressed more in hypertrophied than control muscles and was reduced with resveratrol (P<0.05). ConclusionThe blunted hypertrophic response in old mice was associated with a lower SC density, but there was no evidence for a lower capacity for proliferation. Resveratrol did not rescue the hypertrophic response and even reduced, rather than increased, the number of SCs in hypertrophied muscles.

Alterations of the Murine Gut Microbiome with Age and Allergic Airway Disease.

The gut microbiota plays an important role in the development of asthma. With advanced age the microbiome and the immune system are changing and, currently, little is known about how these two factors contribute to the development of allergic asthma in the elderly. In this study we investigated the associations between the intestinal microbiome and allergic airway disease in young and old mice that were sensitized and challenged with house dust mite (HDM). After challenge, the animals were sacrificed, blood serum was collected for cytokine analysis, and the lungs were processed for histopathology. Fecal pellets were excised from the colon and subjected to 16S rRNA analysis. The microbial community structure changed with age and allergy development, where alterations in fecal communities from young to old mice resembled those after HDM challenge. Allergic mice had induced serum levels of IL-17A and old mice developed a greater allergic airway response compared to young mice. This study demonstrates that the intestinal bacterial community structure differs with age, possibly contributing to the exaggerated pulmonary inflammatory response in old mice. Furthermore, our results show that the composition of the gut microbiota changes with pulmonary allergy, indicating bidirectional gut-lung communications.

Accumulation of 4-1BBL+ B cells in the elderly induces the generation of granzyme-B+ CD8+ T cells with potential antitumor activity

AbstractAlthough the accumulation of highly-differentiated and granzyme B (GrB)-expressing CD8+CD28– T cells has been associated with aging, the mechanism for their enrichment and contribution to immune function remains poorly understood. Here we report a novel B-cell subset expressing 4-1BBL, which increases with age in humans, rhesus macaques, and mice, and with immune reconstitution after chemotherapy and autologous progenitor cell transplantation. These cells (termed 4BL cells) induce GrB+CD8+ T cells by presenting endogenous antigens and using the 4-1BBL/4-1BB axis. We found that the 4BL cells increase antitumor responses in old mice, which may explain in part the paradox of retarded tumor growth in the elderly. 4BL cell accumulation and its capacity to evoke the generation of GrB+CD8+ T cells can be eliminated by inducing reconstitution of B cells in old mice, suggesting that the age-associated skewed cellular immune responses are reversible. We propose that 4BL cells and the 4-1BBL signaling pathway are useful targets for improved effectiveness of natural antitumor defenses and therapeutic immune manipulations in the elderly.

MTORC1 inhibition pronounces CD8 T cell effector defects in aging (P1233)

Abstract Mammalian target of rapamycin complex 1 (mTORC1) is a critical cell nutrient sensor and its role in CD8 T cell development, homeostasis, and fate decisions during infection are becoming increasingly evident. Seminal studies showed that direct mTORC1 inhibition with rapamcyin favored memory CD8 T cell development after infection. Simultaneously, rapamycin was shown to extend lifespan in mice. Whether or not rapamycin treatment alters the CD8 T cell immune response in old mice has not been studied. This question is particularly critical given recent data from our lab that CD8 T cell defects after Listeria monocytogenes (Lm) and West Nile Virus infection are both associated with increased infection mortality in old mice. Therefore, we tested the effect of rapamycin (75μg/kg), when given during the T cell priming phase, on CD8 T cell effector responses in adult (3 months) and old (18 months) mice infected with Lm. We found that mTORC1 inhibition with rapamycin significantly inhibited effector differentiation of antigen-specific CD8 T cells and this was especially pronounced in old mice. Additionally, contrary to previous reports, we measured no benefit on CD8 T cell memory formation in either old or adult mice. Therefore, we conclude that any beneficial effects of rapamycin on memory formation likely come at the cost of effector function, and these effects may be pathogen-specific. Furthermore, rapamycin may not be a suitable candidate for extending lifespan or “Healthspan.”