Aging-independent decrease of complex multi-spine boutons in hippocampal area CA1 after contextual fear conditioning

Limited recovery of striatal dopaminergic fibers by adrenal medullary grafts in MPTP-treated aging mice

Age is the biggest risk factor for developing most forms of cancer. In breast cancer, over 50% of patients are over 60 years old; however, less than 25% of patients enrolled in clinical trials are in this age bracket. Furthermore, most mouse modeling is performed in young mice, meaning that from preclinical through clinical stages patients ages are not accurately reflected in trials designs. Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with no targeted therapies, so many treatment strategies utilize chemotherapy in combination with immunotherapy – which relies on a functional immune response. It is well known that immune function declines as we age, and thus therapeutic responses may differ with age. It is yet unclear how the tumor immune microenvironment changes with age, and how this may impact therapeutic response. Here, we utilize a mouse model which mimics hallmarks of human immune aging to understand how age impacts TNBC immune responses to dictate disease progression and therapeutic response. Immunologically young (8 weeks old) and aged (∼12 months old) FVB mice were orthotopically injected with syngeneic Met1 TNBC cells. Once tumors became palpable, mice were treated with paclitaxel chemotherapy or vehicle control and/or anti-PDL1 immunotherapy to mimic first-line therapeutic strategies. Tumors grew significantly faster in young control mice compared to aged, but young mice had a more robust response to therapy. Notable, only young mice had a significant benefit of combo therapy over monotherapy alone. Despite aged mice having more circulating CD8+ and CD4+ T cells, tumors in aged mice had significantly more infiltration of CD8+ cytotoxic T cells and FOXP3+ inhibitory T-regulatory cells compared to young control mice, yet only tumors in young mice exhibited an increase in T cell infiltration with therapy. Additionally, the ratio of CD8+ T cells and FOXP3+ Tregs was unchanged with age or treatment. Depletion of either CD8+ or CD4+ T cells led to a complete loss of age-associated differences in tumor growth. RNA sequencing analysis suggests that infiltrating T cells in aged mice may be exhausted due to a chronic interferon (IFN) phenotype, indicated by enrichment of IFN-alpha, IFN-gamma, and IL2 pathways, as well as increased expression of immune exhaustion markers PD1, CTLA4, TIM3 and LAG3. Neutralization of IFN-gamma blocks therapeutic response to anti-PDL1 in young mice but has no impact on aged mice. Analysis of published single cell RNAseq data indicated the same IFN response pathways enriched with age in human TNBC. These results demonstrate that the aging tumor immune microenvironment significantly impacts tumor progression and therapeutic response and suggest that targeting IFN signaling may lead to improved therapeutic responses. Together these findings establish a significant impact of immune aging on TNBC disease progression and response to therapy, highlighting the importance of developing age- stratified treatment strategies to improve responses in patients of all ages. Citation Format: Milos Spasic, Adrienne Parsons, Abigail Recko, Elizabeth Mittendorf, Peter van Galen, Sandra McAllister. Age dictates immune response within the tumor microenvironment of triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C016.

Immunosuppression after injury in aged mice is associated with a TH1–TH2 shift, which can be restored by estrogen treatment

This study was designed to define the lipopolysaccharide (LPS) sensitivity of aged mice in terms of lethality and cytokine production and to determine down-regulating responses of corticosterone and interleukin 10 (IL-10). The 50% lethal doses of LPS in young (6- to 7-week-old) and aged (98- to 102-week-old) mice were 601 and 93 microg per mouse (25.6 and 1.6 mg per kg of body weight), respectively. Aged mice were approximately 6.5-fold more sensitive to the lethal toxicity of LPS in micrograms per mouse (16-fold more sensitive in milligrams per kilogram) than young mice. Levels in sera of tumor necrosis factor-alpha (TNF-alpha) IL-1alpha, and IL-6 after intraperitoneal injection of 100 microg of LPS peaked at 1.5, 3, and 3 h, respectively, and declined thereafter in both groups of mice. However, the peak values of these cytokines were significantly higher in aged than in young mice (P < 0.05). Gamma interferon (IFN-gamma) was detectable at 3 h, and sustained high levels were still detected after 12 h in both age groups. Although there were no significant differences in levels of IFN-gamma in sera from both groups, aged mice showed higher IFN-gamma levels throughout the 3- to 12-h study period. Administration of increasing doses of LPS revealed that aged mice had a lower threshold to IL-1alpha production than young mice. In addition, aged mice were approximately 4-fold more sensitive to the lethal toxicity of exogenous TNF in units per mouse (10-fold more sensitive in units per kilogram) than young mice. With regard to down-regulating factors, corticosterone amounts were similar at basal levels and no differences in kinetics after the LPS challenge were observed, whereas IL-10 levels in sera were significantly higher in aged mice at 1.5 and 3 h than in young mice (P < 0.01). These results indicate that aged mice are more sensitive to the lethal toxicities of LPS and TNF than young mice. We conclude that a relatively activated, or primed, state for LPS-induced cytokine production, in spite of full down-regulating responses by corticosterone and IL- 10, may explain at least in part LPS sensitivity in aged mice.

Long-term effect of MPTP in the mouse brain in relation to aging: neurochemical and immunocytochemical analysis

With the failure of so many pre-clinical stroke studies to translate into the clinic, there is a need to find new therapeutics to minimize the extent of cellular damage and aid in functional recovery. Domain V (DV), the c-terminal protein fragment of the vascular basement membrane component, perlecan, was recently shown to afford significant protection in multiple transient middle cerebral artery occlusion stroke models. We sought here to determine whether DV might have similar therapeutic properties in a focal photothrombosis stroke model in both young and aged mice. Young (3-month old) and aged (24-month old) mice underwent photothrombotic stroke to the motor cortex and were then treated with DV or phosphate buffered saline vehicle at different initial time points up to 7days. Stroke volume was analyzed histologically using cresyl violet and functional recovery assessed behaviorally on both the grid-walking and cylinder tasks. In young mice, DV administration resulted in a significant decrease in infarct volume when treatment started 3 or 6h post-stroke. In aged mice, DV administration was only protective when started 3h post-stroke. In addition to a decrease in the area of infarction, DV treatment was effective in significantly decreasing the number of foot-faults on the grid-walking task and improving use of the stroke-affected limb in the cylinder task in both young and aged. Previously, we have shown that DV can alter the expression profile of various astroglial markers. Consistent with our previous finding, treatment groups that showed therapeutic potential in both young and aged mice also showed an elevation in glial fibrillary acidic protein (GFAP) expression in peri-infarct regions. We conclude that DV is neuroprotective and affords significant improvements in functional recovery in both young and aged mice after focal ischemia. These data also highlight a therapeutic time-window shift that is narrower in aged compared with young mice and is associated with an elevation in GFAP expression and heightened astrogliosis.

Rationale: In the United States, chronic non-healing wounds affect more than 6.5 million patients yearly. While advanced age and diabetes are well recognized risk factors for non-healing wounds, little is known about the mechanism by which aging impairs wound healing. We have shown that diabetes impairs wound healing through a hematopoietic stem cell (HSC)-autonomous mechanism. Objective: This study tests the hypothesis that aging impairs wound healing through a hematopoietic stem cell (HSC) autonomous mechanism that disrupts the normal orchestrated immune cell response across the three phases of wound healing. Methods and Results: To test this hypothesis, cutaneous wounds were created in young (2-month-old) and aged mice (24-month-old). The wound closure rate of aged mice was almost two times longer than that in young mice (Young WT vs. Aged WT: 24 vs. 49). To determine if this difference is due to an HSC-autonomous mechanism, we generated chimeric mouse models by transplanting HSCs from either young or aged WT donor mice into lethally irradiated young WT mice. The wound closure rate of young WT mice reconstituted with HSCs of aged mice was similar to that in aged mice, consistent with an HSC-autonomous mechanism. To determine the effects of aging on HSC differentiation towards immune cells and thereby on wound healing, chimeric mouse models were generated by which young WT mice were reconstituted with HSCs from young or aged EGFP mice. In the cutaneous wounds of young WT recipient with HSCs from aged EGFP mice, neutrophils and proinflammatory Ly6Chi monocytes/macrophages were significantly increased during all three phases of wound healing. More than 90% of neutrophils and macrophages in cutaneous wounds were derived from donor HSCs. As for T cells, decreased CD4/CD8 ratio was observed during last phase (tissue remodeling phase) of wound healing, but no difference in CD3e+ T cells. Conclusion: This study shows for the first time that the HSC-derived immune cells are the major cell components dynamically responding to wound healing processes. Aging impairs wound healing by HSC autonomous mechanisms that dysregulates the normal immune cell response. These findings could lead to a novel HSC-based therapy to restore normal wound healing.

Chronic low-grade inflammation is a hallmark of aging, aka "inflammaging", which is linked to a wide range of age-associated diseases. Immune dysfunction increases disease susceptibility, and increases morbidity and mortality of aging. Innate immune cells, including monocytes, macrophages and neutrophils, are the first responders of host defense and the key mediators of various metabolic and inflammatory insults. Currently, the understanding of innate immune programming in aging is largely fragmented. Here we investigated the phenotypic and functional properties of innate immune cells in various peripheral tissues of young and aged mice under normal and endotoxic conditions. Under the steady state, aged mice showed elevated pro-inflammatory monocytes/macrophages in peripheral blood, adipose tissue, liver, and colon. Under lipopolysaccharide (LPS)-induced inflammatory state, the innate immune cells of aged mice showed a different response to LPS stimulus than that of young mice. LPS-induced immune responses displayed differential profiles in different tissues and cell types. In the peripheral blood, when responding to LPS, the aged mice showed higher neutrophils, but lower pro-inflammatory monocytes than that in young mice. In the peritoneal fluid, while young mice exhibited significantly elevated pro-inflammatory neutrophils and macrophages in response to LPS, aged mice exhibited decreased pro-inflammatory neutrophils and variable cytokine responses in macrophages. In the adipose tissue, LPS induced less infiltrated neutrophils but more infiltrated macrophages in old mice than young mice. In the liver, aged mice showed a more robust increase of pro-inflammatory macrophages compared to that in young mice under LPS stimulation. In colon, macrophages showed relatively mild response to LPS in both young and old mice. We have further tested bone-marrow derived macrophages (BMDM) from young and aged mice, we found that BMDM from aged mice have impaired polarization, displaying higher expression of pro-inflammatory markers than those from young mice. These data collectively suggest that innate immunity in peripheral tissues is impaired in aging, and the dysregulation of immunity is tissue- and cell-dependent. Our findings in the rodent model underscore the complexity of aging immunity. Further investigation is needed to determine whether the immune profile observed in aged mice is applicable in age-associated diseases in humans.

1. The contractile force was compared in isolated soleus muscles from young (2.5-8 months old) and aged (28-31 months old) mice. Force was measured at 25 degrees C during isometric tetanic contractions during isovelocity stretching and shortening contractions. 2. The normalized isometric force was lower by 13.3% in muscles from aged mice. Muscles from young and aged mice produced 0.951 +/- 0.031 N mg-1 (n = 12) and 0.824 +/- 0.048 N mg-1 (n = 9) respectively. The relaxation time, from 90 to 10% of the tetanic force, of muscles from aged mice was 102.1 +/- 3.7 ms (n = 6), which was longer than that for muscles from young mice, 84.4 +/- 3.8 ms (n = 6) (means +/- S.E.M.). 3. The force during shortening was also reduced in muscles from aged animals by the same proportion as the isometric force. Therefore the force during shortening relative to the isometric force was the same in muscles from young and aged mice. 4. During rapid stretching soleus muscles from aged mice produced a similar force to those from young mice. Therefore stretch can remove the weakness in muscles of aged mice. 5. These changes in muscles from aged mice are similar to those produced when inorganic phosphate (Pi) levels are raised, in skinned rabbit psoas fibres, or during fatigue or with low intracellular pH (pHi), in frog muscle. It is possible therefore that the force loss due to ageing may be due to a higher Pi level or a lower pHi.

BackgroundRetinal fibrosis affects 40–70% of neovascular age-related macular degeneration patients. This study investigated the effect of ageing on subretinal fibrosis secondary to choroidal neovascularization and the mechanism of action.MethodsSubretinal fibrosis was induced in young (2.5-month) and aged (15–16-month) C57BL/6J mice using the two-stage laser protocol. Five and 30 days later, eyes were collected and stained for CD45 and collagen-1 and observed by confocal microscopy. Fibrocytes (CD45+collagen-1+) were detected in the bone marrow (BM), blood and fibrotic lesions by flow cytometry and confocal microscopy, respectively. BM-derived macrophages (BMDMs) were cultured from young and aged mice with or without TGF-β1 (10 ng/mL) treatment. The expression of mesenchymal marker αSMA (Acta2), fibronectin (Fn1) and collagen-1 (Col1a1) was examined by qPCR and immunocytochemistry, whereas cytokine/chemokine production was measured using the Luminex multiplex cytokine assay. BM were transplanted from 22-month (Ly5.2) aged mice into 2.5-month (Ly5.1) young mice and vice versa. Six weeks later, subretinal fibrosis was induced in recipient mice and eyes were collected for evaluation of fibrotic lesion size.ResultsUnder normal conditions, the number of circulating fibrocytes (CD45+collagen-1+) and the expression levels of Tgfb1, Col1a1, Acta2 and Fn1 in BMDMs were significantly higher in aged mice compared to young mice. Induction of subretinal fibrosis significantly increased the number of circulating fibrocytes, enhanced the expression of Col1a1, Acta2 and Fn1 and the production of soluble urokinase plasminogen activator surface receptor (suPAR) but decreased the production of CXCL10 in BMDMs. BMDMs from aged subretinal fibrosis mice produced significantly higher levels of VEGF, angiopoietin-2 and osteopontin than cells from young subretinal fibrosis mice. The subretinal fibrotic lesion in 15–16-month aged mice was 62% larger than that in 2.5-month young mice. The lesion in aged mice contained a significantly higher number of fibrocytes compared to that in young mice. The number of circulating fibrocytes positively correlated with the size of subretinal fibrotic lesion. Transplantation of BM from aged mice significantly increased subretinal fibrosis in young mice.ConclusionsA retina–BM–blood–retina pathway of fibrocyte/macrophage recruitment exists during retinal injury. Ageing promotes subretinal fibrosis through higher numbers of circulating fibrocytes and profibrotic potential of BM-derived macrophages.

The incidence and mortality of sepsis increase with age, consequently, 80% of the clinical mortality from sepsis occurs in patients over age 65. Despite this aged clinical population, most research models of sepsis use 6- to 16-week-old mice as patient surrogates. This age range of mice corresponds to human ages 10 to 17 years. To assess the influence of age on rodent CLP and on antibiotic therapy, we studied young (4 month), mature (12 month), and aged (24 month) mice. Male C57BL/6 mice (n = 27-30 in each age group) were subjected to cecal ligation and puncture (CLP), two punctures with a 25-gauge needle. Mice were observed untreated for 10 days. Young mice had 20% mortality, mature mice had 70% mortality (P = 0.0013 vs. young), and aged mice had 75% mortality (P = 0.0001 vs. young). To assess the effects of age on antibiotic therapy, mice were subjected to CLP as above (n = 38-40 in each age group). Mice were then randomized to treatment with intraperitoneal injections of ceftriaxone and metronidazole or normal saline. Therapy was initiated 12 h after CLP, and injections were repeated every 12 h for 7 days. Young mice saw a 56% decrease in mortality from CLP with antibiotic therapy (P = 0.001), and mature mice had a 30% decrease in mortality (P = 0.06). Aged mice saw no benefit from antibiotic therapy. We also compared plasma cytokine levels between young and aged mice after CLP. When compared with young mice, aged mice had higher levels of IL-6 and TNF-alpha 24 h after CLP. However, high IL-6 was predictive of mortality at any age. Mice appear to have age-dependent responses to intra-abdominal sepsis and to appropriate therapy.

The role of T cells in the reduced frequency of splenic B cells specific for several antigens in aged mice was studied by assessing B cell responsiveness in (a) aged nude mice and (b) irradiated young mice repopulated with splenic B cells or with Ig- bone marrow cells from young mice and T cells from aged vs. young mice. Using the fragment culture technique to assess B cells specific for 2,4-dinitrophenyl (DNP) and for (4-hydroxy-3,5-dinitrophenyl) acetyl, we found that the frequency of responsive splenic B cells in aged BALB/c nude mice was very similar to that of young nude mice. In addition, we found that in chimeric mice constructed with either bone marrow or splenic B cells from young mice and T cells from aged mice the frequency of DNP-specific splenic B cells was significantly lower than that in control chimeras constructed with T cells from young mice. These results indicate that T cells from aged mice can down regulate B cell responsiveness and that a mature, naive B cell may be its possible target. The results of both experimental approaches are consistent with a role for T cells in the regulation of responsive B cells in aging.

Decreased capacity of aged mice to produce interferon-gamma in Legionella pneumophila infection

&lt;div&gt;Abstract&lt;p&gt;Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;hi&lt;/sup&gt; regulatory T cells (Treg) exhibited equivalent &lt;i&gt;in vitro&lt;/i&gt; T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti–Gr-1 antibody was immunologically and clinically more efficacious than anti–Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti–Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved. &lt;i&gt;Cancer Res; 72(8); 2089–99. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Aging influences peripheral nerve regeneration. Nevertheless, most basic research of bioabsorbable nerve conduits including commercial products have been performed in very young animals. Results from these studies may not provide information about axonal regeneration in aged tissue, because young nerve tissue holds sufficient endogenous potential for axonal regeneration. The clinical target age for nerve conduit application is most likely going to increase with a rapidly growing elderly population. In the present study, we examined axonal regeneration after sciatic nerve defects in aged and young mice. 5-mm sciatic nerve defects in young (6 weeks old) and aged (92 weeks old) mice were reconstructed using nerve conduits (composed of a poly lactide and caprolactone) or autografts. In addition, in aged mice, sciatic nerve defects were reconstructed using nerve conduits coated with mouse induced pluripotent stem cell (iPSc)-derived neurospheres. Using electrophysiological and histological techniques, we demonstrated axonal regeneration was significantly less effective in aged than in young mice both for nerve conduits and for nerve autografts. However, despite the low regenerative capacity of the peripheral nerve in aged mice, axonal regeneration significantly increased when nerve conduits coated with iPSc-derived neurospheres, rather than nerve conduits alone, were used. The present study shows that aging negatively affects peripheral nerve regeneration based on nerve conduits in mice. However, axonal regeneration using nerve conduits was improved when supportive iPSc-derived neurospheres were added in the aged mice. We propose that tissue-engineered bioabsorbable nerve conduits in combination with iPSc-derived neurospheres hold therapeutic potential both in young and elderly patients. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1752-1758, 2018.