Psychosocial Stress and Age Influence Depression and Anxiety-Related Behavior, Drive Tumor Inflammatory Cytokines and Accelerate Prostate Cancer Growth in Mice.

Prostate cancer (PCa) prevalence is higher in older men and poorer coping with psychosocial stressors effect prognosis. Yet, interactions between age, stress and PCa progression are underexplored. Therefore, we characterized the effects of age and isolation combined with restraint (2 h/day) for 14 days post-tumor inoculation on behavior, tumor growth and host defense in the immunocompetent, orthotopic RM-9 murine PCa model. All mice were tumor inoculated. Isolation/restraint increased sympathetic and hypothalamic-pituitary-adrenal cortical activation, based on elevated serum 3-methoxy-4-hydroxyphenylglycol/norepinephrine ratios and corticosterone levels, respectively. Elevated zero maze testing revealed age-related differences in naïve C57Bl/6 mice, and increased anxiety-like behavior in tumor-bearing mice. In open field testing, old stressed mice were less active throughout the 30-min test than young non-stressed and stressed, and old non-stressed mice, suggesting greater anxiety in old stressed mice. Old (18 month) mice demonstrated more depression-like behavior than young mice with tail suspension testing, without effects of isolation/restraint stress. Old mice developed larger tumors, despite similar tumor expression of tumor vascular endothelial growth factor or transforming growth factor-beta1 across age. Tumor chemokine/cytokine expression, commonly prognostic for poorer outcomes, were uniquely age- and stress-dependent, underscoring the need for PCa research in old animals. Macrophages predominated in RM-9 tumors. Macrophages, and CD4+ and CD4+FoxP3+ T-cell tumor infiltration were greater in young mice than in old mice. Stress increased macrophage infiltration in old mice. Conversely, stress reduced intratumoral CD4+ and CD4+FoxP3+ T-cell numbers in young mice. CD8+ T-cell infiltration was similar across treatment groups. Our findings support that age- and psychological stress interacts to affect PCa outcomes by interfering with neural-immune mechanisms and affecting behavioral responses.

Maternal plasma proteomics in a rat model of pregnancy complications reveals immune and pro-coagulant gene pathway activation.

PROBLEM: The Brown Norway (BN) rat is a model of T-helper 2 immune diseases, and also a model of pregnancy disorders that include placental insufficiency, fetal loss, and pre-eclampsia-like symptoms. The aim of this study was to investigate the plasma proteomic/cytokine profile of pregnant BN rats in comparison to that of the Lewis (LEW) rat strain. METHOD OF STUDY: Plasma proteomics differences were studied at day 13 of pregnancy in pooled plasma samples by differential in-gel electrophoresis, and protein identification was performed by mass spectrometry. Key protein findings and predicted cytokine differences were validated by ELISA using plasma from rats at various pregnancy stages. Proteomics data were used for ingenuity pathway analysis (IPA). RESULTS: In-gel analysis revealed 74 proteins with differential expression between BN and LEW pregnant dams. ELISA studies confirmed increased maternal plasma levels of complement 4, prothrombin, and C-reactive protein in BN compared to LEW pregnancies. LEW pregnancies showed higher maternal plasma levels of transthyretin and haptoglobin than BN pregnancies. Ingenuity pathway analysis revealed that BN pregnancies are characterized by activation of pro-coagulant, reactive oxygen species, and immune-mediated chronic inflammation pathways, and suggested increased interleukin 6 and decreased transforming growth factor-ß1 as potential upstream events. Plasma cytokine analysis revealed that pregnant BN dams have a switch from anti- to pro-inflammatory cytokines with the opposite switch observed in pregnant LEW dams. CONCLUSION: Brown Norway rats show a maternal pro-inflammatory response to pregnancy that likely contributes to the reproductive outcomes observed in this rat strain.

Sympathetic neurotransmission in spleens from aging Brown-Norway rats subjected to reduced sympathetic tone.

Senescence of innate and adaptive responses and low-grade inflammation (inflammaging) hallmarks normal aging, which increases vulnerability to infectious diseases, autoimmunity and cancer. In normal aging, sympathetic dysregulation contributes to the dysregulation of innate and adaptive immunity and inflammaging. Sympathetic innervation of immune cells in secondary immune organs regulates immune responses. Previously in Fischer 344 (F344) rats, we reported an age-related increase in sympathetic tone and sympathetic dysfunction in beta-adrenergic receptor (AR) signaling of splenic lymphocytes that contributes to immune senescence, although the responsible mechanisms remains unexplored. In this study, we extend our previous findings using the much longer-lived Brown-Norway (BN) rats, whose behavior and immune response profile differ strikingly from F344 rats. Here, we investigated whether increased sympathetic nerve activity (SNA) in the aging spleen contributes to age-related sympathetic neuropathy and altered neurotransmission in splenic lymphocytes in BN rats. Fifteen-month male BN rats received 0, 0.5 or 1.5 µg/kg/day rilmenidine intraperitoneally for 90 days to lower sympathetic tone. Untreated young and age-matched rats controlled for effects of age. We found that elevated SNA in the aging BN rat spleen does not contribute significantly to sympathetic neuropathy or the aging-induced impairment of canonical ß-AR signal transduction. Despite the rilmenidine-induced increase in ß-AR expression, splenocyte c-AMP production was comparable with age-matched controls, thus dampening nerve activity had no effect on receptor coupling to adenylate cyclase. Understanding how aging affects neuroimmune regulation in healthy aging rodent models may eventually lead to strategies that improve health in aging populations vulnerable to immunosenescence and low-grade systemic inflammation.

Prevention of Mammary Tumor Development through Neuroimmunomodulation in the Spleen and Lymph Nodes of Old Female Sprague-Dawley Rats by L-Deprenyl.

BACKGROUND: Development of mammary tumors is an age-associated phenomenon that is likely due to deficits in the neuroendocrine-immune interactions. Previously, we demonstrated that L-deprenyl, a monoamine oxidase-B (MAO-B) inhibitor, can enhance immune responses and restore noradrenergic (NA) innervation in the spleens of rats with carcinogen-induced and spontaneously developing mammary tumors. OBJECTIVES: To investigate whether (1) treatment of early middle-aged female rats would prevent the spontaneous development of mammary tumors accompanied by restoration of immunity in the spleen and draining lymph nodes (DLN) and sympathetic NA innervation in the spleen and (2) deprenyl can influence the proliferation of estrogen receptor (ER)-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231 and Hs 578T) human breast cancer cells. METHODS: Early middle-aged (8- to 9-month-old) female Sprague-Dawley rats were treated with 0, 1.0 or 2.5 mg of deprenyl/kg body weight (BW) daily i.p. for 12 months. Cells of ER-positive (ER+) and ER-negative (ER-) human breast cancer cell lines were incubated with media or 10(-3) to 10(-8) M deprenyl for 1, 2, 4 or 6 days to examine the proliferation of cells. RESULTS: Tumor incidence increased in saline-treated old female rats, while deprenyl treatment significantly reduced the incidence of mammary tumors in these rats. Saline-treated tumor-bearing rats exhibited reduced splenic NA innervation and norepinephrine (NE) content, splenic interleukin (IL)-2 and interferon (IFN)-γ levels and NK cell activity as well as DLN IL-2 and IFN-γ levels compared to young female rats without tumors. In contrast, treatment with 2.5 mg/kg of deprenyl enhanced IL-2 and IFN-γ production in both the spleen and DLN as well as splenic natural killer (NK) cell activity. Deprenyl treatment also increased concanavalin A (Con A)-induced proliferation of T lymphocytes in the DLN. Deprenyl-induced changes in immune responses were accompanied by enhanced NA innervation and NE content in the spleen. In vitro incubation of various concentrations of deprenyl with ER+ human breast cancer cell lines partly inhibited the proliferation of cells, while it had no effect on the ER- breast cancer cells. CONCLUSIONS: These results suggest that (1) development of mammary tumors is mediated through the loss of immunity and sympathetic NA nerve fibers accompanied by reduced NE levels in the spleen, (2) the prevention of mammary tumor development by deprenyl may involve the reversal of the tumor-associated decline in sympathetic NA activity and cell-mediated immune responses in the spleen and DLN and (3) the antitumor effects of deprenyl may be partially mediated through ER-dependent intracellular signaling pathways.

Chronically lowering sympathetic activity protects sympathetic nerves in spleens from aging F344 rats.

In the present study, we investigated how increased sympathetic tone during middle-age affects the splenic sympathetic neurotransmission. Fifteen-month-old (M) F344 rats received rilmenidine (0, 0.5 or 1.5mg/kg/day, i.p. for 90 days) to lower sympathetic tone. Controls for age were untreated 3 or 18M rats. We report that rilmenidine (1) reduced plasma and splenic norepinephrine concentrations and splenic norepinephrine turnover, and partially reversed the sympathetic nerve loss; and (2) increased ß-adrenergic receptor (ß-AR) density and ß-AR-stimulated cAMP production. Collectively, these findings suggest a protective effect of lowering sympathetic tone on sympathetic nerve integrity, and enhanced sympathetic neurotransmission in secondary immune organs.

Sympathetic innervation of the spleen in male Brown Norway rats: a longitudinal aging study.

Aging leads to reduced cellular immunity with consequent increased rates of infectious disease, cancer, and autoimmunity in the elderly. The sympathetic nervous system (SNS) modulates innate and adaptive immunity via innervation of lymphoid organs. In aged Fischer 344 (F344) rats, noradrenergic (NA) nerve density in secondary lymphoid organs declines, which may contribute to immunosenescence with aging. These studies suggest there is SNS involvement in age-induced immune dysregulation. The purpose of this study was to longitudinally characterize age-related change in sympathetic innervation of the spleen and sympathetic activity/tone in male Brown Norway (BN) rats, which live longer and have a strikingly different immune profile than F344 rats, the traditional animal model for aging research. Splenic sympathetic neurotransmission was evaluated between 8 and 32 months of age by assessing (1) NA nerve fiber density, (2) splenic norepinephrine (NE) concentration, and (3) circulating catecholamine levels after decapitation. We report a decline in NA nerve density in splenic white pulp (45%) at 15 months of age compared with 8-month-old (M) rats, which is followed by a much slower rate of decline between 24 and 32 months. Lower splenic NE concentrations between 15 and 32 months of age compared with 8M rats were consistent with morphometric findings. Circulating catecholamine levels after decapitation stress generally dropped with increasing age. These findings suggest there is a sympathetic-to-immune system dysregulation beginning at middle age. Given the unique T-helper-2 bias in BN rats, altered sympathetic-immune communication may be important for understanding the age-related rise in asthma and autoimmunity.

Sympathetic nervous system and lymphocyte proliferation in the Fischer 344 rat spleen: a longitudinal study.

UNLABELLED: Aging is associated with reduced cellular immunity, which leads to increased rates of infectious disease, cancer and autoimmunity in the elderly. Previous findings from our laboratory revealed an age-related decline in sympathetic innervation of immune organs that affects immunity. These studies suggested potential sympathetic nervous system involvement in age-induced immune dysregulation. OBJECTIVES: The purpose of this study was to longitudinally characterize the effects of age on sympathetic neurotransmission in the spleen and net sympathetic activity/tone in male Fischer 344 rats. METHODS: Splenic sympathetic neurotransmission was evaluated between 8 and 24 months of age by (1) splenic norepinephrine (NE) concentration and turnover, (2) beta-adrenergic receptor (beta-AR) expression and (3) beta-AR-stimulated splenocyte cAMP production. Measures of sympathetic neurotransmission were correlated with age-related changes in Concanavalin A (Con A)-stimulated splenocyte proliferation. RESULTS: Splenic NE turnover increased during middle age, then subsequently declined by 18 months of age compared with 8-month-old controls (young). Splenic NE concentration increased at 10 months and decreased at 18-24 months, compared with young rats; however, plasma NE levels were not affected by age. Plasma epinephrine levels were decreased at 24 months. NE synthesis blockade increased and decreased the rate of plasma catecholamine depletion in middle and old age, respectively. beta-AR-stimulated cAMP production increased in splenocytes by 15 months. An age-related decrease in Con A-induced splenocyte proliferation was apparent by 10 months and persisted through 24 months. The decline in Con A-induced splenocyte proliferation correlated with the age-related increase in cAMP production. CONCLUSIONS: Aging alters sympathetic nervous system metabolism in the spleen to affect beta-AR signaling to splenocytes, suggesting that altered sympathetic-immune modulation changes are evident by early middle age.

Sympathetic modulation of immunity: relevance to disease.

Optimal host defense against pathogens requires cross-talk between the nervous and immune systems. This paper reviews sympathetic-immune interaction, one major communication pathway, and its importance for health and disease. Sympathetic innervation of primary and secondary immune organs is described, as well as evidence for neurotransmission with cells of the immune system as targets. Most research thus far has focused on neural-immune modulation in secondary lymphoid organs, has revealed complex sympathetic modulation resulting in both potentiation and inhibition of immune functions. SNS-immune interaction may enhance immune readiness during disease- or injury-induced 'fight' responses. Research also indicate that dysregulation of the SNS can significantly affect the progression of immune-mediated diseases. However, a better understanding of neural-immune interactions is needed to develop strategies for treatment of immune-mediated diseases that are designed to return homeostasis and restore normal functioning neural-immune networks.

Involvement of lysosomal cathepsins in the cleavage of DNA topoisomerase I during necrotic cell death.

OBJECTIVE: Autoantibodies to DNA topoisomerase I (topo I) are associated with diffuse systemic sclerosis (SSc), appear to be antigen driven, and may be triggered by cryptic epitopes exposed during in vivo topo I fragmentation. These autoantibodies recognize topo I and fragments of this autoantigen generated during apoptosis and necrosis. We undertook this study to determine whether lysosomal cathepsins are involved in topo I fragmentation during necrosis. METHODS: Topo I cleavage during necrosis was assessed by immunoblotting of lysates from L929 fibroblasts exposed to tumor necrosis factor alpha (TNFalpha) and the broad caspase inhibitor Z-VAD-FMK, and by immunoblotting of lysates from endothelial cells treated with HgCl2. Purified topo I and L929 nuclei were incubated with cathepsins B, D, G, H, and L, and topo I cleavage was detected by immunoblotting. The intracellular localization of cathepsin L activity and topo I in necrotic cells was examined using fluorescence microscopy. RESULTS: Treatment of L929 cells with TNFalpha and Z-VAD-FMK induced caspase-independent cell death with necrotic morphology. This cell death involved topo I cleavage into fragments of approximately 70 kd and 45 kd. This cleavage profile was reproduced in vitro by cathepsins L and H and was inhibited by the cathepsin L inhibitor Z-FY-CHO. During necrosis, cathepsin L activity diffused from lysosomes into the cytoplasm and nucleus, whereas topo I partially relocalized to the cytoplasm. Z-FY-CHO delayed necrosis and partially blocked topo I cleavage. The topo I cleavage fragments were also detected in necrotic endothelial cells and recognized by SSc sera containing anti-topo I antibodies. CONCLUSION: These results implicate cathepsins, particularly cathepsin L, in the cleavage of topo I during necrosis. This cleavage may generate potentially immunogenic fragments that could trigger anti-topo I immune responses in SSc.

Fragmentation of Golgi complex and Golgi autoantigens during apoptosis and necrosis.

Anti-Golgi complex autoantibodies are found primarily in patients with Sjögren's syndrome and systemic lupus erythematosus, although they are not restricted to these diseases. Several Golgi autoantigens have been identified that represent a small family of proteins. Common features of all Golgi autoantigens appear to be their distinct structural organization of multiple alpha-helical coiled-coil rods in the central domains flanked by non-coiled-coil N-termini and C-termini, and their localization to the cytoplasmic face of Golgi cisternae. Many autoantigens in systemic autoimmune diseases have distinct cleavage products in apoptosis or necrosis and this has raised the possibility that cell death may play a role in the generation of potentially immunostimulatory forms of autoantigens. In the present study, we examined changes in the Golgi complex and associated autoantigens during apoptosis and necrosis. Immunofluorescence analysis showed that the Golgi complex was altered and developed distinctive characteristics during apoptosis and necrosis. In addition, immunoblotting analysis showed the generation of antigenic fragments of each Golgi autoantigen, suggesting that they may play a role in sustaining autoantibody production. Further studies are needed to determine whether the differences observed in the Golgi complex during apoptosis or necrosis may account for the production of anti-Golgi complex autoantibodies.