RESUMO
Chronic psychosocial stress induced by the chronic subordinate colony housing (CSC, 19 Days) paradigm promotes functional splenic in vitro glucocorticoid (GC) resistance, but only if associated with significant bite wounding or prior abdominal transmitter implantation. Moreover, sensory contact to social defeat of conspecifics represents a social stressor for the observer individual. As the occurence and severity of bite wounding is not adequately controllable, the present study aimed to develop an animal model, allowing a bite wound-independent, more reliable generation of chronically-stressed mice characterized by functional splenic in vitro GC resistance. Therefore, male C57BL/6N mice received a standardized sterile intraperitoneal (i.p.) incision surgery or SHAM treatment one week prior to 19-days of (i) CSC, (ii) witnessing social defeat during CSC exposure in sensory contact (SENS) or (iii) single-housing for control (SHC), before assessing basal and LPS-induced splenic in vitro cell viability and GC resistance. Our results indicate that individually-housed SENS but not CSC mice develop mild signs of splenic in vitro GC resistance, when undergoing prior i.p.-wounding. Taken together and considering that future studies are warranted, our findings support the hypothesis that the combination of repeated standardized i.p.-wounding with chronic sensory stress exposure represents an adequate tool to induce functional splenic in vitro GC resistance independent of the occurrence of uncontrollable bite wounds required in social stress paradigms to induce a comparable phenotype.
Assuntos
Glucocorticoides , Camundongos Endogâmicos C57BL , Baço , Estresse Psicológico , Animais , Masculino , Baço/metabolismo , Camundongos , Modelos Animais de Doenças , Derrota SocialRESUMO
Mitochondria within the adrenal cortex play a key role in synthesizing steroid hormones. The adrenal cortex is organized in three functionally specialized zones (glomerulosa, fasciculata, and reticularis) that produce different classes of steroid hormones in response to various stimuli, including psychosocial stress. Given that the functions and morphology of mitochondria are dynamically related and respond to stress, we applied transmission electron microscopy (TEM) to examine potential differences in mitochondrial morphology under basal and chronic psychosocial stress conditions. We used the chronic subordinate colony housing (CSC) paradigm, a murine model of chronic psychosocial stress. Our findings quantitatively define how mitochondrial morphology differs among each of the three adrenal cortex zones under basal conditions, and show that chronic psychosocial stress mainly affected mitochondria in the zona glomerulosa, shifting their morphology towards the more typical glucocorticoid-producing zona fasciculata mitochondrial phenotype. Analysis of adrenocortical lipid droplets that provide cholesterol for steroidogenesis showed that chronic psychosocial stress altered lipid droplet diameter, without affecting droplet number or inter-organellar mitochondria-lipid droplet interactions. Together, our findings support the hypothesis that each adrenal cortex layer is characterized by morphologically distinct mitochondria and that this adrenal zone-specific mitochondrial morphology is sensitive to environmental stimuli, including chronic psychosocial stressors. Further research is needed to define the role of these stress-induced changes in mitochondrial morphology, particularly in the zona glomerulosa, on stress resilience and related behaviors.
Assuntos
Córtex Suprarrenal , Camundongos , Animais , Córtex Suprarrenal/metabolismo , Corticosteroides/metabolismo , Mitocôndrias , Colesterol/metabolismo , Estresse PsicológicoRESUMO
Psychological stress and traumatic brain injury (TBI) result in long-lasting emotional and behavioral impairments in patients. So far, the interaction of psychological stress with TBI not only in the brain but also in peripheral organs is poorly understood. Herein, the impact of acute stress (AS) occurring immediately before TBI is investigated. For this, a mouse model of restraint stress and TBI was employed, and their influence on behavior and gene expression in brain regions, the hypothalamic-pituitary-adrenal (HPA) axis, and peripheral organs was analyzed. Results demonstrate that, compared to single AS or TBI exposure, mice treated with AS prior to TBI showed sex-specific alterations in body weight, memory function, and locomotion. The induction of immediate early genes (IEGs, e.g., c-Fos) by TBI was modulated by previous AS in several brain regions. Furthermore, IEG upregulation along the HPA axis (e.g., pituitary, adrenal glands) and other peripheral organs (e.g., heart) was modulated by AS-TBI interaction. Proteomics of plasma samples revealed proteins potentially mediating this interaction. Finally, the deletion of Atf3 diminished the TBI-induced induction of IEGs in peripheral organs but left them largely unaltered in the brain. In summary, AS immediately before brain injury affects the brain and, to a strong degree, also responses in peripheral organs.
Assuntos
Lesões Encefálicas Traumáticas , Sistema Hipotálamo-Hipofisário , Humanos , Masculino , Feminino , Camundongos , Animais , Sistema Hipófise-Suprarrenal , Lesões Encefálicas Traumáticas/metabolismo , Hipófise/metabolismo , Estresse Psicológico/genética , Estresse Psicológico/metabolismo , Expressão GênicaRESUMO
Chronic psychosocial stress is a burden of modern society and poses a clear risk factor for a plethora of somatic and affective disorders, of which most are associated with an activated immune status and chronic low-grade inflammation. Preclinical and clinical studies further suggest that a failure in immunoregulation promotes an over-reaction of the inflammatory stress response and, thus, predisposes an individual to the development of stress-related disorders. Therefore, all genetic (i.e., sex) and environmental (i.e., early life adversity; ELA) factors facilitating an adult's inflammatory stress response are likely to increase their stress vulnerability. In the present study we investigated whether repeated subcutaneous (s.c.) administrations with a heat-killed preparation of Mycobacterium vaccae (M. vaccae; National Collection of Type Cultures (NCTC) 11659), an abundant soil saprophyte with immunoregulatory properties, are protective against negative behavioral, immunological and physiological consequences of ELA alone or of ELA followed by chronic psychosocial stress during adulthood (CAS) in male and female mice. ELA was induced by the maternal separation (MS) paradigm, CAS was induced by 19 days of chronic subordinate colony housing (CSC) in males and by a 7-week exposure to the social instability paradigm (SIP) in females. Our data indicate that ELA effects in both sexes, although relatively mild, were to a great extent prevented by subsequent s.c. M. vaccae administrations. Moreover, although the use of different paradigms for males and females impedes a direct comparison, male mice seemed to be more susceptible to CAS than females, with only females benefitting slightly from the stress protective effects of s.c. M. vaccae administrations when given prior to CAS alone. Finally, our data support the hypothesis that female mice are more vulnerable to the additive effects of ELA and CAS than male mice and that s.c. M. vaccae administrations subsequent to ELA but prior to CAS are protective in both sexes. Taken together and considering the limitation that CAS in males and females was induced by different paradigms, our findings are consistent with the hypotheses that murine stress vulnerability during different phases of life is strongly sex dependent and that developing immunoregulatory approaches, such as repeated s.c. administrations with immunoregulatory microorganisms, have potential for prevention/treatment of stress-related disorders.
RESUMO
Ex vivo culturing of isolated PBMCs from individuals vaccinated with the coronavirus disease 2019 (COVID-19) vaccine BNT162b1 revealed a pronounced T cell response in the presence of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. The latter was 10-fold more pronounced than the ex vivo response of PBMCs from the same individuals to other common pathogen T cell epitope pools, suggesting COVID-19 vaccination to induce RBD-specific T cell responses and not to facilitate T cell (re)activity in general. In the current study we investigated whether COVID-19 vaccination long-lastingly affects plasma interleukin (IL)-6 concentrations, complete blood counts, ex vivo IL-6 and IL-10 secretion of PBMCs cultured under basal conditions or in the presence of concanavalin (Con) A and lipopolysaccharide (LPS), salivary cortisol and α-amylase, mean arterial pressure (MAP), heart rate (HR) as well as mental and physical health status. The study was initially designed to investigate whether the presence vs. absence of own pets during urban upbringing has protective effects against psychosocial stress-induced immune activation during adulthood. However, as COVID-19 vaccines were approved while the study was ongoing and as, therefore, both vaccinated and non-vaccinated individuals have been recruited, we were able to stratify our data set with respect to the COVID-19 vaccination status and to assess the long-lasting effects of COVID-19 vaccination on physiological immunological, cardiovascular and psychosomatic health parameters. This data is presented in the current study. We show that isolated PBMCs from individuals vaccinated against COVID-19 show a ~ 600-fold increase in basal and a ~ 6000-fold increase in ConA-induced proinflammatory IL-6 secretion, and a ~ 2-fold increase in basal and ConA-induced antiinflammatory IL-10 secretion, both in comparison with non-vaccinated individuals. In contrast, LPS-induced ex vivo IL-6 and IL-10 secretions were not affected by vaccination status, as were plasma IL-6 concentrations, complete blood counts, salivary cortisol and α-amylase, cardiovascular measures and psychosomatic health. In summary, our findings are of relevance for many clinical studies ran before/during the pandemic, clearly indicating that consideration of participants' vaccination status is critical, at least when assessing ex vivo PBMC functionality.
Assuntos
COVID-19 , Humanos , Adulto , Vacinas contra COVID-19 , SARS-CoV-2 , Interleucina-6 , Vacina BNT162 , Hidrocortisona , Interleucina-10 , Leucócitos Mononucleares , Lipopolissacarídeos , Concanavalina ARESUMO
Mental traumatization is associated with long-bone growth retardation, osteoporosis and increased fracture risk. We revealed earlier that mental trauma disturbs cartilage-to-bone transition during bone growth and repair in mice. Trauma increased tyrosine hydroxylase-expressing neutrophils in bone marrow and fracture callus. Here we show that tyrosine hydroxylase expression in the fracture hematoma of patients correlates positively with acknowledged stress, depression, and pain scores as well as individual ratings of healing-impairment and pain-perception post-fracture. Moreover, mice lacking tyrosine hydroxylase in myeloid cells are protected from chronic psychosocial stress-induced disturbance of bone growth and healing. Chondrocyte-specific ß2-adrenoceptor-deficient mice are also protected from stress-induced bone growth retardation. In summary, our preclinical data identify locally secreted catecholamines in concert with ß2-adrenoceptor signalling in chondrocytes as mediators of negative stress effects on bone growth and repair. Given our clinical data, these mechanistic insights seem to be of strong translational relevance.
Assuntos
Consolidação da Fratura , Fraturas Ósseas , Camundongos , Animais , Catecolaminas/metabolismo , Neutrófilos , Tirosina 3-Mono-Oxigenase/metabolismo , Calo Ósseo , Fraturas Ósseas/metabolismo , Transtornos do Crescimento , Receptores Adrenérgicos/metabolismo , Dor/metabolismoRESUMO
Male C57BL/6N mice exposed to the chronic subordinate colony housing (CSC; 19 days) paradigm, a preclinically validated model of chronic psychosocial stress, are characterized by unaffected basal morning plasma corticosterone (CORT) concentrations despite adrenal and pituitary hyperplasia and increased adrenocorticotropic hormone (ACTH) plasma concentrations, compared with single-housed control (SHC) mice. However, as CSC mice are still able to show an increased CORT secretion towards novel heterotypic stressors, these effects might reflect an adaptation rather than a functional breakdown of general hypothalamus-pituitary-adrenal (HPA) axis functionality. In the present study we used male mice of a genetically modified mouse line, to investigate whether genetically-driven ACTH overexpression compromises adaptational processes occurring at the level of the adrenals during CSC exposure. Experimental mice carried a point mutation in the DNA binding domain of the glucocorticoid (GC) receptor (GR), attenuating dimerization of GR (GRdim), resulting in a congenially compromised negative feedback inhibition at the level of the pituitary. In line with previous studies, CSC mice in both the wild type (WT; GR+/+) and GRdim group developed adrenal enlargement. Moreover, compared with respective SHC and WT mice, CSC GRdim mice show increased basal morning plasma ACTH and CORT concentrations. Quantitative polymerase chain reaction (qPCR) analysis revealed neither a genotype effect, nor a CSC effect on pituitary mRNA expression of the ACTH precursor proopiomelanocortin (POMC). Finally, CSC increased anxiety-related behavior, active coping and splenocyte in vitro (re)activity in both WT and GRdim mice, while a CSC-induced increase in adrenal lipid vesicles and splenic GC resistance was detectable only in WT mice. Of note, lipopolysaccharide (LPS)-stimulated splenocytes of GRdim mice were resistant to the inhibitory effects of CORT. Together our findings support the hypothesis that pituitary ACTH protein concentration is negatively controlled by GR dimerization under conditions of chronic psychosocial stress, while POMC gene transcription is not dependent on intact GR dimerization under both basal and chronic stress conditions. Finally, our data suggest that adrenal adaptations during chronic psychosocial stress (i.e., ACTH desensitization), aiming at the prevention of prolonged hypercorticism, are protective only to a certain threshold of plasma ACTH levels.
RESUMO
Herein we report the structure-activity relationship (SAR) studies and optimization of new highly potent and selective CRTH2 receptor antagonists as potential follow-ups of our previous reported clinical candidate setipiprant (ACT-129968) for the treatment of respiratory diseases. Structural modification of the amide part of setipiprant (ACT-129968) led to the identification of the tetrahydrocarbazole derivative (S)-B-1 (ACT-453859) ((S)-2-(3-((5-chloropyrimidin-2-yl)(methyl)amino)-6-fluoro-1,2,3,4-tetrahydro-9H-carbazol-9-yl)acetic acid). This compound which displayed a substantial improvement in potency in the presence of plasma versus setipiprant (ACT-129968) has exhibited an excellent overall pharmacokinetic profile. Further lead optimization to overcome a safety issue as observed in non-clinical studies with (S)-B-1 (ACT-453859), led to the discovery of the 4-azaindole derivative (S)-72 (ACT-774312) ((S)-2-(8-((5-chloropyrimidin-2-yl)(methyl)amino)-2-fluoro-6,7,8,9-tetrahydro-5H-pyrido[3,2-b]indol-5-yl)acetic acid) which was selected as a potential follow-up of setipiprant (ACT-129968).
Assuntos
Ácido Acético , Relação Estrutura-AtividadeRESUMO
Inflammatory conditions, including allergic asthma and conditions in which chronic low-grade inflammation is a risk factor, such as stress-related psychiatric disorders, are prevalent and are a significant cause of disability worldwide. Novel approaches for the prevention and treatment of these disorders are needed. One approach is the use of immunoregulatory microorganisms, such as Mycobacterium vaccae NCTC 11659, which have anti-inflammatory, immunoregulatory, and stress-resilience properties. However, little is known about how M. vaccae NCTC 11659 affects specific immune cell targets, including monocytes, which can traffic to peripheral organs and the central nervous system and differentiate into monocyte-derived macrophages that, in turn, can drive inflammation and neuroinflammation. In this study, we investigated the effects of M. vaccae NCTC 11659 and subsequent lipopolysaccharide (LPS) challenge on gene expression in human monocyte-derived macrophages. THP-1 monocytes were differentiated into macrophages, exposed to M. vaccae NCTC 11659 (0, 10, 30, 100, 300 µg/mL), then, 24 h later, challenged with LPS (0, 0.5, 2.5, 250 ng/mL), and assessed for gene expression 24 h following challenge with LPS. Exposure to M. vaccae NCTC 11659 prior to challenge with higher concentrations of LPS (250 ng/mL) polarized human monocyte-derived macrophages with decreased IL12A, IL12B, and IL23A expression relative to IL10 and TGFB1 mRNA expression. These data identify human monocyte-derived macrophages as a direct target of M. vaccae NCTC 11659 and support the development of M. vaccae NCTC 11659 as a potential intervention to prevent stress-induced inflammation and neuroinflammation implicated in the etiology and pathophysiology of inflammatory conditions and stress-related psychiatric disorders.
Assuntos
Lipopolissacarídeos , Mycobacterium , Humanos , Lipopolissacarídeos/farmacologia , Doenças Neuroinflamatórias , Inflamação , MacrófagosRESUMO
Stress-related somatic and psychiatric disorders are often associated with a decline in regulatory T cell (Treg) counts and chronic low-grade inflammation. Recent preclinical evidence suggests that the latter is at least partly mediated by stress-induced upregulation of toll-like receptor (TLR)2 in newly generated neutrophils and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), as well as glucocorticoid (GC) resistance in predominantly PMN-MDSCs following stress-induced upregulation of TLR4 expression. Here we show in mice exposed to the chronic subordinate colony housing (CSC) paradigm that repeated intragastric (i.g.) administrations of a heat-killed preparation of Mycobacterium vaccae NCTC 11659, a saprophytic microorganism with immunoregulatory properties, protected against the stress-induced reduction in systemic Tregs, increase in basal and LPS-induced in vitro splenocyte viability, as well as splenic in vitro GC resistance. Our findings further support the hypothesis that i.g. M. vaccae protects against CSC-associated splenic GC resistance via directly affecting the myeloid compartment, thereby preventing the CSC-induced upregulation of TLR4 in newly generated PMN-MDSCs. In contrast, the protective effects of i.g. M. vaccae on the CSC-induced upregulation of TLR2 in neutrophils and the subsequent increase in basal and LPS-induced in vitro splenocyte viability seems to be indirectly mediated via the Treg compartment. These data highlight the potential for use of oral administration of M. vaccae NCTC 11659 to prevent stress-induced exaggeration of inflammation, a risk factor for development of stress-related psychiatric disorders.
Assuntos
Glucocorticoides , Mycobacterium , Camundongos , Animais , Glucocorticoides/farmacologia , Lipopolissacarídeos , Receptor 4 Toll-Like , InflamaçãoRESUMO
Stress-associated somatic and psychiatric disorders are often linked to non-resolving low-grade inflammation, which is promoted at least in part by glucocorticoid (GC) resistance of distinct immune cell subpopulations. While the monocyte/macrophage compartment was in the focus of many clinical and preclinical studies, the role of myeloid-derived suppressor cells (MDSCs) in stress-associated pathologies and GC resistance is less understood. As GC resistance is a clear risk factor for posttraumatic complications in patients on intensive care, the exact interplay of physical and psychosocial traumatization in the development of GC resistance needs to be further clarified. In the current study we employ the chronic subordinate colony housing (CSC) paradigm, a well-characterized mouse model of chronic psychosocial stress, to study the role of myeloid cells, in particular of MDSCs, in innate immune activation and GC resistance following combined psychosocial and physical (e.g., bite wounds) trauma. Our findings support the hypothesis that stress-induced neutrophils, polymorphonuclear (PMN)-MDSCs and monocytes/monocyte-like (MO)-MDSCs get primed and activated locally in the bone marrow as determined by toll-like receptor (TLR)2 upregulation and increased basal and lipopolysaccharide (LPS)-induced in vitro cell viability. These primed and activated myeloid cells emigrate into the peripheral circulation and subsequently, if CSC is accompanied by significant bite wounding, accumulate in the spleen. Here, PMN-MDSCs and monocytes/MO-MDSCs upregulate TLR4 expression, which exclusively in PMN-MDSCs promotes NF-κB hyperactivation upon LPS-stimulation, thereby exceeding the anti-inflammatory capacities of GCs and resulting in GC resistance.
Assuntos
Glucocorticoides , Células Supressoras Mieloides , Estresse Psicológico , Animais , Camundongos , Glucocorticoides/farmacologia , Lipopolissacarídeos , Monócitos , Células Mieloides , Células Supressoras Mieloides/metabolismoRESUMO
The bio-psycho-social model highlights intra-individual and inter-individual interactions, including psychotherapy. The processing of these interactions within a person takes place, among others, in the central autonomic network (CAN). The CAN's autonomic output to the periphery can be indexed by heart rate variability (HRV), representing individual adaptive capacity. Further, the CAN influences the hypothalamus-pituitary-adrenal axis with its product cortisol. The aim consisted in investigating HRV and cortisol as well as their relation to symptom course in response to short-term psychotherapy. A single-arm, uncontrolled, explorative study was conducted at an outpatient psychotherapeutic consultation in the workplace offered to employees with mental or psychosomatic complaints. Questionnaires included symptoms of depression, irritation and functional impairment. Circadian profile of HRV and salivary cortisol concentrations collected pre and post short-term psychotherapeutic intervention (4-12 sessions) were assessed. Multilevel-linear mixed regressions were calculated. Out of 29 participants (mean age 42; 72% female), 24% were on sick leave from work. Cortisol concentrations were neither affected by intervention nor by symptom course. The proportion of individuals showing a vagally mediated HRV in the range of the lowest quartile assessed for age- and sex-matched healthy controls was reduced at follow-up (pre 34%, post 22%; p = .017). Higher vagally mediated HRV at baseline predicted lower symptom burden at follow-up. Thus, the results support the assumption that HRV reflects the capability of an organism to adapt and recover. Patients with reduced HRV might need additional psychotherapeutic sessions to achieve the same symptom improvements than patients with retained HRV.
Assuntos
Psicoterapia Breve , Humanos , Feminino , Adulto , Masculino , Frequência Cardíaca/fisiologia , Hidrocortisona , Local de Trabalho , Sistema Nervoso AutônomoRESUMO
Previous studies have shown that the in vivo administration of soil-derived bacteria with anti-inflammatory and immunoregulatory properties, such as Mycobacterium vaccae NCTC 11659, can prevent a stress-induced shift toward an inflammatory M1 microglial immunophenotype and microglial priming in the central nervous system (CNS). It remains unclear whether M. vaccae NCTC 11659 can act directly on microglia to mediate these effects. This study was designed to determine the effects of M. vaccae NCTC 11659 on the polarization of naïve BV-2 cells, a murine microglial cell line, and BV-2 cells subsequently challenged with lipopolysaccharide (LPS). Briefly, murine BV-2 cells were exposed to 100 µg/mL whole-cell, heat-killed M. vaccae NCTC 11659 or sterile borate-buffered saline (BBS) vehicle, followed, 24 h later, by exposure to 0.250 µg/mL LPS (Escherichia coli 0111: B4; n = 3) in cell culture media vehicle (CMV) or a CMV control condition. Twenty-four hours after the LPS or CMV challenge, cells were harvested to isolate total RNA. An analysis using the NanoString platform revealed that, by itself, M. vaccae NCTC 11659 had an "adjuvant-like" effect, while exposure to LPS increased the expression of mRNAs encoding proinflammatory cytokines, chemokine ligands, the C3 component of complement, and components of inflammasome signaling such as Nlrp3. Among LPS-challenged cells, M. vaccae NCTC 11659 had limited effects on differential gene expression using a threshold of 1.5-fold change. A subset of genes was assessed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR), including Arg1, Ccl2, Il1b, Il6, Nlrp3, and Tnf. Based on the analysis using real-time RT-PCR, M. vaccae NCTC 11659 by itself again induced "adjuvant-like" effects, increasing the expression of Il1b, Il6, and Tnf while decreasing the expression of Arg1. LPS by itself increased the expression of Ccl2, Il1b, Il6, Nlrp3, and Tnf while decreasing the expression of Arg1. Among LPS-challenged cells, M. vaccae NCTC 11659 enhanced LPS-induced increases in the expression of Nlrp3 and Tnf, consistent with microglial priming. In contrast, among LPS-challenged cells, although M. vaccae NCTC 11659 did not fully prevent the effects of LPS relative to vehicle-treated control conditions, it increased Arg1 mRNA expression, suggesting that M. vaccae NCTC 11659 induces an atypical microglial phenotype. Thus, M. vaccae NCTC 11659 acutely (within 48 h) induced immune-activating and microglial-priming effects when applied directly to murine BV-2 microglial cells, in contrast to its long-term anti-inflammatory and immunoregulatory effects observed on the CNS when whole-cell, heat-killed preparations of M. vaccae NCTC 11659 were given peripherally in vivo.
Assuntos
Infecções por Citomegalovirus , Microglia , Mycobacteriaceae , Animais , Camundongos , Lipopolissacarídeos/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Interleucina-6 , Adjuvantes Imunológicos , Adjuvantes Farmacêuticos , Anti-InflamatóriosRESUMO
Catecholamine signaling is known to influence bone tissue as reuptake of norepinephrine released from sympathetic nerves into bone cells declines with age leading to osteoporosis. Further, ß-adrenoceptor-blockers like propranolol provoke osteoprotective effects in osteoporotic patients. However, besides systemic adrenal and sympathetic catecholamine production, it is also known that myeloid cells can synthesize catecholamines, especially under inflammatory conditions. To investigate the effects of catecholamines produced by CD11b+ myeloid cells on bone turnover and regeneration, a mouse line with specific knockout of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis, in CD11b+ myeloid cells (THflox/flox/CD11b-Cre+, referred to as THCD11b-Cre) was generated. For bone phenotyping, male mice were sacrificed at eight and twelve weeks of age and harvested bones were subjected to bone length measurement, micro-computed tomography, fluorescence-activated cell sorting of the bone marrow, gene expression analysis, histology and immunohistochemistry. Support for an age-dependent influence of myeloid cell-derived catecholamines on bone homeostasis is provided by the fact that twelve-week-old, but not eight-week-old THCD11b-Cre mice, developed an osteopenic phenotype and showed increased numbers of neutrophils and T lymphocytes in the bone marrow, while CCL2, IL-6, IL-4 and IL-10 mRNA expression was reduced in sorted myeloid bone marrow cells. To investigate the influence of myeloid cell-derived catecholamines on fracture healing, mice received a diaphyseal femur osteotomy. Three days post-fracture, immunohistochemistry revealed an increased number of macrophages, neutrophils and cytotoxic T lymphocytes in the fracture hematoma of THCD11b-Cre mice. Micro-computed tomography on day 21 showed a decreased tissue mineral density, a reduced bone volume and less trabeculae in the fracture callus indicating delayed fracture healing, probably due to the increased presence of inflammatory cells in THCD11b-Cre mice. This indicates a crucial role of myeloid cell-derived catecholamines in immune cell-bone cell crosstalk and during fracture healing.
Assuntos
Catecolaminas , Fraturas Ósseas , Animais , Remodelação Óssea , Calo Ósseo/metabolismo , Calo Ósseo/patologia , Catecolaminas/metabolismo , Fraturas Ósseas/metabolismo , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Interleucina-6 , Macrófagos , Masculino , Camundongos , Norepinefrina , Propranolol , RNA Mensageiro/metabolismo , Receptores Adrenérgicos/metabolismo , Tirosina 3-Mono-Oxigenase , Microtomografia por Raio-XRESUMO
PURPOSE: Endochondral ossification, which involves transdifferentiation of chondrocytes into osteoblasts, is an important process involved in the development and postnatal growth of most vertebrate bones as well as in bone fracture healing. To study the basic molecular mechanisms of this process, a robust and easy-to-use in vitro model is desirable. Therefore, we aimed to develop a standardized in vitro assay for the transdifferentiation of chondrogenic cells towards the osteogenic lineage. METHODS: Murine chondrogenic ATDC5 cells were differentiated into the chondrogenic lineage for seven days and subsequently differentiated towards the osteogenic direction. Gene expression analysis of pluripotency, as well as chondrogenic and osteogenic markers, cell-matrix staining, and immunofluorescent staining, were performed to assess the differentiation. In addition, the effects of Wnt3a and lipopolysaccharides (LPS) on the transdifferentiation were tested by their addition to the osteogenic differentiation medium. RESULTS: Following osteogenic differentiation, chondrogenically pe-differentiated cells displayed the expression of pluripotency and osteogenic marker genes as well as alkaline phosphatase activity and a mineralized matrix. Co-expression of Col2a1 and Col1a1 after one day of osteogenic differentiation indicated that osteogenic cells had differentiated from chondrogenic cells. Wnt3a increased and LPS decreased transdifferentiation towards the osteogenic lineage. CONCLUSION: We successfully established a rapid, standardized in vitro assay for the transdifferentiation of chondrogenic cells into osteogenic cells, which is suitable for testing the effects of different compounds on this cellular process.
Assuntos
Condrócitos , Osteogênese , Animais , Diferenciação Celular , Transdiferenciação Celular , Células Cultivadas , Condrócitos/metabolismo , Condrogênese/genética , Camundongos , OsteoblastosRESUMO
Inflammatory diseases and stressor-related psychiatric disorders, for which inflammation is a risk factor, are increasing in modern Western societies. Recent studies suggest that immunoregulatory approaches are a promising tool in reducing the risk of suffering from such disorders. Specifically, the environmental saprophyte Mycobacterium vaccae National Collection of Type Cultures (NCTC) 11659 has recently gained attention for the prevention and treatment of stress-related psychiatric disorders. However, effective use requires a sophisticated understanding of the effects of M. vaccae NCTC 11659 and related rapidly growing mycobacteria (RGMs) on microbiome-gut-immune-brain interactions. This historical narrative review is intended as a first step in exploring these mechanisms and provides an overview of preclinical and clinical studies on M. vaccae NCTC 11659 and related RGMs. The overall objective of this review article is to increase the comprehension of, and interest in, the mechanisms through which M. vaccae NCTC 11659 and related RGMs promote stress resilience, with the intention of fostering novel clinical strategies for the prevention and treatment of stressor-related disorders.
Assuntos
Agentes de Imunomodulação , Inflamação/prevenção & controle , Mycobacteriaceae , Estresse Psicológico/complicações , Animais , Humanos , Inflamação/etiologiaRESUMO
Chronic psychosocial stress is a risk factor for the development of numerous disorders, of which most are associated with chronic low-grade inflammation. Given the immunosuppressive effects of glucocorticoids (GC), one underlying mechanism might be the development of stress-induced GC resistance in certain immune cell subpopulations. In line with this hypothesis, male mice exposed to the chronic subordinate colony housing (CSC, 19 days) model develop GC resistance of in vitro lipopolysaccharide (LPS)-stimulated splenocytes, splenomegaly and an increased percentage of splenic CD11b+ cells. Here male C57BL/6N mice were euthanized at different days during CSC, and following 30 days of single housing after stressor termination to assess when CSC-induced splenic GC resistance starts to develop and whether this is a transient effect. Moreover, splenic CD11b, GC receptor (GR) and/or macrophage migration inhibiting factor (MIF) protein levels were quantified at respective days. While mild forms of CSC-induced GC resistance, increased splenic CD11b expression and/or splenomegaly were detectable on days 8 and 9 of CSC, more severe forms took until days 15 and 16 to develop, but normalized almost completely within 30 days following stressor termination (day 51). In contrast, splenic GR expression was decreased in CSC versus single-housed control (SHC) mice at all days assessed. While MIF expression was increased on days 15 and 16 of CSC, it was decreased in CSC versus SHC mice on day 20 despite persisting splenomegaly, increased CD11b expression and functional GC resistance. In summary, our data indicate that GC resistance and CD11b+ cell-mediated splenomegaly develop gradually and in parallel over time during CSC exposure and are transient in nature. Moreover, while we can exclude that CSC-induced reduction in splenic GR expression is sufficient to induce functional GC resistance, the role of MIF in CD11b+ cell-mediated splenomegaly and GC resistance requires further investigation.
Assuntos
Cortisona/farmacologia , Glucocorticoides/farmacologia , Leucócitos/fisiologia , Baço/citologia , Estresse Psicológico/imunologia , Comportamento Agonístico , Animais , Mordeduras e Picadas , Antígeno CD11b/biossíntese , Antígeno CD11b/genética , Doença Crônica , Cortisona/sangue , Aglomeração , Resistência a Medicamentos , Abrigo para Animais , Oxirredutases Intramoleculares/biossíntese , Oxirredutases Intramoleculares/genética , Leucócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fatores Inibidores da Migração de Macrófagos/biossíntese , Fatores Inibidores da Migração de Macrófagos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/genética , Baço/patologia , TerritorialidadeRESUMO
Background: A heightened stress reactivity to mental stress tasks has been shown in hypertensive patients and might contribute to a higher disease risk. We investigated this hyperreactivity with regard to an attachment related stressor that focuses on emotions instead of performance and we examined whether this effect can also be found in patients on antihypertensive drugs. Materials and Methods: Fifty patients with primary hypertension, treated with at least one antihypertensive drug, were compared with 25 healthy individuals. After 10 min of rest, they participated in an attachment-related interview (Adult Attachment Projective picture system, AAP) and were exposed to an attachment-related stressor (Separation Recall, SR), a short-time stressor which activates attachment-related emotions and thoughts by talking 5 min about a personal experience of loneliness. Blood samples to measure adrenocorticotrope hormone (ACTH), cortisol, norepinephrine, epinephrine, and dopamine were taken. Blood pressure, heart rate and arterial stiffness were measured at rest, after AAP, after SR and 10 min after recovery. Standard deviation of normal-to-normal intervals (SDNN) and root mean square of successive differences (RMSSD) were calculated. Parameters were compared using Mann Whitney U-test and linear mixed-effects regression models controlling for age and body mass index (BMI) after logarithmic transformation if appropriate. Results: Healthy test persons were younger and had lower BMI than patients. Comparing the two groups there were no significant differences in blood pressure and heart rate at rest. Both stressors provoked a significant response in almost all parameters. Results of the post-estimation of contrasts from linear mixed-effects regression models showed a steeper rise in systolic BP and arterial stiffness as well as a more pronounced decline in SDNN in hypertensive patients than in healthy controls. Levels of cortisol rose earlier and higher in hypertensive patients than in healthy controls. Conclusion: Vascular, autonomic, and hypothalamic pituitary adrenal axis response is heightened in medicated subjects with hypertension in response to attachment-focused stressors compared to healthy subjects. We conclude that the remaining hyper-reactivity even with sufficient antihypertensive medication still poses a substantial risk for affected patients. New ways to diminish this risk should be developed.
RESUMO
Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.
Assuntos
Proteína FUS de Ligação a RNA/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Células HEK293 , Células HeLa , Humanos , Mapeamento de Interação de Proteínas , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/isolamento & purificaçãoRESUMO
The neuropeptide oxytocin (OXT) has generated considerable interest as potential treatment for psychiatric disorders, including anxiety and autism spectrum disorders. However, the behavioral and molecular consequences associated with chronic OXT treatment and chronic receptor (OXTR) activation have scarcely been studied, despite the potential therapeutic long-term use of intranasal OXT. Here, we reveal that chronic OXT treatment over two weeks increased anxiety-like behavior in rats, with higher sensitivity in females, contrasting the well-known anxiolytic effect of acute OXT. The increase in anxiety was transient and waned 5 days after the infusion has ended. The behavioral effects of chronic OXT were paralleled by activation of an intracellular signaling pathway, which ultimately led to alternative splicing of hypothalamic corticotropin-releasing factor receptor 2α (Crfr2α), an important modulator of anxiety. In detail, chronic OXT shifted the splicing ratio from the anxiolytic membrane-bound (mCRFR2α) form of CRFR2α towards the soluble CRFR2α (sCRFR2α) form. Experimental induction of alternative splicing mimicked the anxiogenic effects of chronic OXT, while sCRFR2α-knock down reduced anxiety-related behavior of male rats. Furthermore, chronic OXT treatment triggered the release of sCRFR2α into the cerebrospinal fluid with sCRFR2α levels positively correlating with anxiety-like behavior. In summary, we revealed that the shifted splicing ratio towards expression of the anxiogenic sCRFR2α underlies the adverse effects of chronic OXT treatment on anxiety.