Immunosuppressed non-responders to two doses of mRNA SARS-CoV-2 vaccines achieve an immune response comparable to those of immunocompetent individuals after a third dose.

The SARS-CoV-2 vaccines trigger the production of neutralizing antibodies to the SARS-CoV-2 spike (S) protein and induce a T cell-mediated immune response. However, the antibody titers that confer protection against the SARS-CoV-2 virus are currently not well-established. While immunocompetent individuals achieve a high level of immune response after SARS-CoV-2 vaccination, it now appears that a high proportion of immunosuppressed or immunocompromised, patients exhibit low or no response to two doses of the vaccines. Most non-responders are on treatment with either glucocorticoids, mycophenolate-mofetil (MMF), the anti-CD20 monoclonal antibody rituximab, calcineurin inhibitors like cyclosporine and tacrolimus, rapamycin (mTOR) signaling cascade inhibitors (i.e., sirolimus and everolimus), azathioprine, or methotrexate given for a variety of diseases including autoimmune disorders, hematological malignancies, and solid cancers, while recipients of solid organ transplants also fall within this category. Recently, several published reports have suggested that a third dose of these vaccines induces an elevated antibody response against the SARS-CoV-2 S protein.

Serum chemerin levels are negatively associated with male fertility and reproductive hormones.

STUDY QUESTION: Are chemerin levels different in subfertile men compared to men from the general population, and how does chemerin relate to reproductive hormonal status? SUMMARY ANSWER: Chemerin is negatively associated to LH, SHBG and estradiol and lower levels of chemerin are detected among subfertile men compared to controls. WHAT IS KNOWN ALREADY: Adipokines have pleiotropic effects on tissue homeostasis and have been shown to affect both sex steroid production and action. Among adipokines the newly characterized chemokine chemerin is suggested to influence testosterone production in males, but whether serum levels associate with testosterone or male subfertility has not yet been reported. STUDY DESIGN, SIZE, DURATION: Case control study comprising a consecutive group of men from infertile couples referred to Reproductive Medicine Centre at Skane University Hospital from 2006 through 2012, and age-matched controls. Participants were enrolled in years 2011-2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: Males from infertile couples (n = 180) aged 18-50 years with sperm concentration <20 × 106/ml and age-matched controls (n = 139) from the general population were enrolled. Serum concentrations of total testosterone (TT), calculated free testosterone (cFT), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2) and sex-hormone binding globuline (SHBG) as well as the adipokines chemerin, adiponectin and leptin were measured. Anthropometrics and biochemical parameters of glucose and lipid metabolism were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Chemerin levels were lower in subfertile men compared to controls (mean diff. 7.1 ng/ml; 95% CI, 3.7; 11 ng/ml; P < 0.001) even after adjustment for BMI. After adjustment for age, BMI, smoking, leptin and adiponectin, chemerin associated negatively with LH (ß = -4.2; P = 0.02), E2 (ß = -10; P = 0.004) and SHBG (ß = -7.4, P = 0.003). Men with elevated LH levels had lower chemerin levels compared to those with LH levels within the normal range (mean diff. 4.8 ng/ml; 95% CI, 0.16; 9.4 ng/ml; P = 0.04). LIMITATIONS, REASONS FOR CAUTION: Single sample blood test with immunoassays for determination of hormone levels. Heterogeneous group of subfertile subjects. WIDER IMPLICATIONS OF THE FINDINGS: Even though chemerin has been positively associated with BMI, inverse association with subfertility suggests that it is independently linked to reproductive function, a hypothesis that warrants further assessment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from EU Interreg V (ReproUnion) program as well as Swedish Governmental Fund for Clinical Research. The authors have no conflicts of interest.

The Multi-faceted Profile of Corticotropin-releasing Factor (CRF) Family of Neuropeptides and of their Receptors on the Paracrine/Local Regulation of the Inflammatory Response.

BACKGROUND: The corticotropin releasing factor (CRF) family of neuropeptides, CRF and the Urocortins, and their receptors are present not only within the central nervous system but also in the periphery at various locations and at the sites of inflammation where they influence its progress in a complex local / paracrine manner. OBJECTIVE AND METHODS: This review summarizes current knowledge regarding the regulation of inflammatory process by CRF family of neuropeptides and receptors with a special sight into their role in inflammatory pain and in chronic low grade inflammation that occurs in obesity. For this purpose, we searched for relevant peer-reviewed research articles using bibliographic databases. RESULTS: The CRF neuropeptides are either produced locally, by components of the inflammatory response or they may reach the inflammation sites via postganglionic sympathetic and sensory afferent nerve transport. It now appears that most immune cells taking part in the inflammatory process express CRF receptor type 1 (CRF1R) and type 2 (CRF2R) and thus represent targets of CRF neuropeptides. Indeed, mast cells, monocytes / macrophages, neutrophils and other types of immune cells express both types of the CRF receptors. In addition to their role in the pathophysiology of inflammation, CRF and its receptors also exert modulatory effects on inflammatory pain. Finally, it now appears that the CRF system is also present in adipose tissue and may play a crucial role in the development of the chronic low grade inflammation, which is characteristic of obesity. CONCLUSION: The local effects of the CRF family of neuropeptides can be either pro- or antiinflammatory depending on concentration of each type of neuropeptide present and the ratio of the local expression of their receptors CRF1R and CRF2R.

Selective and differential interactions of BNN27, a novel C17-spiroepoxy steroid derivative, with TrkA receptors, regulating neuronal survival and differentiation.

Nerve growth factor (NGF) holds a pivotal role in brain development and maintenance, been also involved in the pathophysiology of neurodegenerative diseases. Here, we provide evidence that a novel C17-spiroepoxy steroid derivative, BNN27, specifically interacts with and activates the TrkA receptor of NGF, inducing phosphorylation of TrkA tyrosine residues and down-stream neuronal survival-related kinase signaling. Additionally, BNN27 potentiates the efficacy of low levels of NGF, by facilitating its binding to the TrkA receptors and differentially inducing fast return of internalized TrkA receptors into neuronal cell membranes. Furthermore, BNN27 synergizes with NGF in promoting axonal outgrowth, effectively rescues from apoptosis NGF-dependent and TrkA positive sympathetic and sensory neurons, in vitro, ex vivo and in vivo in NGF null mice. Interestingly, BNN27 does not possess the hyperalgesic properties of NGF. BNN27 represents a lead molecule for the development of neuroprotective TrkA receptor agonists, with potential therapeutic applications in neurodegenerative diseases and in brain trauma.

In prostate cancer, low adiponectin levels are not associated with insulin resistance.

PURPOSE: Adiponectin, an adipose tissue-derived hormone with insulin-sensitizing effect, has been inversely associated with several hormonally dependent malignancies. Prostate cancer is associated with low levels of adiponectin, which have been proposed as an independent risk factor for this malignancy. Aim of this study was to examine whether hypoadiponectinaemia in prostate is associated with insulin resistance. EXPERIMENTAL DESIGN: Plasma samples and covariate data in the context of a case-control study of 300 Greek men were evaluated including 75 patients with prostate cancer, 75 patients with benign prostatic hyperplasia (BPH) and 150 age-matched healthy controls. RESULTS: Patients with prostate cancer had significantly lower plasma adiponectin levels compared with the other two groups, that is BPH patients and healthy controls (7.4 ± 5 ng/mL vs. 11.5 ± 6.4 ng/mL and 12.8 ± 8 ng/mL, respectively). On the other hand, no statistically significant differences were found between patients with prostate cancer and the other two groups for both HOMA-IR and QUICKI (P-value = 0.551). As expected, in all three groups, the levels of adiponectin correlated negatively with HOMA-IR (rho = -0.214, P-value = 0.006), QUICKI (rho = 0.214, P-value = 0.006) and insulin levels (rho = 0.942, P-value < 0.001). CONCLUSION: In spite of what would have been expected from the relevant literature, our data suggest that the hypoadiponectinaemia in prostatic cancer does not appear to be associated with insulin resistance.

Dehydroepiandrosterone: an ancestral ligand of neurotrophin receptors.

Dehydroepiandosterone (DHEA), the most abundant steroid in humans, affects multiple cellular functions of the endocrine, immune, and nervous systems. However, up to quite recently, no receptor has been described specifically for it, whereas most of its physiological actions have been attributed to its conversion to either androgens or estrogens. DHEA interacts and modulate a variety of membrane and intracellular neurotransmitter and steroid receptors. We have recently reported that DHEA protects neuronal cells against apoptosis, interacting with TrkA, the high-affinity prosurvival receptor of the neurotrophin, nerve growth factor. Intrigued by its pleiotropic effects in the nervous system of a variety of species, we have investigated the ability of DHEA to interact with the other two mammalian neurotrophin receptors, ie, the TrkB and TrkC, as well as their invertebrate counterparts (orthologs) in mollusks Lymnaea and Aplysia and in cephalochordate fish Amphioxus. Amazingly, DHEA binds to all Trk receptors, although with lower affinity by 2 orders of magnitude compared with that of the polypeptidic neurotrophins. DHEA effectively induced the first step of the TrkA and TrkC receptors activation (phosphorylation at tyrosine residues), including the vertebrate neurotrophin nonresponding invertebrate Lymnaea and Aplysia receptors. Based on our data, we hypothesize that early in evolution, DHEA may have acted as a nonspecific neurotrophic factor promoting neuronal survival. The interaction of DHEA with all types of neurotrophin receptors offers new insights into the largely unidentified mechanisms of its actions on multiple tissues and organs known to express neurotrophin receptors.

The actin cytoskeleton in rapid steroid hormone actions.

Early actin cytoskeleton reorganization is an important regulatory step of membrane-initiated, non-genomic steroid hormone actions. Specific intracellular signaling cascades control the rapid alterations of actin polymerization in a variety of cell models. Moreover, actin remodeling is a decisive component in the signaling of hormone-induced early and late cellular responses. This article briefly summarizes the current knowledge on the steroid hormone-induced early actin cytoskeleton rearrangements. It focuses on the current progress to characterize the glucocorticoid- and androgen-initiated, tissue-specific actin signaling pathways and discusses the plethora of cellular responses that are regulated by the early actin redistribution. It also provides insights into the potential clinical significance of actin dynamics and actin-specific molecular signaling for nongenomic steroid hormone actions on tumor cells. © 2014 Wiley Periodicals, Inc.

Akt2 deficiency protects from acute lung injury via alternative macrophage activation and miR-146a induction in mice.

Acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure, with limited effective treatments available. Alveolar macrophages participate in the pathogenesis of ARDS. To investigate the role of macrophage activation in aseptic lung injury and identify molecular mediators with therapeutic potential, lung injury was induced in wild-type (WT) and Akt2(-/-) mice by hydrochloric acid aspiration. Acid-induced lung injury in WT mice was characterized by decreased lung compliance and increased protein and cytokine concentration in bronchoalveolar lavage fluid. Alveolar macrophages acquired a classical activation (M1) phenotype. Acid-induced lung injury was less severe in Akt2(-/-) mice compared with WT mice. Alveolar macrophages from acid-injured Akt2(-/-) mice demonstrated the alternative activation phenotype (M2). Although M2 polarization suppressed aseptic lung injury, it resulted in increased lung bacterial load when Akt2(-/-) mice were infected with Pseudomonas aeruginosa. miR-146a, an anti-inflammatory microRNA targeting TLR4 signaling, was induced during the late phase of lung injury in WT mice, whereas it was increased early in Akt2(-/-) mice. Indeed, miR-146a overexpression in WT macrophages suppressed LPS-induced inducible NO synthase (iNOS) and promoted M2 polarization, whereas miR-146a inhibition in Akt2(-/-) macrophages restored iNOS expression. Furthermore, miR-146a delivery or Akt2 silencing in WT mice exposed to acid resulted in suppression of iNOS in alveolar macrophages. In conclusion, Akt2 suppression and miR-146a induction promote the M2 macrophage phenotype, resulting in amelioration of acid-induced lung injury. In vivo modulation of macrophage phenotype through Akt2 or miR-146a could provide a potential therapeutic approach for aseptic ARDS; however, it may be deleterious in septic ARDS because of impaired bacterial clearance.

Sex steroids and personality traits in the middle luteal phase of healthy normally menstruating young professional women.

OBJECTIVE: Sex steroids affect human behavior. The aim of the present study was to determine the associations, if any, between the circulating levels of gonadal and adrenal sex steroids in the mid luteal phase (21st day of a normal menstrual cycle, MC) of young professional women and psychometric parameters as assessed by the Minnesota Multiphasic Personality Inventory (MMPI). RESULTS: Our results are as follows: (a) The metabolic product of activated adrenal and gonadal androgens, 3alpha-diolG, was modestly but significantly associated with the social introversion scale (10-SI) (r=0.36, p<0.05), independently accounting for 13% of its variation across participants (R²=0.13, F(1,45)=6.58, p=0.014). (b) Total testosterone was significantly associated with the paranoia scale (6-Pa) (r=0.27, p<0.05). Multiple regression analyses indicated that 10% of the variability in paranoia scores could be independently explained by total testosterone levels (R²=0.10, F(1,57)=6.23, p=0.016). We were unable to find any association between the circulating androgens and scores on the masculinity-femininity scale (Mf). We were also unable to document any association between the weak adrenal androgens DHEA and DHEA-S and depression in contrast to several published reports. (c) Our data suggest a marginally significant association between progesterone and scores on the 7-Pt (obsessive/compulsive/psychasthenia) scale (r=0.27, p<0.05). However, only 7% of the 7-Pt variance was explained by progesterone (R²=0.071, F(1,50)=3.81, p=0.057). CONCLUSIONS: We have found that total testosterone was associated with the paranoia score, the metabolic product of activated androgens, 3alpha-diolG, to social introversion and, finally, progesterone to obsessive-compulsive behavior.

Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization.

Activated macrophages are described as classically activated or M1 type and alternatively activated or M2 type, depending on their response to proinflammatory stimuli and the expression of genetic markers including iNOS, arginase1, Ym1, and Fizz1. Here we report that Akt kinases differentially contribute to macrophage polarization, with Akt1 ablation giving rise to an M1 and Akt2 ablation resulting in an M2 phenotype. Accordingly, Akt2(-/-) mice were more resistant to LPS-induced endotoxin shock and to dextran sulfate sodium (DSS)-induced colitis than wild-type mice, whereas Akt1(-/-) mice were more sensitive. Cell depletion and reconstitution experiments in a DSS-induced colitis model confirmed that the effect was macrophage-dependent. Gene-silencing studies showed that the M2 phenotype of Akt2(-/-) macrophages was cell autonomous. The microRNA miR-155, whose expression was repressed in naive and in LPS-stimulated Akt2(-/-) macrophages, and its target C/EBPß appear to play a key role in this process. C/EBPß, a hallmark of M2 macrophages that regulates Arg1, was up-regulated upon Akt2 ablation or silencing. Overexpression or silencing of miR-155 confirmed its central role in Akt isoform-dependent M1/M2 polarization of macrophages.

Baseline plasma levels of interleukin-8 in stage IV non-small-cell lung cancer patients: relationship with nutritional status and prognosis.

Interleukin (IL)-8 promotes cellular proliferation and angiogenesis in patients with non-small-cell lung cancer (NSCLC) and may be related to cachexia. Our aim was to investigate the relationship of IL-8 levels with nutritional status, and clinical outcome of patients with NSCLC. Patients with metastatic NSCLC referred for first-line therapy were eligible. Baseline IL-8 levels were measured in plasma. The Mini Nutritional Assessment (MNA) was used for the evaluation of the nutritional status, and patients were classified into 3 groups: A (score 24-30) "well nourished," B (score 17-23.5) "risk of malnutrition," and C (0-16.5) "malnourishment." Response to first-line chemotherapy, time-to-tumor progression (TTP), and overall survival (OS) were also recorded. In total, 114 patients (101 males, 88.5%; mean age = 67.5 yr) were evaluated. Performance status was 0-1 in 62% of the patients. According to the MNA, the majority of patients (71%) was either at nutritional risk or malnourished. IL-8 levels were significantly different between MNA groups (P = 0.023) and correlated with TTP (P = 0.013) and OS (P = 0.001) in univariate analysis. Baseline IL-8 levels correlate with the nutritional status of patients with metastatic NSCLC, suggesting that this cytokine may be related with cachexia.

The calcineurin-nuclear factor of activated T cells signaling pathway mediates the effect of corticotropin releasing factor and urocortins on catecholamine synthesis.

The biological effects of the Corticotropin-releasing factor (CRF) family of neuropeptides are mediated by mobilization of [Ca(2+)]. Aim of the current work was to examine if the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway is involved in the effect of CRF peptides in catecholamine synthesis and secretion from PC12 rat pheochromocytona cells, a model for the study of adrenal catecholamine production. PC12 cells express both types of CRF receptors. Our data are as follows: (a) The calcineurin inhibitor cyclosporine A (CsA) blocked norepinephrine secretion induced by ligands of either CRF type 1 (CRF(1)) or 2 (CRF(2)) receptors on PC12 cells. (b) Silencing NFAT2 expression using a selective NFAT2 siRNA blocked CRF(1) and CRF(2) -induced NE production. (c) CRF ligands induced NFAT transcriptional activity in cells transfected with a luciferase reporter construct controlled by NFAT binding elements (NFAT-Luc). (d) CsA completely blocked the stimulatory effect of CRF(1) and CRF(2) ligands on NFAT activity in NFAT-Luc transfected cells. (e) PKA, PKC, p38-MAPK, Tpl2, Ha-Ras, and AKT1 were crucial intermediates for both CRF(1) and CRF(2)-induced NFAT activation. Interestingly, MEK1/2 and ERK1/2 were crucial only for the CRF(2)-induced NFAT activation. (f) p38-MAPK and Tpl2 were crucial intermediates for both CRF(1) and CRF(2)-induced norepinephrine production, while AKT1 affected only CRF(2)-induced norepinephrine production. In conclusion, our data suggest that CRF(1) and CRF(2) ligands activate the transcription factor NFAT and its activation is prerequisite for CRF-induced catecholamine production from chromaffin cells.

A novel role of peripheral corticotropin-releasing hormone (CRH) on dermal fibroblasts.

Corticotropin-releasing hormone, or factor, (CRH or CRF) exerts important biological effects in multiple peripheral tissues via paracrine/autocrine actions. The aim of our study was to assess the effects of endogenous CRH in the biology of mouse and human skin fibroblasts, the primary cell type involved in wound healing. We show expression of CRH and its receptors in primary fibroblasts, and we demonstrate the functionality of fibroblast CRH receptors by induction of cAMP. Fibroblasts genetically deficient in Crh (Crh-/-) had higher proliferation and migration rates and compromised production of IL-6 and TGF-ß1 compared to the wildtype (Crh+/+) cells. Human primary cultures of foreskin fibroblasts exposed to the CRF(1) antagonist antalarmin recapitulated the findings in the Crh-/- cells, exhibiting altered proliferative and migratory behavior and suppressed production of IL-6. In conclusion, our findings show an important role of fibroblast-expressed CRH in the proliferation, migration, and cytokine production of these cells, processes associated with the skin response to injury. Our data suggest that the immunomodulatory effects of CRH may include an important, albeit not explored yet, role in epidermal tissue remodeling and regeneration and maintenance of tissue homeostasis.

The impact of stress on tumor growth: peripheral CRF mediates tumor-promoting effects of stress.

INTRODUCTION: Stress has been shown to be a tumor promoting factor. Both clinical and laboratory studies have shown that chronic stress is associated with tumor growth in several types of cancer. Corticotropin Releasing Factor (CRF) is the major hypothalamic mediator of stress, but is also expressed in peripheral tissues. Earlier studies have shown that peripheral CRF affects breast cancer cell proliferation and motility. The aim of the present study was to assess the significance of peripheral CRF on tumor growth as a mediator of the response to stress in vivo. METHODS: For this purpose we used the 4T1 breast cancer cell line in cell culture and in vivo. Cells were treated with CRF in culture and gene specific arrays were performed to identify genes directly affected by CRF and involved in breast cancer cell growth. To assess the impact of peripheral CRF as a stress mediator in tumor growth, Balb/c mice were orthotopically injected with 4T1 cells in the mammary fat pad to induce breast tumors. Mice were subjected to repetitive immobilization stress as a model of chronic stress. To inhibit the action of CRF, the CRF antagonist antalarmin was injected intraperitoneally. Breast tissue samples were histologically analyzed and assessed for neoangiogenesis. RESULTS: Array analysis revealed among other genes that CRF induced the expression of SMAD2 and ß-catenin, genes involved in breast cancer cell proliferation and cytoskeletal changes associated with metastasis. Cell transfection and luciferase assays confirmed the role of CRF in WNT- ß-catenin signaling. CRF induced 4T1 cell proliferation and augmented the TGF-ß action on proliferation confirming its impact on TGFß/SMAD2 signaling. In addition, CRF promoted actin reorganization and cell migration, suggesting a direct tumor-promoting action. Chronic stress augmented tumor growth in 4T1 breast tumor bearing mice and peripheral administration of the CRF antagonist antalarmin suppressed this effect. Moreover, antalarmin suppressed neoangiogenesis in 4T1 tumors in vivo. CONCLUSION: This is the first report demonstrating that peripheral CRF, at least in part, mediates the tumor-promoting effects of stress and implicates CRF in SMAD2 and ß-catenin expression.

The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs.

MicroRNAs regulated by lipopolysaccharide (LPS) target genes that contribute to the inflammatory phenotype. Here, we showed that the protein kinase Akt1, which is activated by LPS, positively regulated miRNAs let-7e and miR-181c but negatively regulated miR-155 and miR-125b. In silico analyses and transfection studies revealed that let-7e repressed Toll-like receptor 4 (TLR4), whereas miR-155 repressed SOCS1, two proteins critical for LPS-driven TLR signaling, which regulate endotoxin sensitivity and tolerance. As a result, Akt1(-/-) macrophages exhibited increased responsiveness to LPS in culture and Akt1(-/-) mice did not develop endotoxin tolerance in vivo. Overexpression of let-7e and suppression of miR-155 in Akt1(-/-) macrophages restored sensitivity and tolerance to LPS in culture and in animals. These results indicate that Akt1 regulates the response of macrophages to LPS by controlling miRNA expression.

Corticotropin Releasing Factor promotes breast cancer cell motility and invasiveness.

INTRODUCTION: Cancer cells secrete bioactive peptides that act in an autocrine or paracrine fashion affecting tumor growth and metastasis. Corticotropin-releasing factor (CRF), a hypothalamic neuropeptide that controls the response to stress, has been detected in breast cancer tissues and cell lines. CRF can affect breast cancer cells in an autocrine or paracrine manner via its production from innervating sympathetic neurons or immune cells. METHODS: In the present study we report our findings regarding the impact of CRF on breast cancer cell motility and invasiveness. For this purpose we used the MCF7 breast cancer cell line and evaluated the effect of CRF on motility and invasiveness using the wound-healing and boyden-chamber assays. In addition, we measured the effect of CRF on molecules that mediate motility by western blot, immunofluorescence, ELISA and RT-PCR. RESULTS: Our findings show that: 1. CRF transiently inhibited the apoptosis of MCF7 cells. 2. CRF enhanced MCF7 cell motility in a wound healing assay and their invasiveness through extracellular matrix. 3. CRF increased actin polymerization, phosphorylation of Focal Adhesion Kinase (FAK), providing a potential mechanism for the observed induction of MCF7 motility. 4. CRF induced the expression of Cox-1 but not Cox-2 in MCF7 cells as well as the production of prostaglandins, factors known to promote invasiveness and metastasis. CONCLUSION: Overall, our data suggest that CRF stimulates cell motility and invasiveness of MCF7 cells most probably via induction of FAK phosphorylation and actin filament reorganization and production of prostaglandins via Cox1. Based on these findings we postulate that the stress neuropeptide CRF present in the vicinity of tumors (either produced locally by the tumor cells themselves or by nearby normal cells or secreted from the innervations of surrounding tissues) may play an important role on breast tumor growth and metastatic capacity, providing a potential link between stress and tumor progression.

Adiponectin promotes endotoxin tolerance in macrophages by inducing IRAK-M expression.

High levels of plasma adiponectin are associated with low levels of inflammatory markers and cardioprotection. The mechanism via which adiponectin exerts its anti-inflammatory effect is yet unknown. In the present study, we demonstrate that globular adiponectin (gAd) induces the expression of the inactive isoform of IL-1R-associated kinases (IRAK), IRAK-M. Homologous deletion of IRAK-M in IRAK-M(-/-) mice abolished the tolerogenic properties of gAd because pretreatment of IRAK-M(-/-) macrophages with gAd did not suppress LPS-induced proinflammatory cytokine production. GAd activated the MAPKs MEK1/2 and ERK1/2 in macrophages via their upstream regulator Tpl2. Activation of ERK1/2 via Tpl2 appeared necessary for the induction of IRAK-M because gAd did not induce IRAK-M in Tpl2(-/-) macrophages or in macrophages pretreated with the MEK1/2 inhibitor UO126. In addition, activation of PI3K and Akt1 also appeared necessary for the induction of IRAK-M by gAd, because treatment of Akt1(-/-) macrophages or pretreatment of macrophages with the PI3K inhibitor wortmannin abolished gAd-induced IRAK-M expression. Analysis of IRAK-M expression in human peripheral blood cells confirmed that serum adiponectin was negatively associated with IRAK-M and responsiveness to LPS. In conclusion, our data demonstrate that IRAK-M is a major mediator of gAd-induced endotoxin tolerance in primary macrophages, expression of which depends on the activation of Tpl2/ERK and PI3K/Akt1 signaling pathways.

Neurosteroid dehydroepiandrosterone exerts anti-apoptotic effects by membrane-mediated, integrated genomic and non-genomic pro-survival signaling pathways.

Dehydroepiandrosterone (DHEA) protects neural crest-derived PC12 cells from serum deprivation-induced apoptosis via G protein-associated specific plasma membrane-binding sites (mDBS). Here, we studied the signaling pathways involved in the pro-survival effects of DHEA-mediated activation of the mDBS binding sites. Membrane impermeable DHEA-bovine serum albumin (BSA) conjugate induced an acute phosphorylation of the prosurvival kinases Src, protein kinase A (PKA), MEK1/2/ERK1/2, and PI3K/Akt in serum deprived PC12 cells in parallel to an elevation of intracellular cAMP. The physiological significance of these findings was further assessed in a series of experiments using several selective pro-survival kinase inhibitors. Our combined findings suggest that the following sequence of events may take place following activation of mDBS binding sites: DHEA-BSA induces an acute but transient sequential phosphorylation of the pro-survival kinases Src/PKC(a/b)/MEK1/2/ERK1/2 which, in their turn, activate transcription factors cAMP responsive element binding protein and nuclear factor kappa B which induce the expression of the anti-apoptotic Bcl-2 genes. In parallel, DHEA-BSA increases intracellular cAMP, and the subsequent phosphorylation of PKA kinase and of cAMP responsive element binding protein. Finally, DHEA-BSA induces phosphorylation of PI3K/Akt kinases which, subsequently, lead to phosphorylation/deactivation of the pro-apoptotic Bad. Our findings suggest that the neurosteroid DHEA affects neural crest-derived cell survival by multiple pro-survival signaling pathways comprising an integrated system of non-genomic and genomic mechanisms.

Neurosteroids as modulators of neurogenesis and neuronal survival.

Neurons and glia in the central nervous system express the necessary enzymes for the synthesis of neurosteroids that are produced in concentrations high enough to exert paracrine effects. Synthesis of brain neurosteroids declines with age, during stressful conditions (including major depression, chronic psychological stress), and in chronic inflammatory and neurodegenerative diseases. Recent reports associate the decrease of brain neurosteroids to neuronal dysfunction and degeneration. This review summarizes the recent findings on how the most studied neurosteroids (dehydroepiandrosterone, pregnenolone and their sulphate esters, progesterone and allopregnanolone) affect neuronal survival, neurite outgrowth and neurogenesis; furthermore, this review discusses potential applications of these neurosteroids in the therapeutic management of neurodegenerative conditions, including that of age-related brain atrophy.

Vasoactive intestinal peptide suppresses toll-like receptor 4 expression in macrophages via Akt1 reducing their responsiveness to lipopolysaccharide.

Toll-like receptor 4 (TLR4) recognizes and initiates signals from Gram-negative bacterial lipopolysaccharide (LPS) triggering the inflammatory response. Expression levels of TLR4 on macrophages partly regulate the magnitude of the response to LPS. Vasoactive Intestinal Peptide (VIP) is known to block inflammatory responses by inhibiting pro-inflammatory cytokine production from activated macrophages. In the present report we demonstrate that VIP directly suppressed TLR4 expression on naïve primary mouse macrophages utilizing signalling cascades that control TLR4 transcription. VIP-induced suppression of TLR4 occurred at the transcriptional level by decreasing PU.1 DNA binding. Mutation of the proximal PU.1 but not the AP-1-binding site on the TLR4 promoter abrogated VIP-induced suppression of TLR4 transcription. Moreover, inhibition of PI3K by wortmannin or homologous deletion of the Akt1 isoform, a pathway known to act as a negative regulator of macrophage activation, alleviated the suppressive action of VIP on TLR4 expression. To evaluate the biological significance of VIP effect on TLR4 expression, Raw264.7 macrophages were pre-treated with VIP for 24h and then exposed to LPS. Pre-treatment with VIP rendered macrophages hypo-responsive to LPS resulting in reduced pro-inflammatory cytokine production. Moreover, in vivo administration of VIP in C57BL/6 mice resulted in lower IL-6 production upon treatment with LPS. Overall, the results indicate that VIP promotes endotoxin tolerance by downregulating TLR4 expression via Akt1.