Elevated CRP and TNF-α levels are associated with blunted neural oscillations serving fluid intelligence.

INTRODUCTION: Inflammatory processes help protect the body from potential threats such as bacterial or viral invasions. However, when such inflammatory processes become chronically engaged, synaptic impairments and neuronal cell death may occur. In particular, persistently high levels of C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-α) have been linked to deficits in cognition and several psychiatric disorders. Higher-order cognitive processes such as fluid intelligence (Gf) are thought to be particularly vulnerable to persistent inflammation. Herein, we investigated the relationship between elevated CRP and TNF-α and the neural oscillatory dynamics serving Gf. METHODS: Seventy adults between the ages of 20-66 years (Mean = 45.17 years, SD = 16.29, 21.4% female) completed an abstract reasoning task that probes Gf during magnetoencephalography (MEG) and provided a blood sample for inflammatory marker analysis. MEG data were imaged in the time-frequency domain, and whole-brain regressions were conducted using each individual's plasma CRP and TNF-α concentrations per oscillatory response, controlling for age, BMI, and education. RESULTS: CRP and TNF-α levels were significantly associated with region-specific neural oscillatory responses. In particular, elevated CRP concentrations were associated with altered gamma activity in the right inferior frontal gyrus and right cerebellum. In contrast, elevated TNF-α levels scaled with alpha/beta oscillations in the left anterior cingulate and left middle temporal, and gamma activity in the left intraparietal sulcus. DISCUSSION: Elevated inflammatory markers such as CRP and TNF-α were associated with aberrant neural oscillations in regions important for Gf. Linking inflammatory markers with regional neural oscillations may hold promise in identifying mechanisms of cognitive and psychiatric disorders.

T-lymphocyte tyrosine hydroxylase regulates TH17 T-lymphocytes during repeated social defeat stress.

Posttraumatic stress disorder (PTSD) is a debilitating psychiatric disorder which results in deleterious changes to psychological and physical health. Patients with PTSD are especially susceptible to life-threatening co-morbid inflammation-driven pathologies, such as autoimmunity, while also demonstrating increased T-helper 17 (TH17) lymphocyte-driven inflammation. While the exact mechanism of this increased inflammation is unknown, overactivity of the sympathetic nervous system is a hallmark of PTSD. Neurotransmitters of the sympathetic nervous system (i.e., catecholamines) can alter T-lymphocyte function, which we have previously demonstrated to be partially mitochondrial redox-mediated. Furthermore, we have previously elucidated that T-lymphocytes generate their own catecholamines, and strong associations exist between tyrosine hydroxylase (TH; the rate-limiting enzyme in the synthesis of catecholamines) and pro-inflammatory interleukin 17A (IL-17A) expression within purified T-lymphocytes in a rodent model of psychological trauma. Therefore, we hypothesized that T-lymphocyte-generated catecholamines drive TH17 T-lymphocyte polarization through a mitochondrial superoxide-dependent mechanism during psychological trauma. To test this, T-lymphocyte-specific TH knockout mice (THT-KO) were subjected to psychological trauma utilizing repeated social defeat stress (RSDS). RSDS characteristically increased tumor necrosis factor-α (TNFα), IL-6, IL-17A, and IL-22, however, IL-17A and IL-22 (TH17 produced cytokines) were selectively attenuated in circulation and in T-lymphocytes of THT-KO animals. When activated ex vivo, secretion of IL-17A and IL-22 by THT-KO T-lymphocytes was also found to be reduced, but could be partially rescued with supplementation of norepinephrine specifically. Interestingly, THT-KO T-lymphocytes were still able to polarize to TH17 under exogenous polarizing conditions. Last, contrary to our hypothesis, we found RSDS-exposed THT-KO T-lymphocytes still displayed elevated mitochondrial superoxide, suggesting increased mitochondrial superoxide is upstream of T-lymphocyte TH induction, activity, and TH17 regulation. Overall, these data demonstrate TH in T-lymphocytes plays a critical role in RSDS-induced TH17 T-lymphocytes and offer a previously undescribed regulator of inflammation in RSDS.

Dimers of isatin derived α-methylene-γ-butyrolactone as potent anti-cancer agents.

The IKK-NFκB complex is a key signaling node that facilitates activation of gene expression in response to extracellular signals. The kinase IKKß and the transcription factor RELA have been targeted by covalent modifiers that bind to surface exposed cysteine residues. A common feature in well characterized covalent modifiers of RELA and IKKß is the Michael acceptor containing α-methylene-γ-butyrolactone functionality. Through synthesis and evaluation of a focused set of α-methylene-γ-butyrolactone containing spirocyclic dimers (SpiDs) we identified SpiD3 as an anticancer agent with low nanomolar potency. Using cell-free and cell-based studies we show that SpiD3 is a covalent modifier that generates stable RELA containing high molecular weight complexes. SpiD3 inhibits TNFα-induced IκBα phosphorylation resulting in the blockade of RELA nuclear translocation. SpiD3 induces apoptosis, inhibits colony formation and migration of cancer cells. The NCI-60 cell line screen revealed that SpiD3 potently inhibits growth of leukemia cell lines, making it a suitable pre-therapeutic lead for hematological malignancies.

Neutrophils are mediators of metastatic prostate cancer progression in bone.

Bone metastatic prostate cancer (BM-PCa) significantly reduces overall patient survival and is currently incurable. Current standard immunotherapy showed promising results for PCa patients with metastatic, but less advanced, disease (i.e., fewer than 20 bone lesions) suggesting that PCa growth in bone contributes to response to immunotherapy. We found that: (1) PCa stimulates recruitment of neutrophils, the most abundant immune cell in bone, and (2) that neutrophils heavily infiltrate regions of prostate tumor in bone of BM-PCa patients. Based on these findings, we examined the impact of direct neutrophil-prostate cancer interactions on prostate cancer growth. Bone marrow neutrophils directly induced apoptosis of PCa in vitro and in vivo, such that neutrophil depletion in bone metastasis models enhanced BM-PCa growth. Neutrophil-mediated PCa killing was found to be mediated by suppression of STAT5, a transcription factor shown to promote PCa progression. However, as the tumor progressed in bone over time, neutrophils from late-stage bone tumors failed to elicit cytotoxic effector responses to PCa. These findings are the first to demonstrate that bone-resident neutrophils inhibit PCa and that BM-PCa are able to progress via evasion of neutrophil-mediated killing. Enhancing neutrophil cytotoxicity in bone may present a novel therapeutic option for bone metastatic prostate cancer.

Peripheral inflammation is strongly linked to elevated zero maze behavior in repeated social defeat stress.

Post-traumatic stress disorder (PTSD) is a psychiatric illness that results in an increased risk for a variety of inflammatory diseases. The exact etiology of this increased risk is unknown, and thus several animal models have been developed to investigate the neuroimmune interactions of PTSD. Repeated social defeat stress (RSDS) is an established preclinical model of psychological trauma that recapitulates certain behavioral and inflammatory aspects of human PTSD. Furthermore, RSDS has been utilized to subgroup animals into susceptible and resilient populations based on one specific behavioral phenotype (i.e., social interaction). Herein, we conducted an extensive investigation of circulating inflammatory proteins after RSDS and found significant elevations in various cytokines and chemokines after exposure to RSDS. When categorizing animals into either susceptible or resilient populations based on social interaction, we found no inflammatory or other behavioral differences between these subgroups. Furthermore, correlative analyses found no significant correlation between social interaction parameters and inflammation. In contrast, parameters from the elevated zero maze (EZM) demonstrated significant associations and clustering to five circulating cytokines. When animals were subdivided into susceptible and resilient populations solely based upon combined EZM performance, significant inflammatory differences were evident between these groups. Strikingly, these circulating inflammatory proteins displayed a stronger predictive ability of EZM performance compared to social interaction test performance. These findings provide new insights into inflammatory markers associated with RSDS, and the utility of EZM to effectively group RSDS-exposed mice into populations with differential levels of peripheral inflammation.

Oxidative Stress in Pregnant Women Between 12 and 20 Weeks of Gestation and Preterm Birth.

BACKGROUND: A known relationship exists between oxidative stress and preterm birth (PTB). However, few studies have measured oxidative stress prospectively in early or midpregnancy, and no studies have used electron paramagnetic resonance (EPR) spectroscopy prospectively to predict PTB. OBJECTIVE: The purpose of this study was to identify predictive relationships between antioxidants and reactive oxygen species (ROS), specifically, superoxide (O2), peroxynitrite (OONO), and hydroxyl radical (OH), using EPR spectroscopy, measured between 12 and 20 weeks of gestation and compare with the incidence of PTB. METHODS: Blood was obtained from pregnant women (n = 140) recruited from a tertiary perinatal center. Whole blood was analyzed directly for ROS, O2, OONO, and OH using EPR spectroscopy. Red blood cell lysate was used to measure antioxidants. PTB was defined as parturition at <37 weeks of gestation. RESULTS: No differences were found between ROS, O2, OONO, or OH with the incidence of PTB. Catalase activity, glutathione, and reduced/oxidized glutathione ratio were significantly lower with PTB. Logistic regression suggests decreased catalase activity in pregnant women is associated with increased odds of delivering prematurely. DISCUSSION: We prospectively compared antioxidants and specific ROS using EPR spectroscopy in pregnant women between 12 and 20 weeks of gestation with the incidence of PTB. Results are minimal but do suggest that antioxidants-specifically decreased catalase activity-in early pregnancy may be associated with PTB; however, these findings should be cautiously interpreted and may not have clinical significance.

Sympathoexcitation in response to cardiac and pulmonary afferent stimulation of TRPA1 channels is attenuated in rats with chronic heart failure.

Excessive sympathoexcitation characterizes the chronic heart failure (CHF) state. An exaggerated cardiac sympathetic afferent reflex (CSAR) contributes to this sympathoexcitation. Prior studies have demonstrated that the CSAR to capsaicin [transient receptor potential (TRP) vanilloid 1 agonist] is exaggerated in CHF animal models. We recently discovered that capsaicin application to the lung visceral pleura in anesthetized, vagotomized, open-chested rats increases mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). We named this response the pulmonary spinal afferent reflex (PSAR). Due to the similarities between TRP vanilloid 1 and TRP ankyrin 1 (TRPA1) channels as well as the excessive sympathoexcitation of CHF, we hypothesized that stimulation of the CSAR and PSAR with a specific TRPA1 agonist would result in an augmented response in CHF rats (coronary ligation model) compared with sham control rats. In response to a TRPA1 agonist, both CSAR and PSAR in sham rats resulted in biphasic changes in MAP and increases in HR and RSNA 10-12 wk postmyocardial infarction (post-MI). These effects were blunted in CHF rats. Assessment of TRPA1 expression levels in cardiopulmonary spinal afferents by immunofluorescence, quantitative RT-PCR, and Western blot analysis 10-12 wk post-MI all indicates reduced expression in CHF rats but no reduction at earlier time points. TRPA1 protein was reduced in a dorsal root ganglia cell culture model of inflammation and simulated tissue ischemia, raising the possibility that the in vivo reduction of TRPA1 expression was, in part, caused by CHF-related tissue ischemia and inflammation. These data provide evidence that reflex responses to cardiopulmonary spinal afferent TRPA1 stimulation may be attenuated in CHF rather than enhanced. NEW & NOTEWORTHY Excessive sympathoexcitation characterizes chronic heart failure (CHF). The contribution of transient receptor potential ankyrin 1 (TRPA1) channel-mediated reflexes to this sympathoexcitation is unknown. We found that application of TRPA1 agonist to the heart and lung surface resulted in increased heart rate and sympathetic output and a biphasic change in mean arterial pressure in control rats. These effects were attenuated in CHF rats, decreasing the likelihood that TRPA1 channels contribute to cardiopulmonary afferent sensitization in CHF.

Autonomic regulation of T-lymphocytes: Implications in cardiovascular disease.

The nervous and immune systems both serve as essential assessors and regulators of physiological function. Recently, there has been a great interest in how the nervous and immune systems interact to modulate both physiological and pathological states. In particular, the autonomic nervous system has a direct line of communication with immune cells anatomically, and moreover, immune cells possess receptors for autonomic neurotransmitters. This circumstantial evidence is suggestive of a functional interplay between the two systems, and extensive research over the past few decades has demonstrated neurotransmitters such as the catecholamines (i.e. dopamine, norepinephrine, and epinephrine) and acetylcholine have potent immunomodulating properties. Furthermore, immune cells, particularly T-lymphocytes, have now been found to express the cellular machinery for both the synthesis and degradation of neurotransmitters, which suggests the ability for both autocrine and paracrine signaling from these cells independent of the nervous system. The details underlying the functional interplay of this complex network of neuroimmune communication are still unclear, but this crosstalk is suggestive of significant implications on the pathogenesis of a number of autonomic-dysregulated and inflammation-mediated diseases. In particular, it is widely accepted that numerous forms of cardiovascular diseases possess imbalanced autonomic tone as well as altered T-lymphocyte function, but a paucity of literature exists discussing the direct role of neurotransmitters in shaping the inflammatory microenvironment during the progression or therapeutic management of these diseases. This review seeks to provide a fundamental framework for this autonomic neuroimmune interaction within T-lymphocytes, as well as the implications this may have in cardiovascular diseases.

Interleukin-17A and Chronic Stress in Pregnant Women at 24-28 Weeks Gestation.

BACKGROUND: Allostatic load (AL) is a biopsychosocial model that suggests chronic psychosocial stress leads to physiological dysregulation and poor outcomes. The purpose of this study was to examine AL in pregnant women operationalized using proinflammatory cytokines and psychosocial indicators and perinatal outcomes. OBJECTIVES: The aim of the study was to identify relationships between circulating cytokines/chemokines and the Prenatal Distress Questionnaire, the Maternal Antenatal Attachment Scale, the Emotional Quotient Inventory, the Life Experiences Scale, and demographics in pregnant women. METHODS: A cross-sectional design was used to recruit pregnant women between 24 and 28 weeks of gestation. Blood and stress/emotional indicators were obtained after informed consent. Plasma was abstracted to simultaneously measure 29 cytokines/chemokines using a multiplex array. Cytokine/chemokine levels were compared with continuous variables using Spearman's rho and with categorical variables using Mann-Whitney U. RESULTS: Twenty-five women with medically high-risk (n = 16) and low-risk (n = 9) pregnancies consented. Most women were White (68%) with a mean age of 29 years (SD = 5.9). Although several cytokines and chemokines showed significant correlations with the stress/emotional indicators, only interleukin-17A (IL-17A) was significantly associated with all of the indicators (Prenatal Distress Questionnaire: rs = .528, p = .012; Maternal Antenatal Attachment Scale: rs = -.439, p = .036; Emotional Quotient Inventory total: rs = -.545, p = .007), Life Experiences Scale (rs = .458, p = .032), birth weight (rs = -.499, p = .013), and race (p = .01). DISCUSSION: Increased levels of IL-17A, a known cytokine associated with chronic stress and with poor perinatal outcomes, were associated with high prenatal distress, low maternal attachment, and lower emotional intelligence in pregnant women. Increased levels of IL-17A also were associated with lower birth weight and non-White race. Results support the model of AL in pregnant women and highlight IL-17A as a potential biomarker of AL during pregnancy.

Sympathetic-mediated activation versus suppression of the immune system: consequences for hypertension.

It is generally well-accepted that the immune system is a significant contributor in the pathogenesis of hypertension. Specifically, activated and pro-inflammatory T-lymphocytes located primarily in the vasculature and kidneys appear to have a causal role in exacerbating elevated blood pressure. It has been proposed that increased sympathetic nerve activity and noradrenaline outflow associated with hypertension may be primary contributors to the initial activation of the immune system early in the disease progression. However, it has been repeatedly demonstrated in many different human and experimental diseases that sympathoexcitation is immunosuppressive in nature. Moreover, human hypertensive patients have demonstrated increased susceptibility to secondary immune insults like infections. Thus, it is plausible, and perhaps even likely, that in diseases like hypertension, specific immune cells are activated by increased noradrenaline, while others are in fact suppressed. We propose a model in which this differential regulation is based upon activation status of the immune cell as well as the resident organ. With this, the concept of global immunosuppression is obfuscated as a viable target for hypertension treatment, and we put forth the concept of focused organ-specific immunotherapy as an alternative option.

Overexpression of copper/zinc superoxide dismutase in the median preoptic nucleus improves cardiac function after myocardial infarction in the rat.

Previous reports indicate that overexpression of copper/zinc superoxide dismutase (CuZnSOD), an intracellular superoxide (O2 (•-) ) scavenging enzyme, in the brain subfornical organ improves cardiac function in a mouse model of heart failure (HF). A downstream hypothalamic site, the MnPO, may act as a relay centre for O2 (•-) to serve as a mediator in the pathophysiology of HF. To test the hypothesis that elevated O2 (•-) in the MnPO contributes to the pathophysiology of HF and decreased cardiac function, we injected adenovirus encoding CuZnSOD (AdCuZnSOD, n=7) or control empty adenovirus vector (AdEmpty, n=7) into the MnPO of normal rats. Subsequently, rats were subjected to coronary artery ligation to create a myocardial infarct (MI) of the left ventricle. Cardiac function was monitored via echocardiography. Upon completion, rat brains were examined for CuZnSOD expression in MnPO via immunofluorescence and histopathological analyses of cardiac infarct size were conducted. Baseline (EF) ejection fractions (%) of AdCuZnSOD and AdEmpty rats were 73 ± 1 and 71 ± 1, respectively. Two weeks after MI, EF was significantly decreased in both groups of rats (AdCuZnSOD: 51 ± 3, AdEmpty: 46 ± 1). In contrast, by 4 weeks post MI, EF had improved to 64 ± 2 in AdCuZnSOD rats, yet was only 52 ± 1 in AdEmpty rats, and this was accompanied by lower plasma noradrenaline levels in AdCuZnSOD rats (0.49 ± 0.19 ng/mL) compared to AdEmpty rats (1.20 ± 0.32 ng/mL). In conclusion, despite decreases in EF early after MI, overexpression of CuZnSOD in the MnPO was related to an improvement in left ventricular function and concomitant decreased plasma noradrenaline levels 4 weeks post MI.

Innate and adaptive immune system consequences of post-traumatic stress disorder.

In the field of psychiatry, biological markers are rarely, if ever, used in the diagnosis of mental health disorders. Clinicians rely primarily on patient histories and behavioral symptoms to identify specific psychopathologies, which makes diagnosis highly subjective. Moreover, therapies for mental health disorders are aimed specifically at attenuating behavioral manifestations, which overlooks the pathophysiological indices of the disease. This is highly evident in posttraumatic stress disorder (PTSD) where inflammation and immune system perturbations are becoming increasingly described. Further, patients with PTSD possess significantly elevated risks of developing comorbid inflammatory diseases such as autoimmune and cardiovascular diseases, which are likely linked (though not fully proven) to the apparent dysregulation of the immune system after psychological trauma. To date, there is little to no evidence that demonstrates current PTSD therapies are able to reverse the increased risk for psychological trauma-induced inflammatory diseases, which suggests the behavioral and somatic consequences of PTSD may not be tightly coupled. This observation provides an opportunity to explore unique mechanisms outside of the brain that contribute to the long-term pathology of PTSD. Herein, we provide an overview of neuroimmune mechanisms, describe what is known regarding innate and adaptive immunity in PTSD, and suggest new directions that are needed to advance the understanding, diagnosis, and treatment of PTSD moving forward.

Beta-adrenergic signaling and T-lymphocyte-produced catecholamines are necessary for interleukin 17A synthesis.

Background: Post-traumatic stress disorder (PTSD) is a debilitating psychological disorder that also presents with neuroimmune irregularities. Patients display elevated sympathetic tone and are at an increased risk of developing secondary autoimmune diseases. Previously, using a preclinical model of PTSD, we demonstrated that elimination of sympathetic signaling to T-lymphocytes specifically limited their ability to produce pro-inflammatory interleukin 17A (IL-17A); a cytokine implicated in the development of many autoimmune disorders. However, the mechanism linking sympathetic signaling to T-lymphocyte IL-17A production remained unclear. Methods: Using a modified version of repeated social defeat stress (RSDS) that allows for both males and females, we assessed the impact of adrenergic receptor blockade (genetically and pharmacologically) and catecholamine depletion on T-lymphocyte IL-17A generation. Additionally, we explored the impact of adrenergic signaling and T-lymphocyte-produced catecholamines on both CD4+ and CD8+ T-lymphocytes polarized to IL-17A-producing phenotypes ex vivo. Results: Only pharmacological inhibition of the beta 1 and 2 adrenergic receptors (ß1/2) significantly decreased circulating IL-17A levels after RSDS, but did not impact other pro-inflammatory cytokines (e.g., IL-6, TNF-α, and IL-10). This finding was confirmed using RSDS with both global ß1/2 receptor knock-out mice, as well as by adoptively transferring ß1/2 knock-out T-lymphocytes into immunodeficient hosts. Furthermore, ex vivo polarized T-lymphocytes produced significantly less IL-17A with the blockade of ß1/2 signaling, even in the absence of exogenous sympathetic neurotransmitter supplementation, which suggested T-lymphocyte-produced catecholamines may be involved in IL-17A production. Indeed, pharmacological depletion of catecholamines both in vivo and ex vivo abrogated T-lymphocyte IL-17A production demonstrating the importance of immune-generated neurotransmission in pro-inflammatory cytokine generation. Conclusions: Our data depict a novel role for ß1/2 adrenergic receptors and autologous catecholamine signaling during T-lymphocyte IL-17A production. These findings provide a new target for pharmacological therapy in both psychiatric and autoimmune diseases associated with IL-17A-related pathology.

Mitochondrial-localized NADPH oxidase 4 is a source of superoxide in angiotensin II-stimulated neurons.

Angiotensin II (ANG II) plays an important role in the central regulation of systemic cardiovascular function. ANG II-mediated intraneuronal signaling has been shown to be predicated by an increase in mitochondrial superoxide (O2∙-), yet the source of this reactive oxygen species (ROS) production remains unclear. NADPH oxidase 4 (Nox4), a member of the NADPH oxidase family, has been reported to be localized in mitochondria of various cell types and has been implicated in brain angiotensinergic signaling. However, the subcellular localization and function of Nox4 in neurons has not been fully elucidated. In this study, we hypothesized that Nox4 is expressed in neuron mitochondria and is involved in ANG II-dependent O2∙--mediated intraneuronal signaling. To query this, Nox4 immunofluorescent staining and mitochondrial enrichment were performed in a mouse catecholaminergic neuronal cell model (CATH.a). Nox4 was shown to be present in neuron mitochondria as evidenced by colocalization with both the mitochondrial-localized protein manganese superoxide dismutase (MnSOD) and dye MitoTracker Red. Moreover, Nox4 expression was significantly increased in enriched mitochondrial fractions compared with whole cell lysates. Additionally, adenoviral-encoded small interfering RNA for Nox4 (AdsiNox4) caused a robust knockdown in Nox4 mRNA and protein levels, which led to the attenuation of ANG II-induced mitochondrial O2∙- production. Finally, in the subfornical organ (SFO) of the brain, Nox4 not only demonstrated mitochondrial localization but was induced by chronic, peripheral infusion of ANG II. Collectively, these data suggest that Nox4 is a source of O2∙- in neuron mitochondria that contributes to ANG II intraneuronal signaling.

NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells.

Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formation in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect.

Defining the nuanced nature of redox biology in post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a mental health disorder that arises after experiencing or witnessing a traumatic event. Despite affecting around 7% of the population, there are currently no definitive biological signatures or biomarkers used in the diagnosis of PTSD. Thus, the search for clinically relevant and reproducible biomarkers has been a major focus of the field. With significant advances of large-scale multi-omic studies that include genomic, proteomic, and metabolomic data, promising findings have been made, but the field still has fallen short. Amongst the possible biomarkers examined, one area is often overlooked, understudied, or inappropriately investigated: the field of redox biology. Redox molecules are free radical and/or reactive species that are generated as a consequence of the necessity of electron movement for life. These reactive molecules, too, are essential for life, but in excess are denoted as "oxidative stress" and often associated with many diseases. The few studies that have examined redox biology parameters have often utilized outdated and nonspecific methods, as well as have reported confounding results, which has made it difficult to conclude the role for redox in PTSD. Herein, we provide a foundation of how redox biology may underlie diseases like PTSD, critically examine redox studies of PTSD, and provide future directions the field can implement to enhance standardization, reproducibility, and accuracy of redox assessments for the use of diagnosis, prognosis, and therapy of this debilitating mental health disorder.

Repeated social defeat stress leads to immunometabolic shifts in innate immune cells of the spleen.

Psychosocial stress has been shown to prime peripheral innate immune cells, which take on hyper-inflammatory phenotypes and are implicated in depressive-like behavior in mouse models. However, the impact of stress on cellular metabolic states that are thought to fuel inflammatory phenotypes in immune cells are unknown. Using single cell RNA-sequencing, we investigated mRNA enrichment of immunometabolic pathways in innate immune cells of the spleen in mice subjected to repeated social defeat stress (RSDS) or no stress (NS). RSDS mice displayed a significant increase in the number of splenic macrophages and granulocytes (p < 0.05) compared to NS littermates. RSDS-upregulated genes in macrophages, monocytes, and granulocytes significantly enriched immunometabolic pathways thought to play a role in myeloid-driven inflammation (glycolysis, HIF-1 signaling, MTORC1 signaling) as well as pathways related to oxidative phosphorylation (OXPHOS) and oxidative stress (p < 0.05 and FDR<0.1). These results suggest that the metabolic enhancement reflected by upregulation of glycolytic and OXPHOS pathways may be important for cellular proliferation of splenic macrophages and granulocytes following repeated stress exposure. A better understanding of these intracellular metabolic mechanisms may ultimately help develop novel strategies to reverse the impact of stress and associated peripheral immune changes on the brain and behavior.

S100a9 Protects Against the Effects of Repeated Social Defeat Stress.

Background: Posttraumatic stress disorder, a consequence of psychological trauma, is associated with increased inflammation and an elevated risk of developing comorbid inflammatory diseases. However, the mechanistic link between this mental health disorder and inflammation remains elusive. We previously found that S100a8 and S100a9 messenger RNA, genes that encode the protein calprotectin, were significantly upregulated in T lymphocytes and positively correlated with inflammatory gene expression and the mitochondrial redox environment in these cells. Therefore, we hypothesized that genetic deletion of calprotectin would attenuate the inflammatory and redox phenotype displayed after psychological trauma. Methods: We used a preclinical mouse model of posttraumatic stress disorder known as repeated social defeat stress (RSDS) combined with pharmacological and genetic manipulation of S100a9 (which functionally eliminates calprotectin). A total of 186 animals (93 control, 93 RSDS) were used in these studies. Results: Unexpectedly, we observed worsening of behavioral pathology, inflammation, and the mitochondrial redox environment in mice after RSDS compared with wild-type animals. Furthermore, loss of calprotectin significantly enhanced the metabolic demand on T lymphocytes, suggesting that this protein may play an undescribed role in mitochondrial regulation. This was further supported by single-cell RNA sequencing analysis demonstrating that RSDS and loss of S100a9 primarily altered genes associated with mitochondrial function and oxidative phosphorylation. Conclusions: These data demonstrate that the loss of calprotectin potentiates the RSDS-induced phenotype, which suggests that its observed upregulation after psychological trauma may provide previously unexplored protective functions.

Repeated Social Defeat Stress Induces an Inflammatory Gut Milieu by Altering the Mucosal Barrier Integrity and Gut Microbiota Homeostasis.

Background: Posttraumatic stress disorder (PTSD) is a mental health condition triggered by exposure to traumatic events in an individual's life. Patients with PTSD are also at a higher risk for comorbidities. However, it is not well understood how PTSD affects human health and/or promotes the risk for comorbidities. Nevertheless, patients with PTSD harbor a proinflammatory milieu and dysbiotic gut microbiota. Gut barrier integrity helps to maintain normal gut homeostasis and its dysregulation promotes gut dysbiosis and inflammation. Methods: We used a mouse model of repeated social defeat stress (RSDS), a preclinical model of PTSD. Behavioral studies, metagenomics analysis of the microbiome, gut permeability assay (on mouse colon, using an Ussing chamber), immunoblotting, and immunohistochemical analyses were performed. Polarized intestinal epithelial cells and 3-dimensional crypt cultures were used for mechanistic analysis. Results: The RSDS mice harbor a heightened proinflammatory gut environment and microbiota dysbiosis. The RSDS mice further showed significant dysregulation of gut barrier functions, including transepithelial electrical resistance, mucin homeostasis, and antimicrobial responses. RSDS mice also showed a specific increase in intestinal expression of claudin-2, a tight junction protein, and epinephrine, a stress-induced neurotransmitter. Treating intestinal epithelial cells or 3-dimensional cultured crypts with norepinephrine or intestinal luminal contents (fecal contents) upregulated claudin-2 expression and inhibited transepithelial electrical resistance. Conclusions: Traumatic stress induces dysregulation of gut barrier functions, which may underlie the observed gut microbiota changes and proinflammatory gut milieu, all of which may have an interdependent effect on the health and increased risk of comorbidities in patients with PTSD.

Cytokine levels throughout the perinatal period.

BACKGROUND: Dysregulation of inflammatory processes is linked to perinatal complications yet a comprehensive description of cytokine levels throughout the perinatal period is lacking. We report prospective, serial levels of 29 unique cytokines measured in maternal blood during pregnancy, in the cord blood at birth, and in the neonatal blood. METHODS: Pregnant women (n = 140) for recruited from a Midwest tertiary medical center. Blood was obtained at five timepoints: 12-20 weeks, 24-28 weeks, and at labor in the women, umbilical cord at birth, 24-72 h in the newborn. Cytokine levels were analyzed using an electrochemiluminescence-based immunoassay. RESULTS: Levels for 29 cytokines were measured. The data were separated into two groups: pregnancies with (n = 82) and without major complications (n = 53) (preterm birth, preeclampsia, diabetes mellitus). Eighteen cytokines showed significant changes over time (p < .002). The majority of the cytokines were highest in the newborn. No differences in cytokine levels between complication groups were noted at any timepoint. CONCLUSIONS: This is the first known study to report prospective, serial cytokine levels throughout the perinatal period for pregnancies with/without complications. No differences in maternal cytokine levels between those with/without complications were detected; studies with a larger sample size would be needed to validate our current findings. Results also suggest cytokine dysregulation may be more localized to the placenta making it difficult to measure and predict during pregnancy using maternal systemic blood specimens.