Post-traumatic Stress Disorder: Focus on Neuroinflammation.

Post-traumatic stress disorder (PTSD), gaining increasing attention, is a multifaceted psychiatric disorder that occurs following a stressful or traumatic event or series of events. Recently, several studies showed a close relationship between PTSD and neuroinflammation. Neuroinflammation, a defense response of the nervous system, is associated with the activation of neuroimmune cells such as microglia and astrocytes and with changes in inflammatory markers. In this review, we first analyzed the relationship between neuroinflammation and PTSD: the effect of stress-derived activation of the hypothalamic-pituitary-adrenal (HPA) axis on the main immune cells in the brain and the effect of stimulated immune cells in the brain on the HPA axis. We then summarize the alteration of inflammatory markers in brain regions related to PTSD. Astrocytes are neural parenchymal cells that protect neurons by regulating the ionic microenvironment around neurons. Microglia are macrophages of the brain that coordinate the immunological response. Recent studies on these two cell types provided new insight into neuroinflammation in PTSD. These contribute to promoting comprehension of neuroinflammation, which plays a pivotal role in the pathogenesis of PTSD.

Mediators of systemic sclerosis-associated interstitial lung disease (SSc-ILD): systematic review and meta-analyses.

Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is rare, poorly understood, with heterogeneous characteristics resulting in difficult diagnosis. We aimed to systematically review evidence of soluble markers in peripheral blood or bronchoalveolar lavage fluid (BALF) as biomarkers in SSc-ILD. METHOD: Five databases were screened for observational or interventional, peer-reviewed studies in adults published between January 2000 and September 2021 that assessed levels of biomarkers in peripheral blood or BALF of SSc-ILD patients compared with healthy controls. Qualitative assessment was performed using Critical Appraisal Skills Programme (CASP) checklists. Standardised mean difference (SMD) in biomarkers were combined in random-effects meta-analyses where multiple independent studies reported quantitative data. RESULTS: 768 published studies were identified; 38 articles were included in the qualitative synthesis. Thirteen studies were included in the meta-analyses representing three biomarkers: KL6, SP-D and IL-8. Greater IL-8 levels were associated with SSc-ILD in both peripheral blood and BALF, overall SMD 0.88 (95% CI 0.61 to 1.15; I2=1%). Greater levels of SP-D and KL-6 were both estimated in SSc-ILD peripheral blood compared with healthy controls, at an SMD of 1.78 (95% CI 1.50 to 2.17; I2=8%) and 1.66 (95% CI 1.17 to 2.14; I2=76%), respectively. CONCLUSION: We provide robust evidence that KL-6, SP-D and IL-8 have the potential to serve as reliable biomarkers in blood/BALF for supporting the diagnosis of SSc-ILD. However, while several other biomarkers have been proposed, the evidence of their independent value in diagnosis and prognosis is currently lacking and needs further investigation. PROSPERO REGISTRATION NUMBER: CRD42021282452.

Fibroblast A20 governs fibrosis susceptibility and its repression by DREAM promotes fibrosis in multiple organs.

In addition to autoimmune and inflammatory diseases, variants of the TNFAIP3 gene encoding the ubiquitin-editing enzyme A20 are also associated with fibrosis in systemic sclerosis (SSc). However, it remains unclear how genetic factors contribute to SSc pathogenesis, and which cell types drive the disease due to SSc-specific genetic alterations. We therefore characterize the expression, function, and role of A20, and its negative transcriptional regulator DREAM, in patients with SSc and disease models. Levels of A20 are significantly reduced in SSc skin and lungs, while DREAM is elevated. In isolated fibroblasts, A20 mitigates ex vivo profibrotic responses. Mice haploinsufficient for A20, or harboring fibroblasts-specific A20 deletion, recapitulate major pathological features of SSc, whereas DREAM-null mice with elevated A20 expression are protected. In DREAM-null fibroblasts, TGF-ß induces the expression of A20, compared to wild-type fibroblasts. An anti-fibrotic small molecule targeting cellular adiponectin receptors stimulates A20 expression in vitro in wild-type but not A20-deficient fibroblasts and in bleomycin-treated mice. Thus, A20 has a novel cell-intrinsic function in restraining fibroblast activation, and together with DREAM, constitutes a critical regulatory network governing the fibrotic process in SSc. A20 and DREAM represent novel druggable targets for fibrosis therapy.

Microbial interaction: Prevotella spp. reduce P. aeruginosa induced inflammation in cystic fibrosis bronchial epithelial cells.

BACKGROUND: In Cystic Fibrosis (CF) airways, the dehydrated, thick mucus promotes the establishment of persistent polymicrobial infections and drives chronic airways inflammation. This also predisposes the airways to further infections, the vicious, self-perpetuating cycle causing lung damage and progressive lung function decline. The airways are a poly-microbial environment, containing both aerobic and anaerobic bacterial species. Pseudomonas aeruginosa (P. aeruginosa) infections contribute to the excessive inflammatory response in CF, but the role of anaerobic Prevotella spp., frequently found in CF airways, is not known. MATERIALS: We assessed innate immune signalling in CF airway epithelial cells in response to clinical strains of P. histicola, P. nigresens and P. aeruginosa. CFBE41o- cells were infected with P. aeruginosa (MOI 100, 2h) followed by infection with P. histicola or P. nigrescens (MOI 100, 2h). Cells were incubated under anaerobic conditions for the duration of the experiments. RESULTS: Our study shows that P. histicola and P. nigresens can reduce the growth of P. aeruginosa and dampen the inflammatory response in airway epithelial cells. We specifically illustrate that the presence of the investigated Prevotella spp. reduces Toll-like-receptor (TLR)-4, MAPK, NF-κB(p65) signalling and cytokine release (Interleukin (IL)-6, IL-8) in mixed infections. CONCLUSION: Our work, for the first time, strongly indicates a relationship between P. aeruginosa and anaerobic Prevotella spp.. The observed modified NF-κB and MAPK signalling indicates some mechanisms underlying this interaction that could offer a novel therapeutic approach to combat chronic P. aeruginosa infection in people with CF.

Toll like Receptor signalling by Prevotella histicola activates alternative NF-κB signalling in Cystic Fibrosis bronchial epithelial cells compared to P. aeruginosa.

Cystic Fibrosis (CF), caused by mutations affecting the CFTR gene, is characterised by viscid secretions in multiple organ systems. CF airways contain thick mucus, creating a gradient of hypoxia, which promotes the establishment of polymicrobial infection. Such inflammation predisposes to further infection, a self-perpetuating cycle in mediated by NF-κB. Anaerobic Gram-negative Prevotella spp. are found in sputum from healthy volunteers and CF patients and in CF lungs correlate with reduced levels of inflammation. Prevotella histicola (P. histicola) can suppress murine lung inflammation, however, no studies have examined the role of P. histicola in modulating infection and inflammation in the CF airways. We investigated innate immune signalling and NF-kB activation in CF epithelial cells CFBE41o- in response to clinical stains of P. histicola and Pseudomonas aeruginosa (P. aeruginosa). Toll-Like Receptor (TLR) expressing HEK-293 cells and siRNA assays for TLRs and IKKα were used to confirm signalling pathways. We show that P. histicola infection activated the alternative NF-kB signalling pathway in CF bronchial epithelial cells inducing HIF-1α protein. TLR5 signalling was responsible for the induction of the alternative NF-kB pathway through phosphorylation of IKKα. The induction of transcription factor HIF-1α was inversely associated with the induction of the alternative NF-kB pathway and knockdown of IKKα partially restored canonical NF-kB activation in response to P. histicola. This study demonstrates that different bacterial species in the respiratory microbiome can contribute differently to inflammation, either by activating inflammatory cascades (P. aeruginosa) or by muting the inflammatory response by modulating similar or related pathways (P. histicola). Further work is required to assess the complex interactions of the lung microbiome in response to mixed bacterial infections and their effects in people with CF.

Connectivity mapping (ssCMap) to predict A20-inducing drugs and their antiinflammatory action in cystic fibrosis.

Cystic fibrosis (CF) lung disease is characterized by chronic and exaggerated inflammation in the airways. Despite recent developments to therapeutically overcome the underlying functional defect in the cystic fibrosis transmembrane conductance regulator, there is still an unmet need to also normalize the inflammatory response. The prolonged and heightened inflammatory response in CF is, in part, mediated by a lack of intrinsic down-regulation of the proinflammatory NF-κB pathway. We have previously identified reduced expression of the NF-κB down-regulator A20 in CF as a key target to normalize the inflammatory response. Here, we have used publicly available gene array expression data together with a statistically significant connections' map (sscMap) to successfully predict drugs already licensed for the use in humans to induce A20 mRNA and protein expression and thereby reduce inflammation. The effect of the predicted drugs on A20 and NF-κB(p65) expression (mRNA) as well as proinflammatory cytokine release (IL-8) in the presence and absence of bacterial LPS was shown in bronchial epithelial cells lines (16HBE14o-, CFBE41o-) and in primary nasal epithelial cells from patients with CF (Phe508del homozygous) and non-CF controls. Additionally, the specificity of the drug action on A20 was confirmed using cell lines with tnfαip3 (A20) knockdown (siRNA). We also show that the A20-inducing effect of ikarugamycin and quercetin is lower in CF-derived airway epithelial cells than in non-CF cells.

Cystic Fibrosis from Laboratory to Bedside: The Role of A20 in NF-κB-Mediated Inflammation.

Cystic fibrosis (CF) is a lifelong, inflammatory multi-organ disease and the most common lethal, genetic condition in Caucasian populations, with a median survival rate of 41.5 years. Pulmonary disease, characterized by infective exacerbations, bronchiectasis and increasing airway insufficiency is the most serious manifestation of this disease process, currently responsible for over 80% of CF deaths. Chronic dysregulation of the innate immune and host inflammatory response has been proposed as a mechanism central to this genetic condition, primarily driven by the nuclear factor κB (NF-κB) pathway. Chronic activation of this transcription factor complex leads to the production of pro-inflammatory cytokines and mediators such as IL-6, IL-8 and TNF-α. A20 has been described as a central and inducible negative regulator of NF-κB. This intracellular molecule negatively regulates NF-κB-driven pro-inflammatory signalling upon toll-like receptor activation at the level of TRAF6 activation. Silencing of A20 increases cellular levels of p65 and induces a pro-inflammatory state. We have previously shown that A20 expression positively correlates with lung function (FEV1%) in CF. Despite improvement in survival rates in recent years, advancements in available therapies have been incremental. We demonstrate that the experimental use of naturally occurring plant diterpenes such as gibberellin on lipopolysaccharide-stimulated cell lines reduces IL-8 release in an A20-dependent manner. We discuss how the use of a novel bio-informatics gene expression connectivity-mapping technique to identify small molecule compounds that similarly mimic the action of A20 may lead to the development of new therapeutic approaches capable of reducing chronic airway inflammation in CF.

Toll-like receptor 4 is not targeted to the lysosome in cystic fibrosis airway epithelial cells.

The innate immune response to bacterial infection is mediated through Toll-like receptors (TLRs), which trigger tightly regulated signaling cascades through transcription factors including NF-κB. LPS activation of TLR4 triggers internalization of the receptor-ligand complex which is directed toward lysosomal degradation or endocytic recycling. Cystic fibrosis (CF) patients display a robust and uncontrolled inflammatory response to bacterial infection, suggesting a defect in regulation. This study examined the intracellular trafficking of TLR4 in CF and non-CF airway epithelial cells following stimulation with LPS. We employed cells lines [16hBE14o-, CFBE41o- (CF), and CFTR-complemented CFBE41o-] and confirmed selected experiments in primary nasal epithelial cells from non-CF controls and CF patients (F508del homozygous). In control cells, TLR4 expression (surface and cytoplasmic) was reduced after LPS stimulation but remained unchanged in CF cells and was accompanied by a heightened inflammatory response 24 h after stimulation. All cells expressed markers of the early (EEA1) and late (Rab7b) endosomes at basal levels. However, only CF cells displayed persistent expression of Rab7b following LPS stimulation. Rab7 variants may directly internalize bacteria to the Golgi for recycling or to the lysosome for degradation. TLR4 colocalized with the lysosomal marker LAMP1 in 16 hBE14o- cells, suggesting that TLR4 is targeted for lysosomal degradation in these cells. However, this colocalization was not observed in CFBE41o- cells, where persistent expression of Rab7 and release of proinflammatory cytokines was detected. Consistent with the apparent inability of CF cells to target TLR4 toward the lysosome for degradation, we observed persistent surface and cytoplasmic expression of this pathogen recognition receptor. This defect may account for the prolonged cycle of chronic inflammation associated with CF.

Expression of the nuclear factor-κB inhibitor A20 is altered in the cystic fibrosis epithelium.

A20 is a lipopolysaccharide (LPS)-inducible, cytoplasmic zinc finger protein, which inhibits Toll-like receptor-activated nuclear factor (NF)-κB signalling by deubiquitinating tumour necrosis factor receptor-associated factor (TRAF)-6. The action of A20 is facilitated by complex formation with ring finger protein (RNF)-11, Itch and TAX-1 binding protein-1 (TAX1BP1). This study investigated whether the expression of A20 is altered in the chronically inflamed cystic fibrosis (CF) airway epithelium. Nasal epithelial cells from CF patients (F508del homozygous), non-CF controls and immortalised epithelial cells (16HBE14o- and CFBE41o-) were stimulated with LPS. Cytoplasmic expression of A20 and expression of NF-κB subunits were analysed. Formation of the A20 ubiquitin editing complex was also investigated. In CFBE41o-, peak LPS-induced A20 expression was delayed compared with 16HBE14o- and fell significantly below basal levels 12-24 h after LPS stimulation. This was confirmed in primary CF airway cells. Additionally, a significant inverse relationship between A20 and p65 expression was observed. Inhibitor studies showed that A20 does not undergo proteasomal degradation in CFBE41o-. A20 interacted with TAX1BP1, RNF11 and TRAF6 in 16HBE14o- cells, but these interactions were not observed in CFBE41o-. The expression of A20 is significantly altered in CF, and important interactions with complex members and target proteins are lost, which may contribute to the state of chronic NF-κB-driven inflammation.

Expression of the inflammatory regulator A20 correlates with lung function in patients with cystic fibrosis.

BACKGROUND: A20 and TAX1BP1 interact to negatively regulate NF-κB-driven inflammation. A20 expression is altered in F508del/F508del patients. Here we explore the effect of CFTR and CFTR genotype on A20 and TAX1BP1 expression. The relationship with lung function is also assessed. METHODS: Primary nasal epithelial cells (NECs) from CF patients (F508del/F508del, n=7, R117H/F508del, n=6) and controls (age-matched, n=8), and 16HBE14o- cells were investigated. A20 and TAX1BP1 gene expression was determined by qPCR. RESULTS: Silencing of CFTR reduced basal A20 expression. Following LPS stimulation A20 and TAX1BP1 expression was induced in control NECs and reduced in CF NECs, broadly reflecting the CF genotype: F508del/F508del had lower expression than R117H/F508del. A20, but not TAX1BP1 expression, was proportional to FEV(1) in all CF patients (r=0.968, p<0.001). CONCLUSIONS: A20 expression is reduced in CF and is proportional to FEV1. Pending confirmation in a larger study, A20 may prove a novel predictor of CF inflammation/disease severity.

Effect of fruit and vegetable intake on oxidative stress and inflammation in COPD: a randomised controlled trial.

Epidemiological evidence supports a positive relationship between fruit and vegetable (FV) intake, lung function and chronic obstructive pulmonary disease (COPD). Increasing FV intake may attenuate the oxidative stress and inflammation associated with COPD. An exploratory randomised controlled trial to examine the effect of increased consumption of FV on oxidative stress and inflammation in moderate-to-severe COPD was conducted. 81 symptomatically stable patients with a habitually low FV intake (two or fewer portions of FV per day) were randomised to the intervention group (five or more portions of FV per day) or the control group (two or fewer portions of FV per day). Each participant received self-selected weekly home deliveries of FV for 12 weeks. 75 participants completed the intervention. There was a significant between-group change in self-reported FV intake and biomarkers of FV intake (zeaxanthin (p = 0.034) and ß-cryptoxanthin (p = 0.015)), indicating good compliance; post-intervention intakes in intervention and control groups were 6.1 and 1.9 portions of FV per day, respectively. There were no significant changes in biomarkers of airway inflammation (interleukin-8 and myeloperoxidase) and systemic inflammation (C-reactive protein) or airway and systemic oxidative stress (8-isoprostane). This exploratory study demonstrated that patients with moderate-to-severe COPD were able to comply with an intervention to increase FV intake; however, this had no significant effect on airway or systemic oxidative stress and inflammation.

A20 regulation of nuclear factor-κB: perspectives for inflammatory lung disease.

Persistent activation of NF-κB is central to the pathogenesis of many inflammatory lung disorders, including cystic fibrosis, asthma, and chronic obstructive pulmonary disease. A20 is an endogenous negative regulator of NF-κB signaling, which has been widely described in autoimmune and inflammatory disorders, including diabetes and Crohn's disease, but which has received little attention in terms of chronic lung disorders. This review examines the existing body of research on A20 regulation of NF-κB signaling and details the mechanism and regulation of A20 action focusing, where possible, on pulmonary inflammation. A20 and its associated signaling molecules are highlighted as being of potential therapeutic interest for the treatment of inflammatory disorders, and a proposed model of A20 activity in inflammatory lung disease is provided.

Identifying the spatial distribution of vitamin E, pulmonary surfactant and membrane lipids in cells and tissue by confocal Raman microscopy.

Every organ compromises of several different cell types. When studying the effects of a chosen compound within this organ or tissue uptake, localisation, metabolism, and the effect itself can be expected to differ between cells. Using the example of Vitamin E in pulmonary tissue we introduce confocal Raman Microscopy as a superior method to localise lipid-soluble compounds within tissues and cells. We describe the analyses of vitamin E, its oxidation products, and metabolites as well as pulmonary surfactant phospholipids in fixed lung tissue sections. Examples of main structural membrane lipids (PC, cholesterol) and an example of a lipid-signalling molecule (ceramide) are also included. Confocal Raman microscopy is a non-destructive optical method of analysing chemical and physical composition of solids, liquids, gases, gels, and solutions. The method is rich in information allowing discrimination of chemically similar molecules (including geometric isomers) and sensitive monitoring of subtle physical interactions. Additionally, Raman spectroscopy is relatively insensitive to water allowing the analysis of aqueous solutions and suspensions typical in biochemistry. In contrast, Raman spectroscopy is sensitive to non-polar molecules making it ideal for lipidomics research.

Severe vitamin E deficiency modulates airway allergic inflammatory responses in the murine asthma model.

Allergic asthma is a complex immunologically mediated disease associated with increased oxidative stress and altered antioxidant defenses. It was hypothesized that alpha-tocopherol (alpha-T) decreases oxidative stress and therefore its absence may influence allergic inflammatory process, a pathobiology known to be accompanied by oxidative stress. Therefore, selected parameters of allergic asthma sensitization and inflammation were evaluated following ovalbumin sensitization and re-challenge of alpha-T transfer protein (TTP) knock-out mice (TTP(-/-)) that have greatly reduced lung alpha-T levels (e.g.<5%) compared to their litter mate controls (TTP(+/+)). Results showed that severe alpha-T deficiency result in a blunted lung expression of IL-5 mRNA and IL-5 protein and plasma IgE levels compared with TTP(+/+) mice following immune sensitization and rechallenge, although lung lavage eosinophil levels were comparable in both genomic strains. It is concluded that the initial stimulation of immune responses by the TTP(-/-) mice were generally blunted compared to the TTP(+/+) mice, thus diminishing some aspects of subsequent allergic inflammatory processes.

The use of Raman microscopy to determine and localize vitamin E in biological samples.

Alpha-tocopherol (aT), the predominant form of vitamin E in mammals, is thought to prevent oxidation of polyunsaturated fatty acids. In the lung, aT is perceived to be accumulated in alveolar type II cells and secreted together with surfactant into the epithelial lining fluid. Conventionally, determination of aT and related compounds requires extraction with organic solvents. This study describes a new method to determine and image the distribution of aT and related compounds within cells and tissue sections using the light-scattering technique of Raman microscopy to enable high spatial as well as spectral resolution. This study compared the nondestructive analysis by Raman microscopy of vitamin E, in particular aT, in biological samples with data obtained using conventional HPLC analysis. Raman spectra were acquired at spatial resolutions of 2-0.8 microm. Multivariate analysis techniques were used for analyses and construction of corresponding maps showing the distribution of aT, alpha-tocopherol quinone (aTQ), and other constituents (hemes, proteins, DNA, and surfactant lipids). A combination of images enabled identification of colocalized constituents (heme/aTQ and aT/surfactant lipids). Our data demonstrate the ability of Raman microscopy to discriminate between different tocopherols and oxidation products in biological specimens without sample destruction. By enabling the visualization of lipid-protein interactions, Raman microscopy offers a novel method of investigating biological characterization of lipid-soluble compounds, including those that may be embedded in biological membranes such as aT.

Regulation of the alpha-tocopherol transfer protein in mice: lack of response to dietary vitamin E or oxidative stress.

The alpha-tocopherol transfer protein (TTP) plays an important role in the regulation of plasma alpha-tocopherol concentrations. We hypothesized that hepatic TTP levels would be modulated by dietary vitamin E supplementation and/or by oxidative stress. Mice were fed either a High E (1150 mg RRR-alpha-tocopheryl acetate/kg diet) or a Low E (11.5 mg/kg diet) diet for 2 wk. High E increased plasma and liver alpha-tocopherol concentrations approximately 8- and 40-fold, respectively, compared with Low E-fed mice, whereas hepatic TTP increased approximately 20%. Hepatic TTP concentrations were unaffected by fasting (24 h) in mice fed either diet. To induce oxidative stress, chow-fed mice were exposed for 3 d to environmental tobacco smoke (ETS) for 6 h/d (total suspended particulate, 57.4 +/- 1.8 mg/m3). ETS exposure, while resulting in pulmonary and systemic oxidative stress, had no effect on hepatic alpha-tocopherol concentrations or hepatic TTP. Overall, changes in hepatic TTP concentrations were minimal in response to dietary vitamin E levels or ETS-related oxidative stress. Thus, hepatic TTP concentrations may be at sufficient levels such that they are unaffected by either modulations of dietary vitamin E or by the conditions of environmentally related oxidative stress used in the present studies.

Inhibition of NFkappaB activation and IL-8 expression in human bronchial epithelial cells by acrolein.

Lipid oxidation and environmental pollutants are major sources of alpha,beta-unsaturated aldehydes such as acrolein and 4-hydroxynonenal. Acrolein (2-propenal), a major product of organic combustion such as tobacco smoke, represents the most reactive alpha,beta-unsaturated aldehyde, with high reactivity toward nucleophilic targets such as sulfhydryl groups. To investigate how acrolein affects respiratory tract cell activation, we exposed either primary (NHBE) or immortalized human bronchial epithelial cells (HBE1) to 0-25 microM acrolein, and determined effects on basal and tumor necrosis factor-alpha (TNFalpha)-induced production of the chemokine interleukin (IL)-8. Cell exposure to acrolein dose-dependently suppressed IL-8 mRNA levels in HBE1 cells (26, 40, and 79% at 5, 10, and 25 microM acrolein concentrations, respectively) and resulted in corresponding decreases in IL-8 production. Studies of nuclear factor-kappaB (NFkappaB) activation, an essential event in IL-8 production, showed decreased TNFalpha-induced NFkappaB activation by acrolein, illustrated by inhibition of nuclear translocation of NFkappaB and reduced IkappaBalpha degradation. Immunochemical analysis of IkappaB kinase (IKK), a redox-sensitive regulator of NFkappaB activation, indicated direct modification of the IKK beta-subunit by acrolein, suggesting that acrolein may act directly on IKK. In summary, our results demonstrate that acrolein can suppress inflammatory processes in the airways by inhibiting epithelial IL-8 production through direct or indirect inhibitory effects on NFkappaB activation.