Plasma Proinflammatory Cytokines Research Articles

Pregnancy-induced oxidative stress and inflammation are not associated with impaired maternal neuronal activity or memory function.

Pregnancy is associated with neural and behavioral plasticity, systemic inflammation, and oxidative stress. Yet, the impact of inflammation and oxidative stress on maternal neural and behavioral plasticity during pregnancy are unclear. We hypothesized that healthy pregnancy transiently reduces learning and memory, and these deficits are associated with pregnancy-induced elevations in inflammation and oxidative stress. Cognitive performance was tested using novel object recognition (recollective memory), Morris water maze (spatial memory), and open field (anxiety-like) behavior tasks in female Sprague-Dawley rats of varying reproductive states [non-pregnant (nulliparous), pregnant (near term), and 1-2 months post-pregnancy (primiparous); n = 7-8/group]. Plasma and CA1 proinflammatory cytokines were measured using a MILLIPLEX® magnetic bead assay. Plasma oxidative stress was measured via advanced oxidation protein products (AOPP) assay. CA1 markers of oxidative stress, neuronal activity, and apoptosis were quantified via western blotting. Our results demonstrate CA1 oxidative stress-associated markers were elevated in pregnant compared to nulliparous rats (p ≤ 0.017) but were equivalent levels in pregnant and primiparous rats. In contrast, reproductive state did not impact CA1 inflammatory cytokines, neuronal activity, or apoptosis. Likewise, there was no effect of reproductive state on recollective or spatial memory. Even so, spatial learning was impaired (p ≤ 0.007) while anxiety-like behavior (p ≤ 0.034) was reduced in primiparous rats. Overall, our data suggest maternal hippocampal CA1 is protected from systemic inflammation but vulnerable to peripartum oxidative stress. Peripartum oxidative stress elevations, such as in pregnancy complications, may contribute to peripartum neural and behavioral plasticity.

Elevated Circulating Cell-Free Hemoglobin Drives Endothelial Glycocalyx Destruction and Inflammation in Sepsis

Rationale: Endothelial injury is a hallmark of sepsis-induced acute respiratory distress syndrome (ARDS). Endothelial barrier integrity is regulated by the endothelial glycocalyx, a matrix of proteoglycans and glycosaminoglycans (GAGs) that lines the vascular lumen. In sepsis, this glycocalyx is degraded by heparanase, which cleaves heparan sulfate, the primary glycocalyx GAG. Glycocalyx breakdown is linked to worse sepsis outcomes, but the mechanisms inducing heparanase activity in sepsis are unknown. We previously found that plasma levels of hemoglobin released from red blood cells (cell-free hemoglobin, CFH) are elevated in clinical sepsis and correlate with higher rates of ARDS and death. CFH also worsens endothelial injury and microvascular permeability in murine sepsis. We therefore hypothesized that CFH upregulates heparanase production to drive glycocalyx shedding and inflammation in sepsis. Methods: To test the association between CFH levels, heparanase expression, and glycocalyx degradation, circulating levels of heparanase, heparan sulfate, and total GAGs were measured in 77 sepsis patients and compared across CFH quartiles. In mice with intraabdominal polymicrobial sepsis with elevated CFH (CS [cecal slurry] + intravenous [IV] CFH), plasma heparanase, total GAGs, and syndecan-1 were quantified. Whole lung and plasma pro-inflammatory cytokines were also determined to investigate the impact of CFH on inflammation in sepsis. Results: In sepsis patients, higher circulating CFH levels were associated with increased plasma heparanase (p=0.0304), total GAGs (p=0.0178), and heparan sulfate (p=0.0112). CFH also exacerbated glycocalyx degradation in murine sepsis; circulating syndecan-1 (p=0.0152) and total GAGs (p=0.0094) were elevated in animals that received CS+IV CFH compared to CS alone. This effect may be mediated by upregulation of active heparanase, which was higher in the plasma of CS+IV CFH treated animals (p=0.1333). CFH also worsened illness severity, as CS+IV CFH animals had worse sepsis severity scores (p=0.0074). Whole lung tumor necrosis factor-alpha (TNF-a; p=0.0129) and interferon-gamma (IFN-g; p=0.0264) transcript levels were increased in the CS+IV CFH group, indicating greater lung inflammation. Systemic inflammation was also enhanced in the CS+IV CFH animals, demonstrated by higher plasma TNF-a (p<0.001) and IFN-g (p=0.0221) concentrations. Conclusions: In sepsis, CFH is associated with upregulation of heparanase and endothelial glycocalyx degradation, which may disrupt the endothelial barrier, causing downstream organ injury. Future studies to define the molecular pathways that underlie these findings and their effect on endothelial permeability will be an important next step towards identifying novel treatment targets to improve outcomes for patients with sepsis-associated ARDS. Funding F30HL170483, R01HL164937, R01HL158906, R01HL150783, R01HL125371. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Basophil-Derived IL-4 and IL-13 Protect Intestinal Barrier Integrity and Control Bacterial Translocation during Malaria.

Our previous work demonstrated that basophils regulate a suite of malaria phenotypes, including intestinal mastocytosis and permeability, the immune response to infection, gametocytemia, and parasite transmission to the malaria mosquito Anopheles stephensi. Given that activated basophils are primary sources of the regulatory cytokines IL-4 and IL-13, we sought to examine the contributions of these mediators to basophil-dependent phenotypes in malaria. We generated mice with basophils depleted for IL-4 and IL-13 (baso IL-4/IL-13 (-)) and genotype controls (baso IL-4/IL-13 (+)) by crossing mcpt8-Cre and Il4/Il13fl/fl mice and infected them with Plasmodium yoelii yoelii 17XNL. Conditional deletion was associated with ileal mastocytosis and mast cell (MC) activation, increased intestinal permeability, and increased bacterial 16S levels in blood, but it had no effect on neutrophil activation, parasitemia, or transmission to A. stephensi. Increased intestinal permeability in baso IL-4/IL-13 (-) mice was correlated with elevated plasma eotaxin (CCL11), a potent eosinophil chemoattractant, and increased ileal MCs, proinflammatory IL-17A, and the chemokines MIP-1α (CCL3) and MIP-1β (CCL4). Blood bacterial 16S copies were positively but weakly correlated with plasma proinflammatory cytokines IFN-γ and IL-12p40, suggesting that baso IL-4/IL-13 (-) mice failed to control bacterial translocation into the blood during malaria infection. These observations suggest that basophil-derived IL-4 and IL-13 do not contribute to basophil-dependent regulation of parasite transmission, but these cytokines do orchestrate protection of intestinal barrier integrity after P. yoelii infection. Specifically, basophil-dependent IL-4/IL-13 control MC activation and prevent infection-induced intestinal barrier damage and bacteremia, perhaps via regulation of eosinophils, macrophages, and Th17-mediated inflammation.

EARLY FLUID PLUS NOREPINEPHRINE RESUSCITATION DIMINISHES KIDNEY HYPOPERFUSION AND INFLAMMATION IN SEPTIC NEWBORN PIGS.

Sepsis is the most frequent risk factor for acute kidney injury (AKI) in critically ill infants. Sepsis-induced dysregulation of kidney microcirculation in newborns is unresolved. The objective of this study was to use the translational swine model to evaluate changes in kidney function during the early phase of sepsis in newborns and the impact of fluid plus norepinephrine resuscitation. Newborn pigs (3-7-day-old) were allocated randomly to three groups: 1) sham, 2) sepsis (cecal ligation and puncture) without subsequent resuscitation, and 3) sepsis with lactated Ringer plus norepinephrine resuscitation. All animals underwent standard anesthesia and mechanical ventilation. Cardiac output and glomerular filtration rate were measured noninvasively. Mean arterial pressure, total renal blood flow, cortical perfusion, medullary perfusion, and medullary tissue oxygen tension (mtPO 2 ) were determined for 12 h. Cecal ligation and puncture decreased mean arterial pressure and cardiac output by more than 50%, with a proportional increase in renal vascular resistance and a 60-80% reduction in renal blood flow, cortical perfusion, medullary perfusion, and mtPO 2 compared to sham. Cecal ligation and puncture also decreased glomerular filtration rate by ~79% and increased AKI biomarkers. Isolated foci of tubular necrosis were observed in the septic piglets. Except for mtPO 2 , changes in all these parameters were ameliorated in resuscitated piglets. Resuscitation also attenuated sepsis-induced increases in the levels of plasma C-reactive protein, proinflammatory cytokines, lactate dehydrogenase, alanine transaminase, aspartate aminotransferase, and renal NLRP3 inflammasome. These data suggest that newborn pigs subjected to cecal ligation and puncture develop hypodynamic septic AKI. Early implementation of resuscitation lessens the degree of inflammation, AKI, and liver injury.

Improved levels of checkpoint molecule PD-L1 on peripheral blood monocyte subsets in obstructive sleep apnea syndrome patients upon hypoglossal nerve stimulation.

Oxidative stress in patients suffering from obstructive sleep apnea syndrome (OSAS) is associated with a low-grade systemic inflammation, immune disturbance, and increased invasion of monocytes into the endothelium. Besides continuous positive airway pressure (PAP), hypoglossal nerve stimulation (HNS) has become a promising treatment option for patients with OSAS. We aimed to analyse the influence of HNS therapy on the cellular characteristics relevant for adhesion and immune regulation of circulating CD14/CD16 monocyte subsets. Whole blood flow cytometric measurements were performed to analyse the expression levels of different adhesion molecules and checkpoint molecule PD-L1 (programmed death-ligand 1) in connection with pro-inflammatory plasma cytokine IL-8 and the clinical values of BMI (body mass index), AHI (apnea-hypopnea index), ODI (oxygen desaturation index), and ESS (Epworth sleepiness scale) upon HNS treatment. Hypoglossal nerve stimulation treatment significantly improved the expression of adhesion molecule CD162 (P-selectin receptor) on non-classical monocytes and significantly downregulated the expression of PD-L1 on all three monocyte subsets. We conclude that the holistic improvement of different parameters such as the oxygenation of the peripheral blood, a reduced systemic inflammation, and the individual sleeping situation upon HNS respiratory support, leads to an improved immunologic situation.

Oral post-treatment supplementation with a combination of glutamine, citrulline, and antioxidant vitamins additively mitigates jejunal damage, oxidative stress, and inflammation in rats with intestinal ischemia and reperfusion.

Intestinal ischemia and reperfusion (IIR) injury is closely associated with oxidative stress. Evidence shows that oral supplementation with glutamine and citrulline alleviates IIR-induced jejunal damage. We investigated the effects of a combination of glutamine, citrulline, and antioxidant vitamins on IIR-induced jejunal damage, oxidative stress, and inflammation. Male Wistar rats that underwent 60 min of superior mesenteric artery occlusion were orally administered glutamine plus citrulline (GC), vitamin C plus E (CE), or a combination of GC and CE 15 min before and 3, 9, and 21 h after reperfusion. Healthy rats without IIR were used as controls. After reperfusion for 24 h, rats with IIR showed lower levels of red blood cells, hemoglobin, serum glucose, and jejunal DNA and increased white blood cell counts compared to controls (1-way ANOVA with the least significant difference, P < 0.05). The IIR-induced decrease in serum albumin and increase in plasma interleukin-6 and jejunal thiobarbituric acid-reactive substances (TBARS) were significantly reversed by GC and/or CE. The results of the 2-way ANOVA indicated that GC was the main factor that increased jejunal villus height and muscularis DNA, and CE was the main factor that increased jejunal muscularis protein and decreased jejunal proinflammatory cytokine levels and myeloperoxidase activity. In addition, GC and CE are the main factors that decrease plasma proinflammatory cytokine levels and the jejunal apoptotic index. Oral post-treatment supplementation with glutamine and citrulline, combined with vitamins C and E, may alleviate IIR-induced oxidative stress, inflammation, and jejunal damage.

The use of a systems approach to increase NAD+ in human participants

Reversal or mitigation against an age-related decline in NAD+ has likely benefits, and this premise has driven academic and commercial endeavour to develop dietary supplements that achieve this outcome. We used a systems-based approach to improve on current supplements by targeting multiple points in the NAD+ salvage pathway. In a double-blind, randomised, crossover trial, the supplement – Nuchido TIME+® (NT) - increased NAD+ concentration in whole blood. This was associated with an increase in SIRT1 and an increase in nicotinamide phosphoribosyltransferase (NAMPT) in peripheral blood mononucleocytes, lower concentrations of pro-inflammatory cytokines in plasma, including a reduction in interleukin 2 (IL2), a reduction in glycated serum protein and a shift in the glycosylation profile of immunoglobulin G (IgG) toward a younger biological age, all of which are likely to promote a healthier ageing trajectory.

Epithelium-derived kallistatin promotes CD4+ T-cell chemotaxis to TH2-type inflammation in chronic rhinosinusitis

The function of kallistatin in airway inflammation, particularly chronic rhinosinusitis with nasal polyps (CRSwNP), has not been elucidated. We sought to investigate the role of kallistatin in airway inflammation. Kallistatin and proinflammatory cytokine expression levels were detected in nasal polyps. For the invivo studies, we constructed the kallistatin-overexpressing transgenic mice to elucidate the role of kallistatin in airway inflammation. Furthermore, the levels of plasma IgE and proinflammatory cytokines in the airways were evaluated in the kallistatin-/- rat invivo model under a type 2 inflammatory background. Finally, the Notch signaling pathway was explored to understand the role of kallistatin in CRSwNP. We showed that the expression of kallistatin was significantly higher in nasal polyps than in the normal nasal mucosa and correlated with IL-4 expression. We also discovered that the nasal mucosa of kallistatin-overexpressing transgenic mice expressed higher levels of IL-4 expression, associating to TH2-type inflammation. Interestingly, we observed lower IL-4 levels in the nasal mucosa and lower total plasma IgE of the kallistatin-/- group treated with house dust mite allergen compared with the wild-type house dust mite group. Finally, we observed a significant increase in the expression of Jagged2 in the nasal epithelium cells transduced with adenovirus-kallistatin. This heightened expression correlated with increased secretion of IL-4, attributed to the augmented population of CD4+CD45+Notch1+ T cells. These findings collectively may contribute to the induction of TH2-type inflammation. Kallistatin was demonstrated to be involved in the CRSwNP pathogenesis by enhancing the TH2 inflammation, which was found to be associated with more expression of IL-4, potentially facilitated through Jagged2-Notch1 signaling in CD4+ T cells.

Lack of Annexin A1 Exacerbates Inflammatory Response in Acute Endometritis Model.

Annexin A1 (AnxA1) is a glucocorticoid-inducible protein and an important endogenous modulator of inflammation. However, its effect in the endometrial microenvironment is poorly explained. This study aimed to evaluate the role of endogenous AnxA1 in an endometritis mouse model induced by lipopolysaccharide (LPS). Female C57BL/6 wild-type (WT) and AnxA1-/- mice were divided into two groups: SHAM and LPS. To induce endometritis, mice received a vaginal infusion of 50 μL of LPS (1 mg/mL) dissolved in phosphate-buffered saline. After 24 h, the mice were euthanized, and blood and uteri samples were collected. The endometrium inflammatory scores were significantly increased in the LPS-treated group. AnxA1-/- mice from the LPS group demonstrated a significant increase in the number of degranulated mast cell levels compared to AnxA1-/- SHAM mice. The Western blotting analysis revealed that a lack of AnxA1 promoted the upregulation of NLRP3 and pro-IL-1β in the acute endometritis animal model compared to WT LPS animals. LPS-induced endometritis increased the number of blood peripheral leukocytes in both WT and AnxA1-/- mice compared with SHAM group mice (p < 0.001). AnxA1-/- mice also showed increased plasma levels of IL-1β (p < 0.01), IL-6, IL-10, IL-17, and TNF-α (p < 0.05) following LPS-induced endometritis. In conclusion, a lack of endogenous AnxA1 exacerbated the inflammatory response in an endometritis model via NLRP3 dysregulation, increased uterine mast cell activation, and plasma pro-inflammatory cytokine release.

Effect of the ketone beta-hydroxybutyrate on markers of inflammation and immune function in adults with type 2 diabetes.

Pre-clinical and cell culture evidence supports the role of the ketone beta-hydroxybutyrate (BHB) as an immunomodulatory molecule that may inhibit inflammatory signalling involved in several chronic diseases such as type 2 diabetes (T2D), but studies in humans are lacking. Therefore, we investigated the anti-inflammatory effect of BHB in humans across three clinical trials. To investigate if BHB suppressed pro-inflammatory cytokine secretion, we treated LPS-stimulated leukocytes from overnight-fasted adults at risk for T2D with BHB (Study 1). Next (Study 2), we investigated if exogenously raising BHB acutely in vivo by ketone monoester supplementation (KME) in adults with T2D would suppress pro-inflammatory plasma cytokines. In Study 3, we investigated the effect of BHB on inflammation via ex vivo treatment of LPS-stimulated leukocytes with BHB and in vivo thrice-daily pre-meal KME for 14 days in adults with T2D. Ex vivo treatment with BHB suppressed LPS-stimulated IL-1β, TNF-α, and IL-6 secretion and increased IL-1RA and IL-10 (Study 1). Plasma IL-10 increased by 90 min following ingestion of a single dose of KME in T2D, which corresponded to peak blood BHB (Study 2). Finally, 14 days of thrice-daily KME ingestion did not significantly alter plasma cytokines or leukocyte subsets including monocyte and T-cell polarization (Study 3). However, direct treatment of leukocytes with BHB modulated TNF-α, IL-1β, IFN-γ, and MCP-1 secretion in a time- and glucose-dependent manner (Study 3). Therefore, BHB appears to be anti-inflammatory in T2D, but this effect is transient and is modulated by the presence of disease, glycaemia, and exposure time.

Cysteine-modified PEGylated nanoparticles for targeted delivery of methylprednisolone to pancreatitis

The timely suppression of inflammatory mediator production and mitigation of their effects on pancreatic acinar cells are crucial for the successful management of acute pancreatitis. To achieve effective treatment, we present a novel approach utilizing cysteine modified PEG nanoparticles for both precise accumulation at the site of pancreatitis and specific targeting of acinar cells. Methylprednisolone, a nonsteroidal anti-inflammatory drug, was tailored to enhance its circulation time in the bloodstream, preferentially accumulate in the pancreas and enhance cell uptake efficiency by acinar cells through specifically targeting L-Type amino acid transporter 1. The nanosystem significantly downregulated pro-inflammatory cytokines in plasma, resulting in the effective suppression of inflammation in acinar cells within an acute pancreatitis rat model. The utilization of the dual targeted therapy strategy holds considerable potential for the clinical management of pancreatitis.

Protocatechuic acid alleviates TMAO-aggravated atherosclerosis via mitigating inflammation, regulating lipid metabolism, and reshaping gut microbiota.

Trimethylamine-N-oxide (TMAO) is a risk factor for atherosclerosis. As a natural phenolic acid, protocatechuic acid (PCA) is abundant in various plant foods. The present study investigated the effect of PCA on TMAO-aggravated atherosclerosis in ApoE-/- mice. The mice were randomly divided into five groups and fed one of the following five diets for 12 weeks: namely a low-fat diet (LFD), a western diet (WD), a WD + 0.2% TMAO diet (WDT), a WDT + 0.5% PCA diet (WDT + LPCA), and a WDT + 1.0% PCA diet (WDT + HPCA). Results demonstrated that dietary TMAO exacerbated the development of atherosclerosis by eliciting inflammation and disturbing lipid metabolism. The diet with PCA at 1% reduced TMAO-induced aortic plaque by 30% and decreased the levels of plasma pro-inflammatory cytokines. PCA also improved lipid metabolism by up-regulating the hepatic gene expression of peroxisome proliferator-activated receptor alpha (PPARα). In addition, PCA supplementation enhanced fecal excretion of fatty acids and decreased hepatic fat accumulation. PCA supplementation favorably modulated gut microbiota by increasing the α-diversity with an increase in the abundance of beneficial genera (Rikenella, Turicibacter, Clostridium_sensu_stricto and Bifidobacterium) and a decrease in the abundance of the harmful Helicobacter genus. In summary, PCA could alleviate the TMAO-exacerbated atherosclerosis and inflammation, improve the lipid metabolism, and modulate gut microbiota.

INCREASED LEVELS OF PROINFLAMMATORY CYTOKINES IN BLOOD PLASMA IN PATIENTS WITH CHRONIC THROMBOEMBOLIC PULMONARY HYPERTENSION

HighlightsIL-8 and MCP-1 have a significant role in the CTEPH pathogenesis, which indicates the importance of nonspecific immunity in the formation and progression of CTEPH. The coupling between cytokines and hemodynamic parameters, cardiac structural changes and plasma biochemical parameters were determined. AbstractBackground. Chronic thromboembolic pulmonary hypertension (CTEPH) pathogenesis is complex and not fully understood. Particular attention to the microvascular damage genesis in CTEPH is given to aseptic inflammation, which in turn could be mediated through various molecular mechanisms. According to the conflicting and incomplete data on changes in the profile of factors controlling inflammation in CTEPH, research in this field would identify new therapeutic targets for the prevention and treatment of CTEPH.Aim. To study the profile of plasma proinflammatory cytokines in patients with chronic thromboembolic pulmonary hypertension (CTEPH) and evaluate the coupling of these cytokines with the main morphofunctional and laboratory values of the disease severity.Methods. 34 patients with CTEPH were included in this study. To characterize the group, the following methods were used: echocardiographic examination, catheterization of the right cardiac chambers. Biomarkers of heart failure, systemic inflammation, as well as erythropoiesis and iron metabolism were assessed in all patients. The control group included 10 donors. To study the proinflammatory cytokine profile in plasma, interleukins (IL) 6, 8, 18, monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase 9 were determined using standard enzyme-linked immunosorbent assay (ELISA) kits.Results. Hemodynamic and morphofunctional changes in the pulmonary circulation specific to pulmonary hypertension were determined with catheterization of the right cardiac chambers and echocardiography. During plasma proinflammatory cytokines analysis, a significant increase in the level of IL-8 (p = 0.030) and MCP-1 (p = 0.031) in CTEPH group compared to the control group was observed. No significant differences for other analyzed markers were found. In the elaboration of the correlation analysis, moderate inverse coupling between proinflammatory markers and hemodynamic parameters characterizing the CTEPH severity were revealed, as well as positive correlations with parameters of remodeling of the right cardiac chambers and iron metabolism.Conclusion. The increased levels of IL-8 and MCP-1 in patients with CTEPH identified in the present study indicate a significant role of nonspecific immunity in the formation and progression of CTEPH. The coupling between cytokines and hemodynamic parameters, structural cardiac changes and plasma biochemical parameters were determined. Based on the obtained data, it is possible to develop new medicinal substances, targeting towards proinflammatory cytokines, their receptors and signaling pathways.

Microglial-specific knockdown of iron import gene, Slc11a2, blunts LPS-induced neuroinflammatory responses in a sex-specific manner

Neuroinflammation and microglial iron load are significant hallmarks found in several neurodegenerative diseases. In in vitro systems, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and it has been shown that iron can augment cellular inflammation, suggesting a feed-forward loop between mechanisms involved in iron import and inflammatory signaling. However, it is not understood how microglial iron import mechanisms contribute to inflammation in vivo, or whether altering a microglial iron-related gene affects the inflammatory response. These studies aimed to determine the effect of knocking down microglial iron import gene Slc11a2 on the inflammatory response in vivo. We generated a novel model of tamoxifen-inducible, microglial-specific Slc11a2 knockdown using Cx3cr1Cre-ERT2 mice. Transgenic male and female mice were administered intraperitoneal saline or lipopolysaccharide (LPS) and assessed for sickness behavior post-injection. Plasma cytokines and microglial bulk RNA sequencing (RNASeq) analyses were performed at 4 h post-LPS, and microglia were collected for gene expression analysis after 24 h. A subset of mice was assessed in a behavioral test battery following LPS-induced sickness recovery. Control male, but not female, mice significantly upregulated microglial Slc11a2 at 4 and 24 h following LPS. In Slc11a2 knockdown mice, we observed an improvement in the acute behavioral sickness response post-LPS in male, but not female, animals. Microglia from male, but not female, knockdown animals exhibited a significant decrease in LPS-provoked pro-inflammatory cytokine expression after 24 h. RNASeq data from male knockdown microglia 4 h post-LPS revealed a robust downregulation in inflammatory genes including Il6, Tnfα, and Il1β, and an increase in anti-inflammatory and homeostatic markers (e.g., Tgfbr1, Cx3cr1, and Trem2). This corresponded with a profound decrease in plasma pro-inflammatory cytokines 4 h post-LPS. At 4 h, male knockdown microglia also upregulated expression of markers of iron export, iron recycling, and iron homeostasis and decreased iron storage and import genes, along with pro-oxidant markers such as Cybb, Nos2, and Hif1α. Overall, this work elucidates how manipulating a specific gene involved in iron import in microglia alters acute inflammatory signaling and overall cell activation state in male mice. These data highlight a sex-specific link between a microglial iron import gene and the pro-inflammatory response to LPS in vivo, providing further insight into the mechanisms driving neuroinflammatory disease.

Childhood Treatment with Adeno-Associated Viral Gene Therapy Results in Stable FVIII Expression and Improved Bleeding Phenotype in Adult Severe Hemophilia A Dogs

Introduction: In patients with severe hemophilia A, adeno-associated virus (AAV)-mediated factor VIII (FVIII) gene therapy is associated with reduced bleeding compared with FVIII prophylaxis. AAV5-FVIII gene therapy is approved in the U.S. and conditionally approved in Europe for the treatment of adults with severe hemophilia A. Gene therapy during childhood could potentially prevent the development of arthropathy and improve quality of life. However, there are unknowns regarding long-term safety and whether the predominant episomal nature of AAV persistence would lead to loss of transgene expression with hepatocyte divisions and liver expansion during childhood. The inflammatory response to AAV gene therapy in children is also largely uncharacterized. We have previously reported the treatment of five neonatal or infant dogs with a single vector infusion of a codon-optimized AAV5-B-domain deleted canine FVIII (cFVIII) construct with a hybrid liver promoter (AAV5-cFVIII). Dogs treated at 2 weeks of age demonstrated improved whole blood clot time (WBCT) over a 6-month period despite minimal cFVIII expression (&amp;lt;3%). Dogs treated at 2 months of age demonstrated stable cFVIII expression measured over a 6-month period and decreased WBCT. Aims: To describe the early inflammatory response to AAV5-cFVIII in infant and neonatal dogs, and to provide an update on the safety and efficacy of AAV5-cFVIII in these animals 12-16 months post-treatment. Methods: Hemophilia A dogs were treated with a single vector infusion (dose=2.0e14 vg/kg) of AAV5-cFVIII at 2 weeks (n=2) or 2 months (n=3) of age. Epigenomic data suggests that 2 months of age in dogs is equivalent to 9 months in humans. Liver volume was measured by magnetic resonance imaging at baseline, 3, 6, and 12 months post-AAV5-cFVIII treatment. Plasma cytokine levels were measured using a cytokine array pre-treatment (day 0) and post-treatment (days 2-28). Percutaneous liver biopsy samples were obtained at 2 weeks, 6 months, and 12 months post-treatment. Results: Cytokine array analyses of samples demonstrated that one dog who received AAV5-cFVIII at 2 weeks experienced transient post-treatment plasma elevation of IL-6, KC-like, MCP-1, TGFβ-1, and TGFβ-2 that returned to baseline levels by day 18. Two dogs treated at 2 months experienced a transient increase in GMC-SF, IL-2, IL-6, IL-7, IL-15, and IL-18 that returned to baseline levels by day 21. No changes in levels of IFNγ, IL-8, IL-10, IP-10, TNFα, or TGFβ-3 were observed. Changes in plasma cytokine levels were not associated with an elevation in plasma alanine transaminase levels. Hemophilia A dogs treated with AAV5-cFVIII at 2 weeks of age maintained an improved WBCT 12 to 16 months post-treatment, although cFVIII expression as measured by one-stage (OSA) and chromogenic substrate assays (CSA) remained minimal (&amp;lt;3%). Body weight and liver volume increased by 10.6-fold (measured at 16 months) and 12-fold (measured at 12 months) respectively. Dogs treated with AAV5-cFVIII at 2 months of age demonstrated sustained cFVIII expression by OSA and CSA after 12 to 16 months (8.9-13.39% measured at 16 months by OSA), despite a 3.2-fold increase in body weight (measured at 16 months), and a 3.9-fold increase in liver volume (measured at 12 months). This was associated with improved WBCT observed over the 12 to 16-month period for all three dogs. Overall, a reduction in bleeding events (BEs) was observed in all dogs. No BEs were recorded prior to AAV5-cFVIII treatment for dogs treated at 2 weeks of age. During the 16 months post-treatment, one dog experienced two spontaneous BEs, while no BEs were observed for the second dog. The dogs treated at 2 months experienced 4 (3 spontaneous and 1 traumatic) BEs prior to AAV5-cFVIII treatment, and 1 traumatic BE 48 hours post-treatment prior to the detection of FVIII expression. For the remaining 16 months post-treatment, no BEs were observed in this group. Conclusions: Treatment using AAV5-cFVIII at 2 months of age in a hemophilia A dog model resulted in stable FVIII expression into adult life despite significant liver expansion. For some dogs, AAV-cFVIII treatment resulted in an early transient increase in plasma proinflammatory cytokine levels with no evidence of transaminitis that resolved within three weeks.Further studies on liver biopsy samples are ongoing to evaluate cellular implications, vector genome distribution, and mechanisms of AAV persistence.

Esketamine mitigates cognitive impairment following exposure to LPS by modulating the intestinal flora/subdiaphragmatic vagus nerve/spleen axis

IntroductionSusceptibility to secondary infection often increases after primary infection. Secondary infections can lead to more severe inflammatory injuries; however, the underlying mechanisms are not yet fully elucidated. ObjectiveTo investigate whether esketamine treatment immediately after primary lipopolysaccharide (LPS) exposure could alleviate cognitive impairment caused by secondary infection. MethodsMice were injected intraperitoneally (IP) with LPS (5 mg/kg) 10 days apart. Esketamine (10, 15, or 30 mg/kg) was administered IP immediately after the primary LPS injection. Splenectomy or subdiaphragmatic vagotomy (SDV) was performed 7 days before secondary LPS exposure or broad-spectrum antibiotic administration. ResultsSplenomegaly was observed after the primary LPS injection on Days 3 and 10. Splenomegaly was attenuated by treatment with 30 mg/kg esketamine. Esketamine treatment prevented increased plasma proinflammatory cytokines levels and cognitive dysfunction induced by secondary LPS exposure. Mice that underwent splenectomy or SDV had lower proinflammatory cytokines levels, higher hippocampal brain-derived neurotrophic factor (BDNF) levels, and improved cognitive function 1 day after secondary infection, which was not further improved by esketamine. Fecal microbiota transplantation (FMT) from endotoxic mice treated with esketamine attenuated hippocampal BDNF downregulation and cognitive dysfunction only in pseudo germ-free (PGF) mice without splenectomy. FMT with fecal suspensions from esketamine-treated endotoxic mice abrogated splenomegaly only in PGF mice without SDV. Blocking BDNF signaling blocked esketamine’s ameliorating effects on secondary LPS exposure-induced cognitive dysfunction. ConclusionThe intestinal flora/subdiaphragmatic vagus nerve/spleen axis-mediated hippocampal BDNF downregulation significantly affected secondary LPS-induced systemic inflammation and cognitive dysfunction. Esketamine preserves cognitive function via this mechanism.

Does liraglutide alleviate inflammation in brain-dead donors? A randomized clinical trial.

Brain death triggers an inflammatory cascade that damages organs before procurement, adversely affecting the quality of grafts. This randomized clinical trial aimed to compare the efficacy of liraglutide compared to placebo in attenuating brain death-induced inflammation, endoplasmic reticulum stress, and oxidative stress. We conducted a double-blinded, placebo-controlled, randomized clinical trial with brain-dead donors. Fifty brain-dead donors were randomized to receive subcutaneous liraglutide or placebo. The primary outcome was the reduction in IL-6 plasma levels. Secondary outcomes were changes in other plasma pro-inflammatory (IL-1β, interferon-γ, TNF) and anti-inflammatory cytokines (IL-10), expression of antiapoptotic ( BCL2 ), endoplasmic reticulum stress markers ( DDIT3/CHOP , HSPA5/BIP ), and antioxidant ( superoxide dismutase 2 , uncoupling protein 2 ) genes, and expression TNF, DDIT3, and superoxide dismutase 2 proteins in liver biopsies. The liraglutide group showed lower cytokine levels compared to the placebo group during follow-up: Δ IL-6 (-28 [-182, 135] vs. 32 [-10.6, 70.7] pg/mL; p = 0.041) and Δ IL-10 (-0.01 [-2.2, 1.5] vs. 1.9 [-0.2, 6.1] pg/mL; p = 0.042), respectively. The administration of liraglutide did not significantly alter the expression of inflammatory, antiapoptotic, endoplasmic reticulum stress, or antioxidant genes in the liver tissue. Similar to gene expression, expressions of proteins in the liver were not affected by the administration of liraglutide. Treatment with liraglutide did not increase the organ recovery rate [OR = 1.2 (95% CI: 0.2-8.6), p = 0.82]. Liraglutide administration reduced IL-6 and prevented the increase of IL-10 plasma levels in brain-dead donors without affecting the expression of genes and proteins related to inflammation, apoptosis, endoplasmic reticulum stress, or oxidative stress.

Humanin ameliorates TBI-related cognitive impairment by attenuating mitochondrial dysfunction and inflammation

Traumatic brain injury (TBI) often results in a reduction of the capacity of cells to sustain energy demands, thus, compromising neuronal function and plasticity. Here we show that the mitochondrial activator humanin (HN) counteracts a TBI-related reduction in mitochondrial bioenergetics, including oxygen consumption rate. HN normalized the disruptive action of TBI on memory function, and restored levels of synaptic proteins (synapsin 1 and p-CREB). HN also counteracted TBI-related elevations of pro-inflammatory cytokines in plasma (TNF-α, INF-y, IL 17, IL 5, MCP 5, GCSF, RANNETS, sTNFRI) as well as in the hippocampus (gp-130 and p-STAT3). Gp-130 is an integral part of cytokine receptor impinging on STAT3 (Tyr-705) signaling. Furthermore, HN reduced astrocyte proliferation in TBI. The overall evidence suggests that HN plays an integral role in normalizing fundamental aspects of TBI pathology which are central to energy balance, brain function, and plasticity.

Multimodal nanoparticle-containing modified suberoylanilide hydroxamic acid polymer conjugates to mitigate immune dysfunction in severe inflammation.

Excessive immune activation and immunosuppression are opposing factors that contribute to the dysregulated innate and adaptive immune responses seen in severe inflammation and sepsis. Here, a novel analog of the histone deacetylase inhibitor (HDACi), suberoylanilide hydroxamic acid (SAHA-OH), was incorporated into immunomodulatory poly(lactic acid)-based nanoparticles (iNP-SAHA) by employing a prodrug approach through the covalent modification of poly(lactic-co-glycolic acid) (PLGA) with SAHA-OH. iNP-SAHA formulation allowed for controlled incorporation and delivery of SAHA-OH from iNP-SAHA and treatment led to multimodal biological responses including significant reductions in proinflammatory cytokine secretions and gene expression, while increasing the survival of primary macrophages under lipopolysaccharide (LPS) challenge. Using a lethal LPS-induced endotoxemia mouse model of sepsis, iNP-SAHA administration improved the survival of mice in a dose-dependent manner and tended to improve survival at the lowest doses compared to iNP control. Further, iNP-SAHA reduced the levels of plasma proinflammatory cytokines and chemokines associated with sepsis more significantly than iNP and similarly improved inflammation-induced spleen and liver toxicity as iNP, supporting its potential polypharmacological activity. Collectively, iNP-SAHA offers a potential drug delivery approach to modulate the multifaceted inflammatory responses observed in diseases such as sepsis.

No evidence for systemic low-grade inflammation in adult patients with early-treated phenylketonuria: The INGRAPH study.

The question of an increased cardiovascular risk has been recently raised in adults with phenylketonuria (PKU). As low-grade systemic inflammation increases cardiovascular risk, the INGRAPH study aimed to evaluate low-grade inflammation in adult PKU patients compared to healthy controls and to determine the potential influence of Phe-controlled diet on inflammation. Twenty early-treated adult PKU patients, including a subgroup of 15 classical PKU patients, and 20 healthy volunteers were included. PKU patients and healthy subjects were matched on age, sex and body mass index class. Plasma concentrations of CRP, IFNg, IL1a, IL1b, IL2, IL6, IL10, and TNFα were measured in PKU patients and compared to controls. Plasma CRP was not different in the PKU group as compared to controls. No significant differences were observed between the two groups concerning plasma cytokines concentrations. Plasma CRP and cytokine profile were not different between "on diet" and "off diet" PKU patients. All these results were similar considering only the classical PKU subgroup. No differences were shown in plasma CRP and pro-inflammatory cytokines between adult PKU patients and healthy controls. Further studies are needed, including more patients and extensive characterization of systemic low-grade inflammation, as cardiovascular risk appears to be a new concern in adult PKU population.