Azithromycin reduces hemoglobin-induced innate neuroimmune activation.

Neonatal intraventricular hemorrhage (IVH) releases blood products into the lateral ventricles and brain parenchyma. There are currently no medical treatments for IVH and surgery is used to treat a delayed effect of IVH, post-hemorrhagic hydrocephalus. However, surgery is not a cure for intrinsic brain injury from IVH, and is performed in a subacute time frame. Like many neurological diseases and injuries, innate immune activation is implicated in the pathogenesis of IVH. Innate immune activation is a pharmaceutically targetable mechanism to reduce brain injury and post-hemorrhagic hydrocephalus after IVH. Here, we tested the macrolide antibiotic azithromycin, which has immunomodulatory properties, to reduce innate immune activation in an in vitro model of microglial activation using the blood product hemoglobin (Hgb). We then utilized azithromycin in our in vivo model of IVH, using intraventricular blood injection into the lateral ventricle of post-natal day 5 rat pups. In both models, azithromycin modulated innate immune activation by several outcome measures including mitochondrial bioenergetic analysis, cytokine expression and flow cytometric analysis. This suggests that azithromycin, which is safe for neonates, could hold promise for modulating innate immune activation after IVH.

Restoring a balanced pool of host-derived and microbiota-derived ligands of the aryl hydrocarbon receptor is beneficial after stroke.

Background: Stroke is a major cause of morbidity and mortality, and its incidence increases with age. While acute therapies for stroke are currently limited to intravenous thrombolytics and endovascular thrombectomy, recent studies have implicated an important role for the gut microbiome in post-stroke neuroinflammation. After stroke, several immuno-regulatory pathways, including the aryl hydrocarbon receptor (AHR) pathway, become activated. AHR is a master regulatory pathway that mediates neuroinflammation. Among various cell types, microglia (MG), as the resident immune cells of the brain, play a vital role in regulating post-stroke neuroinflammation and antigen presentation. Activation of AHR is dependent on a dynamic balance between host-derived and microbiota-derived ligands. While previous studies have shown that activation of MG AHR by host-derived ligands, such as kynurenine, is detrimental after stroke, the effects of post-stroke changes in microbiota-derived ligands of AHR, such as indoles, is unknown. Our study builds on the concept that differential activation of MG AHR by host-derived versus microbiome-derived metabolites affects outcomes after ischemic stroke. We examined the link between stroke-induced dysbiosis and loss of essential microbiota-derived AHR ligands. We hypothesize that restoring the balance between host-derived (kynurenine) and microbiota-derived (indoles) ligands of AHR is beneficial after stroke, offering a new potential avenue for therapeutic intervention in post-stroke neuroinflammation. Method: We performed immunohistochemical analysis of brain samples from stroke patients to assess MG AHR expression after stroke. We used metabolomics analysis of plasma samples from stroke and non-stroke control patients with matched comorbidities to determine the levels of indole-based AHR ligands after stroke. We performed transient middle cerebral artery occlusion (MCAO) in aged (18 months) wild-type (WT) and germ-free (GF) mice to investigate the effects of post-stroke treatment with microbiota-derived indoles on outcome. To generate our results, we employed a range of methodologies, including flow cytometry, metabolomics, and 16S microbiome sequencing. Results: We found that MG AHR expression is increased in human brain after stroke and after ex vivo oxygen-glucose deprivation and reperfusion (OGD/R). Microbiota-derived ligands of AHR are decreased in the human plasma at 24 hours after ischemic stroke. Kynurenine and indoles exhibited differential effects on aged WT MG survival after ex vivoOGD/R. We found that specific indole-based ligands of AHR (indole-3-propionic acid and indole-3-aldehyde) were absent in GF mice, thus their production depends on the presence of a functional gut microbiota. Additionally, a time-dependent decrease in the concentration of these indole-based AHR ligands occurred in the brain within the first 24 hours after stroke in aged WT mice. Post-stroke treatment of GF mice with a cocktail of microbiota-derived indole-based ligands of AHR regulated MG-mediated neuroinflammation and molecules involved in antigen presentation (increased CD80, MHC-II, and CD11b). Post-stroke treatment of aged WT mice with microbiota-derived indole-based ligands of AHR reduced both infarct volume and neurological deficits at 24 hours. Conclusion: Our novel findings provide compelling evidence that the restoration of a well-balanced pool of host-derived kynurenine-based and microbiota-derived indole-based ligands of AHR holds considerable therapeutic potential for the treatment of ischemic stroke.

Iron overload induces cerebral endothelial senescence in aged mice and in primary culture in a sex-dependent manner.

Iron imbalance in the brain negatively affects brain function. With aging, iron levels increase in the brain and contribute to brain damage and neurological disorders. Changes in the cerebral vasculature with aging may enhance iron entry into the brain parenchyma, leading to iron overload and its deleterious consequences. Endothelial senescence has emerged as an important contributor to age-related changes in the cerebral vasculature. Evidence indicates that iron overload may induce senescence in cultured cell lines. Importantly, cells derived from female human and mice generally show enhanced senescence-associated phenotype, compared with males. Thus, we hypothesize that cerebral endothelial cells (CEC) derived from aged female mice are more susceptible to iron-induced senescence, compared with CEC from aged males. We found that aged female mice, but not males, showed cognitive deficits when chronically treated with ferric citrate (FC), and their brains and the brain vasculature showed senescence-associated phenotype. We also found that primary culture of CEC derived from aged female mice, but not male-derived CEC, exhibited senescence-associated phenotype when treated with FC. We identified that the transmembrane receptor Robo4 was downregulated in the brain vasculature and in cultured primary CEC derived from aged female mice, compared with those from male mice. We discovered that Robo4 downregulation contributed to enhanced vulnerability to FC-induced senescence. Thus, our study identifies Robo4 downregulation as a driver of senescence induced by iron overload in primary culture of CEC and a potential risk factor of brain vasculature impairment and brain dysfunction.

Heterochronic Parabiosis Causes Dacryoadenitis in Young Lacrimal Glands.

Aging is associated with inflammation and oxidative stress in the lacrimal gland (LG). We investigated if heterochronic parabiosis of mice could modulate age-related LG alterations. In both males and females, there were significant increases in total immune infiltration in isochronic aged LGs compared to that in isochronic young LGs. Male heterochronic young LGs were significantly more infiltrated compared to male isochronic young LGs. While both females and males had significant increases in inflammatory and B-cell-related transcripts in isochronic and heterochronic aged LGs compared to levels isochronic and heterochronic young LGs, females had a greater fold expression of some of these transcripts than males. Through flow cytometry, specific subsets of B cells were increased in the male heterochronic aged LGs compared to those in male isochronic aged LGs. Our results indicate that serum soluble factors from young mice were not enough to reverse inflammation and infiltrating immune cells in aged tissues and that there were specific sex-related differences in parabiosis treatment. This suggests that age-related changes in the LG microenvironment/architecture participate in perpetuating inflammation, which is not reversible by exposure to youthful systemic factors. In contrast, male young heterochronic LGs were significantly worse than their isochronic counterparts, suggesting that aged soluble factors can enhance inflammation in the young host. Therapies that aim at improving cellular health may have a stronger impact on improving inflammation and cellular inflammation in LGs than parabiosis.

CD11bhigh B Cells Increase after Stroke and Regulate Microglia.

Recent studies have highlighted the deleterious contributions of B cells to post-stroke recovery and cognitive decline. Different B cell subsets have been proposed on the basis of expression levels of transcription factors (e.g., T-bet) as well as specific surface proteins. CD11b (α-chain of integrin) is expressed by several immune cell types and is involved in regulation of cell motility, phagocytosis, and other essential functions of host immunity. Although B cells express CD11b, the CD11bhigh subset of B cells has not been well characterized, especially in immune dysregulation seen with aging and after stroke. Here, we investigate the role of CD11bhigh B cells in immune responses after stroke in young and aged mice. We evaluated the ability of CD11bhigh B cells to influence pro- and anti-inflammatory phenotypes of young and aged microglia (MG). We hypothesized that CD11bhigh B cells accumulate in the brain and contribute to neuroinflammation in aging and after stroke. We found that CD11bhigh B cells are a heterogeneous subpopulation of B cells predominantly present in naive aged mice. Their frequency increases in the brain after stroke in young and aged mice. Importantly, CD11bhigh B cells regulate MG phenotype and increase MG phagocytosis in both ex vivo and in vivo settings, likely by production of regulatory cytokines (e.g., TNF-α). As both APCs and adaptive immune cells with long-term memory function, B cells are uniquely positioned to regulate acute and chronic phases of the post-stroke immune response, and their influence is subset specific.

Compuestos fenólicos y actividad antioxidante de los extractos de la hoja de chirimoya (Annona cherimola Mill) / Phenolic compounds and antioxidant activity of extracts from chirimoya (Annona cherimola Mill) leaf / Compostos fenólicos e atividade antioxidante dos extratos da folha de cherimoya (Annona cherimola Mill)

Resumen Después de la cosecha del fruto chirimoya, los árboles liberan enormes cantidades de hojas, las cuales son quemadas por los agricultores. Este trabajo muestra que las hojas pueden ser usadas para nuestro beneficio como antioxidante. Se determinaron los compuestos fenólicos (CF) y la actividad antioxidante (AA) de los extractos de la hoja seca de Annona cherimola Mill en etanol al 70% v/v, agua a 80 °C, y agua subcrítica (AS) a 110, 120 y 130 °C, siguiendo un diseño factorial con el programa Minitab. Los CF se cuantificaron con la metodología de Folin Ciocalteu y la AA con el 2,2-difenil-1-picrilhidrazilo (DPPH) y con el poder antioxidante reductor del hierro (FRAP). Los resultados indicaron que el tipo de solvente y el tiempo de extracción presentaron un efecto significativo en el contenido de CF y AA de los extractos. Se concluyó que el extracto de hoja de chirimoya es un potencial antioxidante. El extracto de AS a 130 °C presentó el mayor contenido de CF (5,6 g EAG/100 g de hoja seca) y el extracto etanólico presentó mayor AA (0,86 mg equivalente trolox/mg extracto seco; IC50=0,020 mg de extracto seco/mL de extracto de hoja seca y FRAP de 1710,14 μmol equivalente trolox /g de hoja seca) y los extractos obtenidos con AS a menor temperatura presentaron mayores valores de AA.

Abstract After harvesting cherimoya fruit, the trees release huge amounts of leaves, which are burned by farmers. This work shows that the leaves can be used as a source of antioxidants. Total phenolic compounds (TPC) and antioxidant activity (AA) of extracts of the dry leaf of Annona cherimola Mill in 70% v/v ethanol, water at 80 °C, subcritical water (SW) at 110 °C, 120 °C and 130 °C were determined, following a factorial design with the Minitab program. The TPC was quantified with the Folin Ciocalteu methodology, and the AA with 2,2-diphenyl-1-picrilhidrazil (DPPH) and Ferric Reducing Antioxidant Power (FRAP). Results indicate that the type of solvent and the extraction time had a significant effect on the TPC and AA of the extracts. Extracts of cherimoya leaves were found to be a potential antioxidant. The extract of SW at 130 °C presented the highest content of TPC (5.6 g EAG/100 g dry leaves) and the ethanolic extract had the highest AA (0.86 mg trolox equivalent/mg dry extract, IC50 = 0.020 mg dry extract/mL extract of dry leaves and FRAP of 1710.14 μmol ET/g dry leaves) and the extracts obtained with SW at a lower temperature presented a higher AA value.

Resumo Depois da colheita da fruta Cherimoya, as árvores liberam grandes quantidades de folhas, que são queimadas pelos agricultores. Este trabalho mostra que as folhas podem ser usadas para nosso benefício como um antioxidante. Compostos fenólicos (FC) e a actividade antioxidante (AA) de extractos de folha seca Annona cherimola Mill em etanol a 70% v/v água a 80 °C, água subcrítica (AS) 110, 120, 130 °C, foram determinados seguindo um planejamento fatorial com o programa Minitab. Os FC foram quantificados com a metodologia Folin Ciocalteu; e a capacidade antioxidante (AA) com 2,2-difenil-1-picrilhidrazil (DPPH) e poder antioxidante redutor de ferro (FRAP). Os resultados indicaram que o tipo de solvente e o tempo de extração tiveram um efeito significativo no conteúdo deCFeAA dos extratos. Conclui-se que o extracto de folha de cherimoya é um potencial antioxidante, o extracto de AS 130 °C tinha o maior conteúdo de CF (5,6 g EAG/100 g folha seca) e o extracto etanólico mostraram maior AA (0,86 mg equivalente Trolox/mg extrato seco, IC50 = 0,020 mg extrato seco/mL extracto de folha seca e FRAP de 1710,14 μmol ET/g folha seca). Os extratos obtido com AS a uma temperatura mais baixa apresentaram um maior valor de AA.

Serum folate concentrations, asthma, atopy, and asthma control in Peruvian children.

BACKGROUND: The relationship between folate status and asthma-related outcomes has not been carefully examined in low- and middle-income countries where folate deficiency is common. METHODS: Ancillary analysis of an unmatched case-control study in which we analyzed serum folate concentrations in 412 children with asthma and 342 controls living in peri-urban communities in Lima, Peru. We examined baseline associations between folate and asthma, atopy, total serum IgE, pulmonary function, and fractional exhaled nitric oxide. We then followed children with asthma longitudinally for 6-9 months and assessed associations between folate and odds of uncontrolled asthma (defined as Asthma Control Test score ≤ 19) and of ≥1 emergency visits during follow-up. RESULTS: A 10 ng/mL decrease in serum folate was associated with 45% higher adjusted odds of asthma (OR = 1.45, 95% CI 1.05-2.02). The folate-asthma relationship differed by atopic status: a 10 ng/mL decrease in serum folate was associated with a 2.4-fold higher odds of asthma among children without atopy (2.38, 1.20-4.72) and 23% higher odds of asthma in children with atopy (1.23, 0.85-1.80). Among children with asthma, a 10 ng/mL decrease in serum folate was associated with 62% higher odds of uncontrolled asthma (1.62, 1.02-2.56) and 73% higher odds of ≥1 emergency visits during follow-up (1.73, 1.05-2.85). CONCLUSIONS: Serum folate concentrations were inversely associated with asthma, but this effect was stronger in children without atopy. Among children with asthma, lower serum folate concentrations were associated with higher risk of uncontrolled asthma.

Associations between serum 25(OH)D concentrations and prevalent asthma among children living in communities with differing levels of urbanization: a cross-sectional study.

BACKGROUND: Prior evidence suggests that vitamin D deficiency may increase the risk of asthma and atopy and impair pulmonary function in children. METHODS: In this cross-sectional analysis nested in a case-control study, we analyzed serum 25(OH)D concentrations in 413 children with asthma and 471 children without asthma living in two geographically adjacent study communities (Pampas and Villa El Salvador). We measured total and antigen-specific IgE levels, pulmonary function, asthma control, and exhaled nitric oxide. RESULTS: Mean 25(OH)D concentrations were 25.2 ng/mL (SD 10.1) in children with asthma and 26.1 ng/mL (SD 13.7) in children without asthma (p = 0.28). Vitamin D deficiency (25(OH)D < 20 ng/ml) was more common in Pampas than in Villa El Salvador (52.7% vs. 10.5%; p < 0.001). In the overall study population, a 10 ng/ml decrease in serum 25(OH)D concentrations was not significantly associated with odds of asthma (OR 1.09, 95% CI: 0.94 to 1.25). However, vitamin D deficiency was associated with a 1.6-fold increase in odds of asthma in the overall cohort (95% CI: 1.14 to 2.25). After stratifying by site, a 10 ng/mL decrease in serum 25(OH)D concentrations was associated with 18% higher odds of having asthma in Pampas (OR = 1.18, 95% CI 1.02 to 1.38), whereas there was no significant association between 25(OH)D concentrations and asthma in Villa El Salvador (OR = 0.95, 95% CI 0.87 to 1.05). Combined data from these geographically adjacent populations suggests a possible threshold for the relationship between 25(OH)D levels and asthma at approximately 27.5 ng/ml. Serum 25(OH)D concentrations were not clearly associated with asthma control, total serum IgE, atopy, or airway inflammation. CONCLUSION: Serum 25(OH)D concentrations were inversely associated with asthma in one study community with a high prevalence of deficiency. Studies are needed to investigate a possible threshold 25(OH)D concentration after which higher vitamin D levels show no further benefit for asthma.

Free 25(OH)D concentrations are associated with atopy and lung function in children with asthma.

BACKGROUND: Evidence suggests free mono-hydroxyvitamin D (25[OH]D) concentrations are more strongly linked to certain outcomes than total concentrations; however, no studies have examined the relation between free 25(OH)D and respiratory or allergic disease. OBJECTIVE: To examine associations between total and free 25(OH)D concentrations and asthma outcomes. METHODS: We quantified total and free 25(OH)D concentrations in 137 Peruvian children with asthma and 152 children without asthma and examined associations with asthma outcomes. RESULTS: Mean age ± SD was 13 ± 2.5 years, and 50.2% were boys. Mean total and measured free 25(OH)D concentrations were 29 ± 9.5 ng/mL and 5.0 ± 1.3 pg/mL, respectively. Lower free but not total 25(OH)D concentrations were significantly associated with atopy in all children (total, odds ratio [OR] 1.3 per 10-ng/mL decrease, 95% confidence interval [CI] 0.95-1.7, P = .12; vs free, OR 1.3 per 1-pg/mL decrease, 95% CI 1.0-1.6, P = .02) and children with asthma (total, OR 1.1 per 10-ng/mL decrease, 95% CI 0.75-1.7, P = .57; vs free, OR 1.6 per 1-pg/mL decrease, 95% CI 1.0-2.5, P = .04). Free but not total 25(OH)D levels were significantly associated with pre-bronchodilator forced expiratory volume in 1 second (total, 0.11 L, -0.12 to 0.34, P = .34; vs free, 0.20 L, 0.021-0.39, P = .03) and forced vital capacity (total, 0.13 L, -0.12 to 0.37, P = .31; vs free, 0.22 L, 0.026-0.42, P = .03) Z-scores in children with asthma. CONCLUSION: Atopy, forced expiratory volume in 1 second, and forced vital capacity were more strongly linked to free than to total 25(OH)D concentrations, suggesting the free form might be more relevant in modulating allergic disease risk and pulmonary function in children with asthma.