Vaccination after developing long COVID: Impact on clinical presentation, viral persistence, and immune responses.

BACKGROUND: Vaccination protects against severe COVID-19 manifestations. For those with post-COVID-19 conditions (PCC) or long COVID, the impact of COVID-19 vaccination on the evolution of symptoms, immune responses, and viral persistence is unclear. METHODS: In this prospective observational cohort study, we evaluated the number of PCC symptoms, affected organ systems, and psychological well-being scores before and after patients with PCC received COVID-19 vaccination. We simultaneously evaluated biomarkers of systemic inflammation and levels of plasma cytokines/chemokines. We measured plasma and intracellular levels of SARS-CoV-2 antigens, and immunoreactivity to SARS-CoV-2 antigens in blood. RESULTS: COVID-19 vaccination was associated with decreases in number of PCC symptoms (pre-vaccination: 6.56 ± 3.1 vs post-vaccination: 3.92 ± 4.02; P <0.001) and affected organ systems (pre-vaccination: 3.19 ± 1.04 vs post-vaccination: 1.89 ± 1.12; P <0.001), and increases in World Health Organization (WHO)-5 Well-Being Index Scores (pre-vaccination: 42.67 ± 22.76 vs post-vaccination: 56.15 ± 22.83; P <0.001). Patients with PCC also had significantly decreased levels of several pro-inflammatory plasma cytokines/chemokines after COVID-19 vaccination including sCD40L, GRO-⍺, macrophage inflammatory protein (MIP)-1⍺, interleukin (IL)-12p40, G-colony stimulating factor (CSF), M-CSF, IL-1ß, and stem cell factor (SCF). PCC participants presented a certain level of immunoreactivity toward SARS-CoV-2, that was boosted with vaccination. SARS-CoV-2 S1 antigen persisted in the blood of PCC participants, mostly in non-classical monocytes, regardless of participants receiving vaccination. CONCLUSIONS: Our study shows higher pro-inflammatory responses associated with PCC symptoms and brings forward a possible role for vaccination in mitigating PCC symptoms by decreasing systemic inflammation. We also observed persistence of viral products independent of vaccination that could be involved in perpetuating inflammation through non-classical monocytes.

Impact of ENaC downregulation in transgenic mice on the outcomes of acute lung injury induced by bleomycin.

NEW FINDINGS: What is the central question of this study? How does the downregulation of ENaC, the major driving force for alveolar fluid clearance, impact acute lung injury outcomes induced by bleomycin, featuring alveolar damage, as observed during ARDS exudative phase? What is the main finding and its importance? ENaC downregulation in αENaC(-/-)Tg+ mice did not elicit a substantial worsening impact on the main bleomycin outcomes. In ARDS patients, both ENaC alteration and alveolar damage are observed. Thus, novel therapeutic avenues, favouring alveolar integrity restauration, in addition to lung oedema resolution capacity, mainly driven by ENaC, would be essential. ABSTRACT: The exudative phase of acute respiratory distress syndrome (ARDS) is characterized by extended alveolar damage, resulting in accumulation of protein-rich inflammatory oedematous fluid in the alveolar space. Na+ reabsorption through ENaC channels is a major driving force for alveolar fluid clearance (AFC) in physiological and pathological conditions. It has previously been shown that partial αENaC impairment in transgenic (αENaC(-/-)Tg+) mice results in reduced AFC in basal conditions and increased wet/dry ratio after thiourea-induced lung oedema, a model in which the integrity of the alveolar epithelium is preserved. The goal of this study was to further investigate the impact of αENaC downregulation in αENaC(-/-)Tg+ mice using an experimental model of acute lung injury induced by bleomycin. A non-significant trend in enhanced weight loss and mortality rates was observed after the bleomycin challenge in αENaC(-/-)Tg+ compared to wild-type (WT) mice. Bronchoalveolar lavage analyses revealed increased TNFα levels and protein concentrations, as indexes of lung inflammation and alveolar damage, in αENaC(-/-)Tg+ mice, compared to WT, at day 3 post-bleomycin, although a statistical difference was no longer measured at day 7. Differential immune cell counts were similar in WT and αENaC(-/-)Tg+ mice challenged with bleomycin. Moreover, lung weight measurements indicated similar oedema levels in WT mice and in transgenic mice with impaired ENaC channels. Altogether, our data indicated that change in ENaC expression does not elicit a significant impact on lung oedema level/resolution in the bleomycin model, featuring alveolar damage.

YKL-40 as a clinical biomarker in adult patients with CF: Implications of a CHI3L1 single nucleotide polymorphism in disease severity.

BACKGROUND: YKL-40 (chitinase 3-like 1 gene; CHI3L1) is an inflammatory marker that is increased in the blood of patients with inflammatory diseases, including cystic fibrosis (CF). The objective of our study was to explore the relationship between circulating levels of YKL-40, selected CHI3L1 single nucleotide polymorphisms (SNPs) and the severity of CF disease. METHODS: A prospective cohort of 188 adult patients with CF was established in 2015. Blood samples and clinical data were collected over 2 years to analyze the circulating levels of YKL-40 and to genotype selected CHI3L1 SNPs. We also looked for an association between these factors and clinical parameters. RESULTS: We found that according to the serum YKL-40 concentration, the patients could be categorized into two distinct groups: low and high YKL-40. Compared to the patients in the low YKL-40 group, the patients in the high YKL-40 group had lower lung function (P < 0.001), a higher proportion of delF508 homozygote mutations (P= 0.027) and dysglycemia (P= 0.015). They were also more colonized with Pseudomonas aeruginosa (P= 0.003) and required more frequent antibiotic intravenous courses (P < 0.001). We also observed that patients expressing the C/C-rs4950928 genotype had higher levels of YKL-40 in their blood and were more frequently dysglycemic. CONCLUSION: Our study suggests that YKL-40 could be a potential biomarker of CF disease severity. Furthermore, the CHI3L1 rs4950928 SNP could be a susceptible gene that could be used by CF health professionals to identify patients who are the most at risk of having a severe clinical profile.

Effect of adrenalectomy on pulmonary edema resolution.

PURPOSE: The capacity of the lung to clear edema fluid has been shown to be one of the factors that can influence the prognosis of cardiogenic and noncardiogenic pulmonary edema. Active Na+ transport across the alveolar epithelium is the main driving force involved in this physiological process. Since endogenous catecholamines are known to activate the sodium-dependent mechanism of alveolar edema clearance, the objective of the present study was to explore if adrenalectomy, which prevents the release of endogenous catecholamines and other hormones, such as corticosterone, into circulation, would affect edema resolution in a model of lung injury induced by thiourea. METHODS: A high-permeability pulmonary edema was induced in adult male Sprague-Dawley rats using a thiourea-induced pulmonary edema model. To determine if the release of adrenalin and corticosterone is essential for resolution of the thiourea-induced edema, we measured 1) the release of adrenalin and corticosterone in urine and 2) edema resolution in control animals and adrenalectomized animals. RESULTS: The administration of thiourea significantly increased the wet-to-dry ratio after four and eight hours. After 12 and 24 hours, the wet-to-dry ratio gradually returned to baseline. Although thiourea-induced pulmonary edema was associated with a significant increase in urine adrenalin and corticosterone, the absence of adrenalin and corticosterone response in adrenalectomized animals did not prevent the resolution of the edema. CONCLUSIONS: These experiments demonstrated that resolution of thioureainduced pulmonary edema can occur in the absence of hormonal secretion by the adrenal glands.

Alveolar liquid clearance in lung injury: Evaluation of the impairment of the ß2-adrenergic agonist response in an ischemia-reperfusion lung injury model.

While alveolar liquid clearance (ALC) mediated by the ß2-adrenergic receptor (ß2-AR) plays an important role in lung edema resolution in certain models of lung injury, in more severe lung injury models, this response might disappear. Indeed, we have shown that in an ischemia-reperfusion-induced lung injury model, ß2-agonists do not enhance ALC. The objective of this study was to determine if downregulation of the ß2-AR could explain the lack of response to ß2-agonists in this lung injury model. In an in vivo canine model of lung transplantation, we observed no change in ß2-AR concentration or affinity in the injured transplanted lungs compared to the native lungs. Furthermore, we could not enhance ALC in transplanted lungs with dcAMP + aminophylline, a treatment that bypasses the ß2-adrenergic receptor and is known to stimulate ALC in normal lungs. However, transplantation decreased αENaC expression in the lungs by 50%. We conclude that the lack of response to ß2-agonists in ischemia-reperfusion-induced lung injury is not associated with significant downregulation of the ß2-adrenergic receptors but is attributable to decreased expression of the ENaC channel, which is essential for sodium transport and alveolar liquid clearance in the lung.

DNA Methylation Regulates RGS2-induced S100A12 Expression in Airway Epithelial Cells.

RGS2 is a key modulator of stress in human airway epithelial cells, especially of hyperresponsiveness and mucin hypersecretion, both of which are features of cystic fibrosis (CF). Because its expression can be modulated through the DNA methylation pathway, we hypothesize that RGS2 is downregulated by DNA hypermethylation in CF airway epithelial cells. This downregulation would then lead to an enhanced inflammatory response. We demonstrated RGS2 transcript and protein downregulation in cultured airway epithelial cells from patients with CF and validated our findings in two CF epithelial cell lines. A methylated DNA immunoprecipitation array showed the presence of methylated cytosine on 13 gene promoters in CF. Among these genes, we confirmed that the RGS2 promoter was hypermethylated by using bisulfite conversion coupled with a methylation-specific PCR assay. Finally, we showed that downregulation of RGS2 in non-CF cells increased the expression of S100A12, a proinflammatory marker. These results highlight the importance of epigenetic regulation in gene expression in CF and show that RGS2 might modulate the inflammatory response in CF through DNA methylation control.

Neutrophils as a Potential Source of Chitinase-3-like Protein 1 in Cystic Fibrosis.

The chitinase-3-like protein 1, also known as YKL-40, is an inflammatory marker increased in blood of patients with cystic fibrosis (CF). Systemic levels of YKL-40 are increased in dysglycemic patients with CF. Our objective is to determine if YKL-40 is expressed and released by CF neutrophils. We also assessed if specific stimulus, such as glucose and lipopolysaccharide (LPS), can induce the secretion of YKL-40. Neutrophil cells of healthy adults and patients with CF were isolated. Immunostaining of whole blood and neutrophils was done. CF and healthy neutrophils were cultured with either LPS or varying concentrations of glucose. YKL-40 protein was measured using specific immunoassay ELISA. Isolated neutrophil cells from 11 patients with CF (32.3 ± 8.0 years) were compared to five age-matched healthy individuals (28.3 ± 5.5 years). Although there is a significant increase in the concentration of YKL-40 in CF neutrophils compared to healthy neutrophils (P = 0.027), the spontaneous release of YKL-40 into the media is similar in CF and healthy neutrophils. CF neutrophils stimulated with LPS or glucose do not stimulate the release of YKL-40 (P = 0.995 for glucose and P = 0.624 for LPS). CF neutrophils have higher intracellular level of YKL-40 than neutrophils from healthy volunteers, but they do not release more YKL-40 when stimulated with exogenous stimulus. These results suggest that the increased levels of circulating YKL-40 in CF patients might originate from another cellular source.

S100A8/A9 and sRAGE kinetic after polytrauma; an explorative observational study.

BACKGROUND: Following tissue injury after trauma, the activation of innate immune pathways results in systemic inflammation, organ failure and an increased risk of infections. The objective of this study was to characterize the kinetics of the S100A8/S100A9 complex, a new-recognized alarmin, as well as its soluble receptor sRAGE, over time after trauma as potential early biomarkers of the risk of organ damage. METHODS: We collected comprehensive data from consenting patients admitted to an ICU following severe trauma. The blood samples were taken at Day 0 (admission), Day1, 3 and 5 S100A8/A9 and sRAGE were measured by ELISA. Biomarkers levels were reported as median (IQR). RESULTS: Thirty-eight patients sustaining in majority a blunt trauma (89%) with a median ISS of 39 were included. In this cohort, the S100A8/A9 complex increased significantly over time (p = 0.001), but its levels increment over time (D0 to D5) was significantly smaller in patients developing infection (7.6 vs 40.1 mcg/mL, p = 0.011). The circulating level of sRAGE circulating levels decreased over time (p < 0.0001) and was higher in patients who remained in shock on day 3 (550 vs 918 pg/mL; p = 0.02) or 5 (498 vs 644 pg/mL; p = 0.045). Admission sRAGE levels were significantly higher in non-survivors (1694 vs 745 pg/mL; p = 0.015) and was higher in patients developing renal failure (1143 vs 696 pg/mL, p = 0.011). DISCUSSION: Our findings reveal an interesting association between the biomarker S100A8/9 least increase over time and the presence of infectious complication after trauma. We describe that the sRAGE decline over time is in relation with shock and markers of ischemic injury. We also confirm the association of sRAGE levels measured at admission with mortality and the development of renal failure. CONCLUSIONS: This work illustrates the importance of following the circulating level of biomarker overtime. The utilization of S1008/9 as a tool to stratify infection risk and trigger early interventions need to be validated prospectively.

Oxidative stress modulates the expression of genes involved in cell survival in ΔF508 cystic fibrosis airway epithelial cells.

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 µM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients.

Dexamethasone inhibits the action of TNF on ENaC expression and activity.

We have reported that TNF, a proinflammatory cytokine present in several lung pathologies, decreases the expression and activity of the epithelial Na(+) channel (ENaC) by approximately 70% in alveolar epithelial cells. Because dexamethasone has been shown to upregulate ENaC mRNA expression and is well known to downregulate proinflammatory genes, we tested if it could alleviate the effect of TNF on ENaC expression and activity. In cotreatment with TNF, we found that dexamethasone reversed the inhibitory effect of TNF and upregulated alpha, beta, and gammaENaC mRNA expression. When the cells were pretreated for 24 h with TNF before cotreatment, dexamethasone was still able to increase alphaENaC mRNA expression to 1.8-fold above control values. However, in these conditions, beta and gammaENaC mRNA expression was reduced to 47% and 14%, respectively. The potential role of TNF and dexamethasone on alphaENaC promoter activity was tested in A549 alveolar epithelial cells. TNF decreased luciferase (Luc) expression by approximately 25% in these cells, indicating that the strong diminution of alphaENaC mRNA must be related to posttranscriptional events. Dexamethasone raised Luc expression by fivefold in the cells and augmented promoter activity by 2.77-fold in cotreatment with TNF. In addition to its effect on alphaENaC gene expression, dexamethasone was able to maintain amiloride-sensitive current as well as the liquid clearance abilities of TNF-treated cells within the normal range. All these results suggest that dexamethasone alleviates the downregulation of ENaC expression and activity in TNF-treated alveolar epithelial cells.

Modulation of Na+ transport and epithelial sodium channel expression by protein kinase C in rat alveolar epithelial cells.

Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na+ transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased alpha, beta, and gammaENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of alphaENaC expression. The PKC inhibitors bisindolylmaleimide at 2 micromol/L and Gö6976 at 2 micromol/L diminished the PMA-induced suppression of alphaENaC expression, while rottlerin at 1 micromol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na+-K+-ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na+ entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca2+-dependent PKC is potentially involved in this response.

Downregulation of ENaC activity and expression by TNF-alpha in alveolar epithelial cells.

Sodium absorption by an amiloride-sensitive channel is the main driving force of lung liquid clearance at birth and lung edema clearance in adulthood. In this study, we tested whether tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine involved in several lung pathologies, could modulate sodium absorption in cultured alveolar epithelial cells. We found that TNF-alpha decreased the expression of the alpha-, beta-, and gamma-subunits of epithelial sodium channel (ENaC) mRNA to 36, 43, and 16% of the controls after 24-h treatment and reduced to 50% the amount of alpha-ENaC protein in these cells. There was no impact, however, on alpha(1) and beta(1) Na(+)-K(+)-ATPase mRNA expression. Amiloride-sensitive current and ouabain-sensitive Rb(+) uptake were reduced, respectively, to 28 and 39% of the controls. A strong correlation was found at different TNF-alpha concentrations between the decrease of amiloride-sensitive current and alpha-ENaC mRNA expression. All these data show that TNF-alpha, a proinflammatory cytokine present during lung infection, has a profound influence on the capacity of alveolar epithelial cells to transport sodium.