Kynurenine Pathway in Respiratory Diseases.

The kynurenine pathway is the primary route for tryptophan catabolism and has received increasing attention as its association with inflammation and the immune system has become more apparent. This review provides a broad overview of the kynurenine pathway in respiratory diseases, from the initial observations to the characterization of the different cell types involved in the synthesis of kynurenine metabolites and the underlying immunoregulatory mechanisms. With a focus on respiratory infections, the various attempts to characterize the kynurenine/tryptophan (K/T) ratio as an inflammatory marker are reviewed. Its implication in chronic lung inflammation and its exacerbation by respiratory pathogens is also discussed. The emergence of preclinical interventional studies targeting the kynurenine pathway opens the way for the future development of new therapies.

Untargeted metabolomic profiling in children identifies novel pathways in asthma and atopy.

BACKGROUND: Asthma and other atopic disorders can present with varying clinical phenotypes marked by differential metabolomic manifestations and enriched biological pathways. OBJECTIVE: We sought to identify these unique metabolomic profiles in atopy and asthma. METHODS: We analyzed baseline nonfasted plasma samples from a large multisite pediatric population of 470 children aged <13 years from 3 different sites in the United States and France. Atopy positivity (At+) was defined as skin prick test result of ≥3 mm and/or specific IgE ≥ 0.35 IU/mL and/or total IgE ≥ 173 IU/mL. Asthma positivity (As+) was based on physician diagnosis. The cohort was divided into 4 groups of varying combinations of asthma and atopy, and 6 pairwise analyses were conducted to best assess the differential metabolomic profiles between groups. RESULTS: Two hundred ten children were classified as At-As-, 42 as At+As-, 74 as At-As+, and 144 as At+As+. Untargeted global metabolomic profiles were generated through ultra-high-performance liquid chromatography-tandem mass spectroscopy. We applied 2 independent machine learning classifiers and short-listed 362 metabolites as discriminant features. Our analysis showed the most diverse metabolomic profile in the At+As+/At-As- comparison, followed by the At-As+/At-As- comparison, indicating that asthma is the most discriminant condition associated with metabolomic changes. At+As+ metabolomic profiles were characterized by higher levels of bile acids, sphingolipids, and phospholipids, and lower levels of polyamine, tryptophan, and gamma-glutamyl amino acids. CONCLUSION: The At+As+ phenotype displays a distinct metabolomic profile suggesting underlying mechanisms such as modulation of host-pathogen and gut microbiota interactions, epigenetic changes in T-cell differentiation, and lower antioxidant properties of the airway epithelium.

NLRP6 controls pulmonary inflammation from cigarette smoke in a gut microbiota-dependent manner.

Chronic obstructive pulmonary disease (COPD) is a major health issue primarily caused by cigarette smoke (CS) and characterized by breathlessness and repeated airway inflammation. NLRP6 is a cytosolic innate receptor controlling intestinal inflammation and orchestrating the colonic host-microbial interface. However, its roles in the lungs remain largely unexplored. Using CS exposure models, our data show that airway inflammation is strongly impaired in Nlrp6-deficient mice with drastically fewer recruited neutrophils, a key cell subset in inflammation and COPD. We found that NLRP6 expression in lung epithelial cells is important to control airway and lung tissue inflammation in an inflammasome-dependent manner. Since gut-derived metabolites regulate NLRP6 inflammasome activation in intestinal epithelial cells, we investigated the link between NLRP6, CS-driven lung inflammation, and gut microbiota composition. We report that acute CS exposure alters gut microbiota in both wild-type (WT) and Nlrp6-deficient mice and that antibiotic treatment decreases CS-induced lung inflammation. In addition, gut microbiota transfer from dysbiotic Nlrp6-deficient mice to WT mice decreased airway lung inflammation in WT mice, highlighting an NLRP6-dependent gut-to-lung axis controlling pulmonary inflammation.

IL-20 Cytokines Are Involved in the Repair of Airway Epithelial Barrier: Implication in Exposure to Cigarette Smoke and in COPD Pathology.

BACKGROUND: Dysregulated inflammation as seen in chronic obstructive pulmonary disease (COPD) is associated with impaired wound healing. IL-20 cytokines are known to be involved in wound healing processes. The purpose of this study was to use ex vivo and in vitro approaches mimicking COPD to evaluate the potential modulatory role of interleukin-20 (IL-20) on the inflammatory and healing responses to epithelial wounding. METHODS: The expression of IL-20 cytokines and their receptors was investigated in lung-derived samples collected from non-COPD and COPD patients, from mice chronically exposed to cigarette smoke and from airway epithelial cells from humans and mice exposed in vitro to cigarette smoke. To investigate the role of IL-20 cytokines in wound healing, experiments were performed using a blocking anti-IL-20Rb antibody. RESULTS: Of interest, IL-20 cytokines and their receptors were expressed in bronchial mucosa, especially on airway epithelial cells. Their expression correlated with the disease severity. Blocking these cytokines in a COPD context improved the repair processes after a lesion induced by scratching the epithelial layer. CONCLUSIONS: Collectively, this study highlights the implication of IL-20 cytokines in the repair of the airway epithelium and in the pathology of COPD. IL-20 subfamily cytokines might provide therapeutic benefit for patients with COPD to improve epithelial healing.

Removal of senescent cells reduces the viral load and attenuates pulmonary and systemic inflammation in SARS-CoV-2-infected, aged hamsters.

Older age is one of the strongest risk factors for severe COVID-19. In this study, we determined whether age-associated cellular senescence contributes to the severity of experimental COVID-19. Aged golden hamsters accumulate senescent cells in the lungs, and the senolytic drug ABT-263, a BCL-2 inhibitor, depletes these cells at baseline and during SARS-CoV-2 infection. Relative to young hamsters, aged hamsters had a greater viral load during the acute phase of infection and displayed higher levels of sequelae during the post-acute phase. Early treatment with ABT-263 lowered pulmonary viral load in aged (but not young) animals, an effect associated with lower expression of ACE2, the receptor for SARS-CoV-2. ABT-263 treatment also led to lower pulmonary and systemic levels of senescence-associated secretory phenotype factors and to amelioration of early and late lung disease. These data demonstrate the causative role of age-associated pre-existing senescent cells on COVID-19 severity and have clear clinical relevance.

Genes Involved in miRNA Biogenesis Are Not Downregulated in SARS-CoV-2 Infection.

miRNAs, small non-coding RNAs that regulate gene expression, are involved in various pathological processes, including viral infections. Virus infections may interfere with the miRNA pathway through the inhibition of genes involved in miRNA biogenesis. A reduction in the number and the levels of miRNAs expressed in nasopharyngeal swabs of patients with severe COVID-19 was lately observed by us, pointing towards the potential of miRNAs as possible diagnostic or prognostic biomarkers for predicting outcomes among patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The objective of the present study was to investigate whether SARS-CoV-2 infection influences the expression levels of messenger RNAs (mRNAs) of key genes involved in miRNA biogenesis. mRNA levels of AGO2, DICER1, DGCR8, DROSHA, and Exportin-5 (XPO5) were measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in nasopharyngeal swab specimens from patients with COVID-19 and controls, as well as in cells infected with SARS-CoV-2 in vitro. Our data showed that the mRNA expression levels of AGO2, DICER1, DGCR8, DROSHA, and XPO5 were not significantly different in patients with severe COVID-19 when compared to patients with non-severe COVID-19 and controls. Similarly, the mRNA expression of these genes was not affected by SARS-CoV-2 infection in NHBE and Calu-3 cells. However, in Vero E6 cells, AGO2, DICER1, DGCR8, and XPO5 mRNA levels were slightly upregulated 24 h after infection with SARS-CoV-2. In conclusion, we did not find evidence for downregulation of mRNA levels of miRNA biogenesis genes during SARS-CoV-2 infection, neither ex vivo nor in vitro.

Gut colonisation with multidrug-resistant Klebsiella pneumoniae worsens Pseudomonas aeruginosa lung infection.

Carbapenemase-producing Enterobacterales (CPE) are spreading rapidly in hospital settings. Asymptomatic CPE gut colonisation may be associated with dysbiosis and gut-lung axis alterations, which could impact lung infection outcomes. In this study, in male C57BL/6JRj mice colonised by CPE, we characterise the resulting gut dysbiosis, and analyse the lung immune responses and outcomes of subsequent Pseudomonas aeruginosa lung infection. Asymptomatic gut colonisation by CPE leads to a specific gut dysbiosis and increases the severity of P. aeruginosa lung infection through lower numbers of alveolar macrophages and conventional dendritic cells. CPE-associated dysbiosis is characterised by a near disappearance of the Muribaculaceae family and lower levels of short-chain fatty acids. Faecal microbiota transplantation restores immune responses and outcomes of lung infection outcomes, demonstrating the involvement of CPE colonisation-induced gut dysbiosis in altering the immune gut-lung axis, possibly mediated by microbial metabolites such as short-chain fatty acids.

Progressive in vivo development of resistance to cefiderocol in Pseudomonas aeruginosa.

We report in vivo development of cefiderocol (FDC) resistance among four sequential Pseudomonas aeruginosa clinical isolates ST244 recovered from a single patient, without exposure to FDC, which raises concern about the effectiveness of this novel drug. The first recovered P. aeruginosa isolate (P-01) was susceptible to FDC (2 µg/mL), albeit this MIC value was higher than that of a wild-type P. aeruginosa (0.12-0.25 µg/ml). The subsequent isolated strains (P-02, P-03, P-04) displayed increasing levels of FDC MICs (8, 16, and 64 µg/ml, respectively). Those isolates also showed variable and gradual increasing levels of resistance to most ß-lactams tested in this study. Surprisingly, no acquired ß-lactamase was identified in any of those isolates. Whole-genome sequence analysis suggested that this resistance was driven by multifactorial mechanisms including mutational changes in iron transporter proteins associated with FDC uptake, ampC gene overproduction, and mexAB-oprM overexpression. These findings highlight that a susceptibility testing to FDC must be performed prior to any prescription.

Role of T CD4+ cells, macrophages, C-low threshold mechanoreceptors and spinal Cav 3.2 channels in inflammation and related pain-like symptoms in murine inflammatory models.

BACKGROUND AND PURPOSE: T-type calcium channels, mainly the Cav 3.2 subtype, are important contributors to the nociceptive signalling pathway. We investigated their involvement in inflammation and related pain-like symptoms. EXPERIMENTAL APPROACH: The involvement of Cav 3.2 and T-type channels was investigated using genetic and pharmacological inhibition to assess mechanical allodynia/hyperalgesia and oedema development in two murine inflammatory pain models. The location of Cav 3.2 channels involved in pain-like symptoms was studied in mice with Cav 3.2 knocked out in C-low threshold mechanoreceptors (C-LTMR) and the use of ABT-639, a peripherally restricted T-type channel inhibitor. The anti-oedema effect of Cav 3.2 channel inhibition was investigated in chimeric mice with immune cells deleted for Cav 3.2. Lymphocytes and macrophages from either green fluorescent protein-targeted Cav 3.2 or KO mice were used to determine the expression of Cav 3.2 protein and the functional status of the cells. KEY RESULTS: Cav 3.2 channels contributed to the development of pain-like symptoms and oedema in the two murine inflammatory pain models. Our results provided evidence of the involvement of Cav 3.2 channels located on C-LTMRs and spinal cord in inflammatory pain. Cav 3.2 channels located in T cells and macrophages contribute to the inflammatory process. CONCLUSION AND IMPLICATIONS: Cav 3.2 channels play crucial roles in inflammation and related pain, implying that targeting of Cav 3.2 channels with pharmacological agents could be an attractive and readily evaluable strategy in clinical trials, to relieve chronic inflammatory pain in patients.

The Nicotinic Receptor Polymorphism rs16969968 Is Associated with Airway Remodeling and Inflammatory Dysregulation in COPD Patients.

Genome-wide association studies unveiled the associations between the single nucleotide polymorphism rs16969968 of CHRNA5, encoding the nicotinic acetylcholine receptor alpha5 subunit (α5SNP), and nicotine addiction, cancer, and COPD independently. Here, we investigated α5SNP-induced epithelial remodeling and inflammatory response in human COPD airways. We included 26 α5SNP COPD patients and 18 wild-type α5 COPD patients in a multi-modal study. A comparative histologic analysis was performed on formalin-fixed paraffin-embedded lung tissues. Isolated airway epithelial cells from bronchial brushings were cultivated in the air-liquid interface. Broncho-alveolar fluids were collected to detect inflammatory mediators. Ciliogenesis was altered in α5SNP COPD bronchial and bronchiolar epithelia. Goblet cell hyperplasia was exacerbated in α5SNP small airways. The broncho-alveolar fluids of α5SNP COPD patients exhibited an increase in inflammatory mediators. The involvement of the rs16969968 polymorphism in airway epithelial remodeling and related inflammatory response in COPD prompts the development of innovative personalized diagnostic and therapeutic strategies.

Characterization of the Achromobacter xylosoxidans Type VI Secretion System and Its Implication in Cystic Fibrosis.

Bacteria of the genus Achromobacter are environmental germs, with an unknown reservoir. It can become opportunistic pathogens in immunocompromised patients, causing bacteremia, meningitis, pneumonia, or peritonitis. In recent years, Achromobacter xylosoxidans has emerged with increasing incidence in patients with cystic fibrosis (CF). Recent studies showed that A. xylosoxidans is involved in the degradation of the respiratory function of patients with CF. The respiratory ecosystem of patients with CF is colonized by bacterial species that constantly fight for space and access to nutrients. The type VI secretion system (T6SS) empowers this constant bacterial antagonism, and it is used as a virulence factor in several pathogenic bacteria. This study aimed to investigate the prevalence of the T6SS genes in A. xylosoxidans isolated in patients with CF. We also evaluated clinical and molecular characteristics of T6SS-positive A. xylosoxidans strains. We showed that A. xylosoxidans possesses a T6SS gene cluster and that some environmental and clinical isolates assemble a functional T6SS nanomachine. A. xylosoxidans T6SS is used to target competing bacteria, including other CF-specific pathogens. Finally, we demonstrated the importance of the T6SS in the internalization of A. xylosoxidans in lung epithelial cells and that the T6SS protein Hcp is detected in the sputum of patients with CF. Altogether, these results suggest for the first time a role of T6SS in CF-lung colonization by A. xylosoxidans and opens promising perspective to target this virulence determinant as innovative theranostic options for CF management.

Prionoids in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the third most frequent neurodegenerative disease after Alzheimer's and Parkinson's disease. ALS is characterized by the selective and progressive loss of motoneurons in the spinal cord, brainstem and cerebral cortex. Clinical manifestations typically occur in midlife and start with focal muscle weakness, followed by the rapid and progressive wasting of muscles and subsequent paralysis. As with other neurodegenerative diseases, the condition typically begins at an initial point and then spreads along neuroanatomical tracts. This feature of disease progression suggests the spreading of prion-like proteins called prionoids in the affected tissues, which is similar to the spread of prion observed in Creutzfeldt-Jakob disease. Intensive research over the last decade has proposed the ALS-causing gene products Cu/Zn superoxide dismutase 1, TAR DNA-binding protein of 43 kDa, and fused in sarcoma as very plausible prionoids contributing to the spread of the pathology. In this review, we will discuss the molecular and cellular mechanisms leading to the propagation of these prionoids in ALS.

Comparison of the in vivo genotoxicity of electronic and conventional cigarettes aerosols after subacute, subchronic and chronic exposures.

Tobacco smoking is classified as a human carcinogen. A wide variety of new products, in particular electronic cigarettes (e-cigs), have recently appeared on the market as an alternative to smoking. Although the in vitro toxicity of e-cigs is relatively well known, there is currently a lack of data on their long-term health effects. In this context, the aim of our study was to compare, on a mouse model and using a nose-only exposure system, the in vivo genotoxic and mutagenic potential of e-cig aerosols tested at two power settings (18 W and 30 W) and conventional cigarette (3R4F) smoke. The standard comet assay, micronucleus test and Pig-a gene mutation assay were performed after subacute (4 days), subchronic (3 months) and chronic (6 months) exposure. The generation of oxidative stress was also assessed by measuring the 8-hydroxy-2'-deoxyguanosine and by using the hOGG1-modified comet assay. Our results show that only the high-power e-cig and the 3R4F cigarette induced oxidative DNA damage in the lung and the liver of exposed mice. In return, no significant increase in chromosomal aberrations or gene mutations were noted whatever the type of product. This study demonstrates that e-cigs, at high-power setting, should be considered, contrary to popular belief, as hazardous products in terms of genotoxicity in mouse model.

Whole and fractionated human platelet lysate biomaterials-based biotherapy induces strong neuroprotection in experimental models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease of motor neurons leading to death within 3 years and without a curative treatment. Neurotrophic growth factors (NTFs) are pivotal for cell survival. A reason for the lack of patient efficacy with single recombinant NTF brain infusion is likely to be due to the synergistic neuroprotective action of multiple NTFs on a diverse set of signaling pathways. Fractionated (protein size <50, <30, <10, <3 kDa) heat-treated human platelet lysate (HHPL) preparations were adapted for use in brain tissue with the aim of demonstrating therapeutic value in ALS models and further elucidation of the mechanisms of action. In neuronal culture all fractions induced Akt-dependent neuroprotection as well as a strong anti-apoptotic and anti-ferroptotic action. In the <3 kDa fraction anti-ferroptotic properties were shown to be GPX4 dependent highlighting a role for other platelet elements associated with NTFs. In the SOD1G86R mouse model, lifespan was strongly increased by intracerebroventricular delivery of HHPL or by intranasal administration of <3 kDa fraction. Our results suggest that the platelet lysate biomaterials are neuroprotective in ALS. Further studies would now validate theragnostic biomarker on its antiferroptotic action, for further clinical development.

Nicotinic receptors as SARS-CoV-2 spike co-receptors?

Nicotinic acetylcholine receptors (nAChRs) play an important role in homeostasis and respiratory diseases. Controversies regarding the association between COVID-19 hospitalizations and smoking suggest that nAChRs may contribute to SARS-CoV-2 respiratory syndrome. We recently detailed the expression and localization of all nAChR subunits in the human lung. Since virus association with nAChRs has been shown in the past, we hypothesize that nAChR subunits act as SARS-CoV-2 Spike co-receptors. Based on sequence alignment analysis, we report domains of high molecular similarities in nAChRs with the binding domain of hACE2 for SARS-CoV-2 Spike protein. This hypothesis supported by in silico pilot data provides a rational for the modelling and the in vitro experimental validation of the interaction between SARS-CoV-2 and the nAChRs.

Astrogliosis and compensatory neurogenesis after the first ethanol binge drinking-like exposure in the adolescent rat.

BACKGROUND: Multiple ethanol binge drinking-like exposures during adolescence in the rat induce neuroinflammation, loss of neurogenesis, and cognitive deficits in adulthood. Interestingly, the first ethanol binge drinking-like exposure during adolescence also induces short- term impairments in cognition and synaptic plasticity in the hippocampus though the cellular mechanisms of these effects are unclear. Here, we sought to determine which of the cellular effects of ethanol might play a role in the disturbances in cognition and synaptic plasticity observed in the adolescent male rat after two binge-like ethanol exposures. METHODS: Using immunochemistry, we measured neurogenesis, neuronal loss, astrogliosis, neuroinflammation, and synaptogenesis in the hippocampus of adolescent rats 48 h after two binge-like ethanol exposures (3 g/kg, i.p., 9 h apart). We used flow cytometry to analyze activated microglia and identify the TLR4-expressing cell types. RESULTS: We detected increased hippocampal doublecortin immunoreactivity in the subgranular zone (SGZ) of the dentate gyrus (DG), astrogliosis in the SGZ, and a reduced number of mature neurons in the DG and in CA3, suggesting compensatory neurogenesis. Synaptic density decreased in the stratum oriens of CA1 revealing structural plasticity. There was no change in microglial TLR4 expression or in the number of activated microglia, suggesting a lack of neuroinflammatory processes, although neuronal TLR4 was decreased in CA1 and DG. CONCLUSIONS: Our findings demonstrate that the cognitive deficits associated with hippocampal synaptic plasticity alterations that we previously characterized 48 h after the first binge-like ethanol exposures are associated with hippocampal structural plasticity, astrogliosis, and decreased neuronal TLR4 expression, but not with microglia reactivity.

IL-20 Cytokines Are Involved in Epithelial Lesions Associated with Virus-Induced COPD Exacerbation in Mice.

(1) Background: viral infections are a frequent cause of chronic obstructive pulmonary disease (COPD) exacerbations, which are responsible for disease progression and mortality. Previous reports showed that IL-20 cytokines facilitate bacterial lung infection, but their production and their role in COPD and viral infection has not yet been investigated. (2) Methods: C57BL/6 WT and IL-20 Rb KO mice were chronically exposed to air or cigarette smoke (CS) to mimic COPD. Cytokine production, antiviral response, inflammation and tissue damages were analyzed after PVM infection. (3) Results: CS exposure was associated with an increase in viral burden and antiviral response. PVM infection in CS mice enhanced IFN-γ, inflammation and tissue damage compared to Air mice. PVM infection and CS exposure induced, in an additive manner, IL-20 cytokines expression and the deletion of IL-20 Rb subunit decreased the expression of interferon-stimulated genes and the production of IFN-λ2/3, without an impact on PVM replication. Epithelial cell damages and inflammation were also reduced in IL-20 Rb-/- mice, and this was associated with reduced lung permeability and the maintenance of intercellular junctions. (4) Conclusions: PVM infection and CS exposure additively upregulates the IL-20 pathway, leading to the promotion of epithelial damages. Our data in our model of viral exacerbation of COPD identify IL-20 cytokine as a potential therapeutic target.

An innate contribution of human nicotinic receptor polymorphisms to COPD-like lesions.

Chronic Obstructive Pulmonary Disease is a generally smoking-linked major cause of morbidity and mortality. Genome-wide Association Studies identified a locus including a non-synonymous single nucleotide polymorphism in CHRNA5, rs16969968, encoding the nicotinic acetylcholine receptor α5 subunit, predisposing to both smoking and Chronic Obstructive Pulmonary Disease. Here we report that nasal polyps from rs16969968 non-smoking carriers exhibit airway epithelium remodeling and inflammation. These hallmarks of Chronic Obstructive Pulmonary Disease occur spontaneously in mice expressing human rs16969968. They are significantly amplified after exposure to porcine pancreatic elastase, an emphysema model, and to oxidative stress with a polymorphism-dependent alteration of lung function. Targeted rs16969968 expression in epithelial cells leads to airway remodeling in vivo, increased proliferation and production of pro-inflammatory cytokines through decreased calcium entry and increased adenylyl-cyclase activity. We show that rs16969968 directly contributes to Chronic Obstructive Pulmonary Disease-like lesions, sensitizing the lung to the action of oxidative stress and injury, and represents a therapeutic target.