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Allergens and Th2 cytokines affect the homeostatic environment in the airways, leading to increased mucus production by goblet cells associated with altered adherens junctional complex (AJC) and tight junction (TJ) proteins responsible for maintaining epithelial barrier function. Circadian clock-dependent regulatory mechanisms such as inflammation and epithelial barrier function are gaining more attention due to their therapeutic potential against allergic inflammatory lung diseases. Currently, there are no studies to support whether REV-ERBα activation can attenuate Th2 cytokine-induced epithelial barrier dysfunction in human bronchial epithelial cells. We hypothesized that Th2 cytokine-induced epithelial barrier dysfunction may be protected by activating REV-ERBα. Treatment with Th2 cytokines or HDM significantly reduced the cell impedance, as confirmed by transepithelial electrical resistance (TEER). However, pre-treatment with SR10067 attenuated Th2 cytokine-induced barrier dysfunction, such as decreased permeability, improved TEER, localization of AJC and TJ proteins, and mRNA and protein levels of selected epithelial barrier and circadian clock targets. Overall, we showed for the first time that REV-ERBα activation regulates altered epithelial barrier function that may have direct implications for the treatment of asthma and other allergic diseases., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

House dust mite (HDM) is a common aeroallergen that can disrupt the airway epithelial barrier leading to dysregulated immune response, resulting in allergic lung diseases such as asthma. Cryptochrome (CRY), a circadian clock gene, plays an important role in the regulation of metabolism, and immune response. It remains unclear whether stabilizing CRY using KL001 can attenuate HDM/Th2 cytokine-induced epithelial barrier dysfunction in 16-HBE cells. We evaluate the effect of KL001 (20 µM) pre-treatment (4 hrs) in HDM/Th2 cytokine (IL-4 or IL-13)-mediated change in epithelial barrier function. HDM and Th2 cytokine-induced changes in transepithelial electrical resistance (TEER) were determined by an xCELLigence real-time cell analyzer and delocalization of adherens junction complex (AJC: E-cadherin and β-catenin) and tight junction proteins (TJP: Occludin and Zonula occludens-1) by immunostaining and confocal microscopy. Finally, quantitative real-time PCR (qRT-PCR) and Western blotting were used to measure altered gene expression and protein abundance of the epithelial barrier function and core clock genes, respectively. HDM and Th2 cytokine treatment significantly decreased TEER associated with altered gene expression and protein abundance of the selected epithelial barrier function and circadian clock genes. However, pre-treatment with KL001 attenuated HDM and Th2 cytokine-induced epithelial barrier dysfunction as early as 12-24 hrs. KL001 pre-treatment showed attenuation of HDM and Th2 cytokine-induced alteration in the localization and gene expression of AJP and TJP ( Cdh1, Ocln, and Zo1 ) and core clock genes ( Clock, Arntl/Bmal1, Cry1/2, Per1/2, Nr1d1/Rev-erbα, and Nfil3 ). We demonstrate, for the first time, the protective role of KL001 in HDM and Th2 cytokine-mediated epithelial barrier dysfunction.

Background: Human cytomegalovirus (HCMV) reactivation is a major cause of morbidity and mortality among stem cell transplant recipients post-transplantation., Aim: HCMV immediate-early messenger RNA (IE-mRNA) was evaluated as marker of post-transplant HCMV reactivation in bone marrow transplant recipients., Method: ology: An in-house real-time reverse transcriptase PCR targeting IE-mRNA was developed to estimate HCMV mRNA levels post-transplantation. Blood samples collected in K2-EDTA tubes from patients (n = 162) admitted with Department of Clinical Hematology were transported in cold condition for routine HCMV DNA screening. For HCMV IE-mRNA quantification, peripheral blood mononuclear cells (PBMCs) were separated from whole blood and stored in RNA later at -70 °C until testing. Samples were collected weekly once for first 3 weeks post-transplantation and thereafter from week 4-12, samples were collected twice weekly. A total of 2467 samples were collected from 162 study participants., Results: Thirty five patients (21.6 %) had post-transplant HCMV reactivation. Twenty five patients with complete follow-up were selected for monitoring HCMV DNA. HCMV IE-mRNA PCR was performed for 15 patients and 7(46.6 %) patients had detectable mRNA levels. HCMV IE-mRNA was detected in all patients with increasing HCMV DNA levels except for one patient in whom IE-mRNA appeared 3 days before HCMV DNA was detected. One patient had detectable HCMV IE-mRNA during declining HCMV DNA level. However the patient showed an increased HCMV DNA one week later, indicating the importance of HCMV mRNA in predicting HCMV replication., Conclusion: Quantification of HCMV IE-mRNA may be a valuable tool to predict progression of HCMV infection post-transplantation, with further prospective studies needed for validation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Circadian rhythms have an established role in regulating physiological processes, such as inflammation, immunity, and metabolism. Ozone, a common environmental pollutant with strong oxidative potential, is implicated in lung inflammation/injury in asthmatics. However, whether O 3 exposure affects the expression of circadian clock genes in the lungs is not known. In this study, changes in the expression of core clock genes are analyzed in the lungs of adult female and male mice exposed to filtered air (FA) or O 3 using qRT-PCR. The findings are confirmed using an existing RNA-sequencing dataset from repeated FA- and O 3 -exposed mouse lungs and validated by qRT-PCR. Acute O 3 exposure significantly alters the expression of clock genes in the lungs of females (Per1, Cry1, and Rora) and males (Per1). RNA-seq data revealing sex-based differences in clock gene expression in the airway of males (decreased Nr1d1/Rev-erbα) and females (increased Skp1), parenchyma of females and males (decreased Nr1d1 and Fbxl3 and increased Bhlhe40 and Skp1), and alveolar macrophages of males (decreased Arntl/Bmal1, Per1, Per2, Prkab1, and Prkab2) and females (increased Cry2, Per1, Per2, Csnk1d, Csnk1e, Prkab2, and Fbxl3). These findings suggest that lung inflammation caused by O 3 exposure affects clock genes which may regulate key signaling pathways., (© 2023 Wiley-VCH GmbH.)

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that impairs lung mechanical properties due to dysregulated extracellular matrix remodeling. Lung function assessment is an important physiological endpoint in the mouse model of pulmonary fibrosis (PF) that has gained a broader scientific acceptance in the field. IPF pathophysiology shows sex-based differences, disproportionately affecting more men compared to women. Prior reports suggest that the circadian clock is perturbed during the pathogenesis of PF. We have comprehensively assessed the sex-based differences and time-of-day response (at Zeitgeber time: ZT0/6:00 a.m. or ZT12/6 p.m.) in lung mechanics among sham (control) versus bleomycin (BLM)-challenged female and male (C57BL/6: WT) mice using Flexi-vent. BLM challenge altered lung function significantly in males in both total lung (reduced dynamic compliance, and increased resistance and elastance) as well as lung tissue-specific parameters (increased tissue elastance and tissue damping) but less pronounced in females. BLM-challenged mice showed a time-of-day response in lung function at ZT0 versus ZT12, which was pronounced in the ZT0 BLM group. Overall, these findings provide a comprehensive analysis of altered lung function in female and male mice and the time-of-day difference in lung function parameters following BLM-induced lung fibrosis., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by excessive scarring of the lungs that can lead to respiratory failure and death. Lungs of patients with IPF demonstrate excessive deposition of extracellular matrix (ECM) and an increased presence of pro-fibrotic mediators such as transforming growth factor-beta 1 (TGFβ1), which is a major driver of fibroblast-to-myofibroblast transition (FMT). Current literature supports that circadian clock dysfunction plays an essential role in the pathophysiology of various chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and IPF. The circadian clock transcription factor Rev-erbα is encoded by Nr1d1 that regulates daily rhythms of gene expression linked to immunity, inflammation, and metabolism. However, investigations into the potential roles of Rev-erbα in TGFβ-induced FMT and ECM accumulation are limited. In this study, we utilized several novel small molecule Rev-erbα agonists (GSK41122, SR9009, and SR9011) and a Rev-erbα antagonist (SR8278) to determine the roles of Rev-erbα in regulating TGFβ1-induced FMT and pro-fibrotic phenotypes in human lung fibroblasts. WI-38 cells were either pre-treated/co-treated with or without Rev-erbα agonist/antagonist along with TGFβ1. After 48 h, the following parameters were evaluated: secretion of COL1A1 (Slot-Blot analysis) and IL-6 (ELISA) into condition media, expressions of α-smooth muscle actin (αSMA: immunostaining and confocal microscopy), and pro-fibrotic proteins (αSMA and COL1A1 by immunoblotting), as well as gene expression of pro-fibrotic targets (qRT-PCR: Acta2, Fn1, and Col1a1). Results revealed that Rev-erbα agonists inhibited TGFβ1-induced FMT (αSMA and COL1A1), and ECM production (reduced gene expression of Acta2, Fn1, and Col1a1), and decreased pro-inflammatory cytokine IL-6 release. The Rev-erbα antagonist promoted TGFβ1-induced pro-fibrotic phenotypes. These findings support the potential of novel circadian clock-based therapeutics, such as Rev-erbα agonist, for the treatment and management of fibrotic lung diseases and disorders., Competing Interests: Declaration of competing interest The authors report no declarations of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Circadian rhythms and sex differences are involved in the pathophysiology of asthma. Yet, there are no reports that simultaneously address the role of the circadian clock and sex-based differences in chronic house dust mite (HDM)-induced asthma. Here, we sought to determine if chronic HDM exposure during the resting phase (zeitgeber time: ZT0/6:00 a.m.) versus the active phase (ZT12/6:00 p.m.) differentially affects the circadian clock and alters asthma pathobiology in female and male mice. HDM exposure at ZT12 exaggerated infiltration of eosinophil subtypes and associated chemokines in females compared to males. Furthermore, HDM exposure augmented eosinophil chemokines, Th2 gene expression and cytokine release, and humoral immune response in females compared to males at ZT12. Concurrently, histopathological evaluation confirmed increased airway inflammation at ZT12 in both females and males. Overall, we showed a time-of-day response and sex-based differences in HDM-induced exaggerated asthmatic phenotypes (inflammation/remodeling) and circadian clock disruption in females compared to males., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Background: Asthma is a chronic inflammatory disease that shows a time-of-day response to variations in symptoms/severity. However, how the lung circadian clock influences time-of-day response and sex-based differences in house dust mite (HDM)-induced airway inflammation and remodeling has not been thoroughly investigated., Objective: We sought to determine whether acute HDM exposure in wild-type mice shows time-of-day response and sex-based differences in allergic airway inflammation and circadian clock disruption in the lungs., Methods: Wild-type (C57BL/6J) and Rev-erbα knockout (KO) mice were exposed to either PBS or HDM (for 10 days) intranasally at Zeitgeber time (ZT0: 6 am; ZT12: 6 pm) and euthanized 48 hours after the last exposure. Acute HDM-induced time-of-day response and sex-based differences in lung inflammation, gated cytokines/chemokines, humoral and hormonal responses, and circadian clock gene expression were analyzed., Results: Acute HDM-exposed mice showed a time-of-day response and sex-based differences in exaggerated lung inflammation (inflammatory eosinophils and interstitial macrophages) at ZT12 when compared with ZT0. HDM-exposed female mice showed increased inflammatory response at ZT12, but HDM-exposed male mice showed comparatively lower inflammation with no time-of-day response. HDM-exposed female and male mice showed augmented IgE levels at ZT12 when compared with ZT0. Myeloid innate immunity panel, cytokines/chemokines, and mucin genes showed a time-of-day gating response at ZT0 and ZT12 in the HDM group. In addition, HDM exposure altered the expression of circadian clock genes in the lung, which was evident in female mice at ZT12. Overall, female mice showed significant time-of-day responses to all these parameters compared with male mice. Rev-erbα KO mice exposed to acute HDM showed exaggerated lung inflammation associated with increased IgE and proinflammatory cytokines in bronchoalveolar lavage fluid. Interestingly, HDM exposure causes reduced expression of clock genes in flow-sorted resident eosinophils but not alveolar macrophages. Acute HDM exposure reduced the nocturnal locomotor activity in mice 5 days post-HDM exposure until day 10., Conclusions: This study shows a time-of-day response to acute HDM exposure and sex-based differences in the severity of lung inflammation and humoral immune response associated with circadian clock disruption. Our findings support the use of separate female and male mice cohorts for preclinical studies to understand the molecular heterogeneity in asthma pathophysiology., (© 2023 The Authors.)

Background: Quantitative Cytomegalovirus (CMV) polymerase chain reactions are increasingly being used for monitoring CMV DNAemia in haematopoietic stem cell transplants and solid organ transplants., Objective: In this study, a commercial CMV viral load assay was compared with an in-house viral load assay., Materials and Methods: A total of 176 whole-blood samples were tested for CMV DNAemia using both assays., Results: Our evaluation showed a difference of 1 log 10 copies/ml between the two assay systems in determining CMV viral loads in the clinical samples., Conclusion: The in-house viral load assay had a better correlation with clinical findings compared to the commercial assay. Quality assessment of these assays was done by the United Kingdom National External Quality Assessment Scheme (UKNEQAS), an external proficiency testing programme, and by the National Institute for Biological Standard and Control (NIBSC) standard. For UKNEQAS and NIBSC standards, the bias between the assays was 0.73 log 10 and 0.85 log 10 , respectively. This difference is well within the acceptable range already reported in the literature., Competing Interests: There are no conflicts of interest