Lactobacillus rhamnosus Restores Antiviral Signaling and Attenuates Cytokines Secretion from Human Bronchial Epithelial Cells Exposed to Cigarette Smoke and Infected with SARS-CoV-2.

Individuals with chronic obstructive pulmonary disease (COPD) are more susceptible to exacerbation crisis triggered by secondary lung infections due to the dysfunction of antiviral signaling, principally via suppression of IFN-γ. Although the probiotic is known for controlling pulmonary inflammation in COPD, the influence of the Lactobacillus rhamnosus (Lr) on antiviral signaling in bronchial epithelium exposed to cigarette smoke extract (CSE) and viruses, remains unknown. Thus, the present study investigated the Lr effect on the antiviral signaling and the secretion of inflammatory mediators from bronchial epithelial cells (16HBE cells) exposed to CSE and SARS-CoV-2. The 16HBE cells were cultured, treated with Lr, stimulated with CSE, and infected with SARS-CoV-2. The cellular viability was evaluated using the MTT assay and cytotoxicity measured by lactate dehydrogenase (LDH) activity. The viral load, TLR2, TLR3, TLR4, TLR7, TLR8, MAVS, MyD88, and TRIF were quantified using specific PCR. The pro-inflammatory mediators were measured by a multiplex biometric immunoassay, and angiotensin converting enzyme 2 (ACE2) activity, NF-κB, RIG-I, MAD5, and IRF3 were measured using specific ELISA kits. Lr decreased viral load, ACE2, pro-inflammatory mediators, TLR2, TLR4, NF-κB, TLR3, TLR7, and TLR8 as well as TRIF and MyD88 expression in CSE and SARS-CoV-2 -exposed 16HBE cells. Otherwise, RIG-I, MAD5, IRF3, IFN-γ, and the MAVS expression were restored in 16HBE cells exposed to CSE and SARS-CoV-2 and treated with Lr. Lr induces antiviral signaling associated to IFN-γ secreting viral sensors and attenuates cytokine storm associated to NF-κB in bronchial epithelial cells, supporting its emerging role in prevention of COPD exacerbation.

The MAPKinase Signaling and the Stimulatory Protein-1 (Sp1) Transcription Factor Are Involved in the Phototherapy Effect on Cytokines Secretion from Human Bronchial Epithelial Cells Stimulated with Cigarette Smoke Extract.

The present study was aimed to investigate the phototherapy effect with low-level laser on human bronchial epithelial cells activated by cigarette smoke extract (CSE). Phototherapy has been reported to actuate positively for controlling the generation/release of anti-inflammatory and pro-inflammatory mediators from different cellular type activated by distinct stimuli. It is not known whether the IL-8 and IL-10 release from CSE-stimulated human bronchial epithelium (BEAS) cells can be influenced by phototherapy. Human bronchial epithelial cell (BEAS) line was cultured in a medium with CSE and irradiated (660 nm) at 9 J. Apoptosis index was standardized with Annexin V and the cellular viability was evaluated by MTT. IL-8, IL-10, cAMP, and NF-κB were measured by ELISA as well as the Sp1, JNK, ERK1/2, and p38MAPK. Phototherapy effect was studied in the presence of mithramycin or the inhibitors of JNK or ERK. The IL-8, cAMP, NF-κB, JNK, p38, and ERK1/2 were downregulated by phototherapy. Both the JNK and the ERK inhibitors potentiated the phototherapy effect on IL-8 as well as on cAMP secretion from BEAS. On the contrary, IL-10 and Sp1 were upregulated by phototherapy. The mithramycin blocked the phototherapy effect on IL-10. The results suggest that phototherapy has a dual effect on BEAS cells because it downregulates the IL-8 secretion by interfering with CSE-mediated signaling pathways, and oppositely upregulates the IL-10 secretion through of Sp1 transcription factor. The manuscript provides evidence that the phototherapy can interfere with MAPK signaling via cAMP in order to attenuate the IL-8 secretion from CSE-stimulated BEAS. In addition, the present study showed that phototherapy effect is driven to downregulation of the both the IL-8 and the ROS secretion and at the same time the upregulation of IL-10 secretion. Besides it, the increase of Sp-1 transcription factor was crucial for laser effect in upregulating the IL-10 secretion. The dexamethasone corticoid produces a significant inhibitory effect on IL-8 as well as ROS secretion, but on the other hand, the corticoid blocked the IL-10 secretion. Taking it into consideration, it is reasonable to suggest that the beneficial effect of laser therapy on lung diseases involves its action on unbalance between pro-inflammatory and anti-inflammatory mediators secreted by human bronchial epithelial cells through different signaling pathway.

Effects of airway surface liquid height on the kinetics of extracellular nucleotides in airway epithelia.

Experimental techniques aimed at measuring the concentration of signaling molecules in the airway surface liquid (ASL) often require an unrealistically large ASL volume to facilitate sampling. This experimental limitation, prompted by the difficulty of pipetting liquid from a very shallow layer (~15 µm), leads to dilution and the under-prediction of physiologic concentrations of signaling molecules that are vital to the regulation of mucociliary clearance. Here, we use a computational model to describe the effect of liquid height on the kinetics of extracellular nucleotides in the airway surface liquid coating respiratory epithelia. The model consists of a reaction-diffusion equation with boundary conditions that represent the enzymatic reactions occurring on the epithelial surface. The simulations reproduce successfully the kinetics of extracellular ATP following hypotonic challenge for ASL volumes ranging from 25 µl to 500 µl in a 12-mm diameter cell culture. The model reveals that [ATP] and [ADO] reach 1200 nM and 2200 nM at the epithelial surface, respectively, while their volumetric averages remain less than 200 nM at all times in experiments with a large ASL volume (500 µl). These findings imply that activation of P2Y2 and A2B receptors is robust after hypotonic challenge, in contrast to what could be concluded based on experimental measurements of volumetric concentrations in large ASL volumes. Finally, given the central role that ATP and ADO play in regulating mucociliary clearance, we investigated which enzymes, when inhibited, provide the greatest increase in ATP and ADO concentrations. Our findings suggest that inhibition of NTPDase1/highTNAP would cause the greatest increase in [ATP] after hypotonic challenge, while inhibition of the transporter CNT3 would provide the greatest increase in [ADO].