DNA Methylation Profiles of Immune Cells From Tuberculosis-Exposed Individuals Overlap With BCG-Induced Epigenetic Changes

The mechanism of protection of the only approved tuberculosis (TB) vaccine, Bacillus Calmette Guérin (BCG) is poorly understood. In recent years, epigenetic modifications induced by BCG have been demonstrated to reflect a state of trained immunity. The concept of trained immunity is now explored as a potential prevention strategy for a variety of infections. Studies on human TB immunity are dominated by those using peripheral blood as surrogate markers for immunity. Here, we instead studied the lung compartment by obtaining induced sputum from subjects included in a TB contact tracing. CD3- and HLA-DR-positive cells were isolated from the collected sputum and DNA methylome analyses performed. Unsupervised cluster analysis revealed that DNA methylomes of cells from TB-exposed individuals and controls appeared as separate clusters, and the numerous genes that were differentially methylated were functionally connected. The enriched pathways were strongly correlated to previously reported epigenetic changes and trained immunity in immune cells exposed to the BCG vaccine in human and animal studies. We further demonstrated that similar pathways were epigenetically modified in human macrophages trained with BCG in vitro. Altogether, our study demonstrates that similar epigenetic changes are induced by M. tuberculosis and BCG.

DNA methylation profiling of immune cells from tuberculosis-exposed individuals overlaps with BCG-induced epigenetic changes and correlates with the emergence of anti-mycobacterial ‘corralling cells’

The mechanism of protection of the only approved tuberculosis (TB) vaccine, Bacillus Calmette Guérin (BCG) is poorly understood. In recent years, epigenetic modifications induced by BCG have been demonstrated to reflect a state of trained immunity. The concept of trained immunity is now explored as a potential prevention strategy for a variety of infections. Studies on human TB immunity are dominated by those using peripheral blood as surrogate markers for immunity. Here, we instead studied the lung compartment by obtaining induced sputum from subjects included in a TB contact tracing. CD3- and HLA-DR-positive cells were isolated from the collected sputum and DNA methylome analyses performed. Unsupervised cluster analysis revealed that DNA methylomes of cells from TB-exposed individuals and controls appeared as separate clusters, and the numerous genes that were differentially methylated were functionally connected. The enriched pathways were strongly correlated to previously reported epigenetic changes and trained immunity in immune cells exposed to the BCG vaccine in human and animal studies. We further demonstrated that similar pathways were epigenetically modified in human macrophages trained with BCG in vitro. Finally, we found evidence of an M. tuberculosis-triggered emergence of a non-macrophage cell population from BCG-trained macrophage cultures. These cells did not phagocytose M. tuberculosis, but ‘corralled’ the bacteria into focal points, resulting in limitation of bacterial growth. Altogether, our study demonstrates that similar epigenetic changes are induced by M. tuberculosis and BCG and suggests that the modifications promote transformation of macrophages (or an unknown progenitor) to establish a yet undescribed cellular defense mechanism which we term ‘corralling’, based on the metaphorical resemblance to sheepdog herding.

A DNA methylome biosignature in alveolar macrophages from TB-exposed individuals predicts exposure to mycobacteria

Several studies have identified biomarkers for tuberculosis (TB) diagnosis based on blood cell transcriptomics. Here, we instead studied epigenomics in the lung compartment by obtaining induced sputum from subjects included in a TB contact tracing. CD3- and HLA-DR-positive cells were isolated from the collected sputum and DNA methylome analyses performed. Unsupervised cluster analysis revealed that DNA methylomes of cells from TB-exposed individuals and controls appeared as separate clusters and the numerous genes that were differentially methylated were functionally connected. The enriched pathways were strongly correlated to data from published work on protective heterologous immunity to TB. Taken together, our results demonstrate that epigenetic changes related to trained immunity occurs in the pulmonary immune cells of TB-exposed individuals and that a DNA methylation signature can be derived from the DNA methylome. Such a signature can be further developed for clinical use as a marker of TB exposure.

Lung retention and particokinetics of silver and gold nanoparticles in rats following subacute inhalation co-exposure

Background Inhalation exposure to nanomaterials in workplaces can include a mixture of multiple nanoparticles. Such ambient nanoparticles can be of high dissolution or low dissolution in vivo and we wished to determine whether co-exposure to particles with different dissolution rates affects their biokinetics. Methods and Results Rats were exposed to biosoluble silver nanoparticles (AgNPs, 10.86 nm) and to biopersistent gold nanoparticles (AuNPs, 10.82 nm) for 28 days (6-h/day, 5-days/week for 4 weeks) either with separate NP inhalation exposures or with combined co-exposure. The separate NPs mass concentrations estimated by the differential mobility analyzer system (DMAS) were determined to be 17.68 ± 1.69 μg/m3 for AuNP and 10.12 ± 0.71 μg/m3 for AgNP. In addition, mass concentrations analyzed by atomic absorption spectrometer (AAS) via filter sampling were for AuNP 19.34 ± 2.55 μg/m3 and AgNP 17.38 ± 1.88 μg/m3 for separate exposure and AuNP 8.20 ± 1.05 μg/m3 and AgNP 8.99 ± 1.77 μg/m3 for co-exposure. Lung retention and clearance were determined on day 1 (6-h) of exposure (E-1) and on post-exposure days 1, 7, and 28 (PEO-1, PEO-7, and PEO-28, respectively). While the AgNP and AuNP deposition rates were determined to be similar due to the similarity of NP size of both aerosols, the retention half-times and clearance rates differed due to the difference in dissolution rates. Thus, when comparing the lung burdens following separate exposures, the AgNP retention was 10 times less than the AuNP retention at 6-h (E-1), and 69, 89, and 121 times lower less than the AuNP retention at PEO-1, PEO-7, and PEO-28, respectively. In the case of AuNP+AgNP co-exposure, the retained AgNP lung burden was 14 times less than the retained AuNP lung burden at E-1, and 26, 43, and 55 times less than the retained AuNP lung burden at PEO-1, PEO-7, and PEO-28, respectively. The retention of AuNP was not affected by the presence of AgNP, but AgNP retention was influenced in the presence of AuNP starting at 24 h after the first day of post day of exposure. The clearance of AgNPs of the separate exposure showed 2 phases; fast (T1/2 3.1 days) and slow (T1/2 48.5 days), while the clearance of AuNPs only showed one phase (T1/2 .81.5 days). For the co-exposure of AuNPs+AgNPs, the clearance of AgNPs also showed 2 phases; fast (T1/2 2.2 days) and slow (T1/2 28.4 days), while the clearance of AuNPs consistently showed one phase (T1/2 54.2 days). The percentage of Ag lung burden in the fast and slow clearing lung compartment was different between separate and combined exposure. For the combined exposure, the slow and fast compartments were each 50% of the lung burden. For the single exposure, 1/3 of the lung burden was cleared by the fast rate and 2/3 of the lung burden by the slow rate. Conclusions The clearance of AgNPs follows a two- phase model of fast and slow dissolution rates while the clearance of AuNPs could be described by a one- phase model with a longer half-time. The co-exposure of AuNPs+AgNPs showed that the clearance of AgNPs was altered by the presence of AuNPs perhaps due to some interaction between AgNP and AuNP affecting dissolution and/or mechanical clearance of AgNP in vivo.

SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species

SummaryThere are no known cures or vaccines for COVID-19, the defining pandemic of this era. Animal models are essential to fast track new interventions and nonhuman primate (NHP) models of other infectious diseases have proven extremely valuable. Here we compare SARS-CoV-2 infection in three species of experimentally infected NHPs (rhesus macaques, baboons, and marmosets). During the first 3 days, macaques developed clinical signatures of viral infection and systemic inflammation, coupled with early evidence of viral replication and mild-to-moderate interstitial and alveolar pneumonitis, as well as extra-pulmonary pathologies. Cone-beam CT scans showed evidence of moderate pneumonia, which progressed over 3 days. Longitudinal studies showed that while both young and old macaques developed early signs of COVID-19, both groups recovered within a two-week period. Recovery was characterized by low-levels of viral persistence in the lung, suggesting mechanisms by which individuals with compromised immune systems may be susceptible to prolonged and progressive COVID-19. The lung compartment contained a complex early inflammatory milieu with an influx of innate and adaptive immune cells, particularly interstitial macrophages, neutrophils and plasmacytoid dendritic cells, and a prominent Type I-interferon response. While macaques developed moderate disease, baboons exhibited prolonged shedding of virus and extensive pathology following infection; and marmosets demonstrated a milder form of infection. These results showcase in critical detail, the robust early cellular immune responses to SARS-CoV-2 infection, which are not sterilizing and likely impact development of antibody responses. Thus, various NHP genera recapitulate heterogeneous progression of COVID-19. Rhesus macaques and baboons develop different, quantifiable disease attributes making them immediately available essential models to test new vaccines and therapies.

An Experimental Study on the Establishment of Pulmonary Hypertension Model in Rats induced by Monocrotaline

Pulmonary hypertension is called PH for short. It is caused by the pulmonary artery vascular disease leading to pulmonary vascular resistance, and the increase right lung compartment load, which resulting in weakening or even collapse of the right ventricular function. The establishment of rat PH model under the action of monocrotaline is a repeatable, simple and accessible operation technique, which has been widely used in the treatment of pulmonary hypertension. This paper discusses the principle and properties of the PH model on rats under the monocrotaline action.

Epithelial Alarmins in Serum and Exhaled Breath in Patients with Idiopathic Pulmonary Fibrosis: A Prospective One-Year Follow-Up Cohort Study

Background: Recently, epithelial alarmins have been shown to play important roles in non-allergen driven respiratory diseases like idiopathic pulmonary fibrosis (IPF). Little is known about the expression of the epithelial alarmins in IPF. Methods: This study aimed to prospectively examine interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP) levels in the serum and exhaled breath condensate (EBC) in patients with IPF before and after one-year of antifibrotic treatment. A total of 82 volunteers, including 52 patients diagnosed with IPF that qualified for antifibrotic therapy as well as 30 controls, were examined. All study participants underwent baseline peripheral blood and EBC sampling. In 35 out of 52 IPF subjects, a follow-up sampling was performed after 12 months of antifibrotic treatment. Concentrations of alarmins in the serum and EBC were evaluated by means of ELISA. Results: Baseline TSLP concentrations were significantly elevated in patients with IPF compared to controls both in the serum (p < 0.05) and EBC (p < 0.0001). Baseline IL-25 and IL-33 serum and EBC levels did not differ significantly between IPF subjects and controls. Prospective analysis of changes in the epithelial alarmin levels showed significantly decreased IL-25 and TSLP EBC concentrations after 12 months of antifibrotic treatment (p < 0.05), which was observed in the subgroup of IPF patients treated with pirfenidone, but not in those treated with nintedanib. In stable patients with IPF over a study period (absolute forced vital capacity (FVC) % of predicted decline/year ≤ 5%, n = 25), a significant decrease in the EBC levels of both IL-25 and TSLP after 12 months of antifibrotic treatment was noted (p < 0.05), whereas, in progressor IPF patients (absolute FVC % of predicted decline/year > 5%, n = 10), a significant decrease was noted in the IL-25 EBC levels only (p < 0.05). Conclusions: Elevated TSLP levels in patients with IPF and their significant decrease in the lung compartment during antifibrotic therapy in stable patients with IPF, but not in progressors, support its significant contribution to pro-fibrotic type 2 immune responses in IPF. Noted changes in the epithelial alarmins concentration in the lung compartment during pirfenidone therapy may suggest its possible interaction with epithelial alarmins pathways in IPF.

Intrapulmonary Pharmacokinetics of Relebactam, a Novel β-Lactamase Inhibitor, Dosed in Combination with Imipenem-Cilastatin in Healthy Subjects

ABSTRACT This phase I study assessed the intrapulmonary pharmacokinetic profiles of relebactam (MK-7655), a novel β-lactamase inhibitor, and imipenem. Sixteen healthy subjects received 250 mg relebactam with 500 mg imipenem-cilastatin, given intravenously every 6 h for 5 doses, and were randomized to bronchoscopy/bronchoalveolar lavage at 0.5, 1, 1.5, or 3 h after the last dose (4 subjects per time point). Both drugs penetrated the epithelial lining fluid (ELF) to a similar degree, with the profiles being similar in shape to the corresponding plasma profiles and with the apparent terminal half-lives in plasma and ELF being 1.2 and 1.3 h, respectively, for relebactam and 1.0 h in both compartments for imipenem. The exposure (area under the concentration-time curve from time zero to infinity) in ELF relative to that in plasma was 54% for relebactam and 55% for imipenem, after adjusting for protein binding. ELF penetration for relebactam was further analyzed by fitting the data to a two-compartment pharmacokinetic model to capture its behavior in plasma, with a partitioning coefficient capturing its behavior in the lung compartment. In this model, the time-invariant partition coefficient for relebactam was found to be 55%, based on free drug levels. These results support the clinical evaluation of relebactam with imipenem-cilastatin for the treatment of bacterial pneumonia.

Leptin Matures Aspects of Lung Structure and Function in the Ovine Fetus

In human and ovine fetuses, glucocorticoids stimulate leptin secretion, although the extent to which leptin mediates the maturational effects of glucocorticoids on pulmonary development is unclear. This study investigated the effects of leptin administration on indices of lung structure and function before birth. Chronically catheterized singleton sheep fetuses were infused iv for 5 days with either saline or recombinant ovine leptin (0.5 mg/kg · d leptin (LEP), 0.5 LEP or 1.0 mg/kg · d, 1.0 LEP) from 125 days of gestation (term ∼145 d). Over the infusion, leptin administration increased plasma leptin, but not cortisol, concentrations. On the fifth day of infusion, 0.5 LEP reduced alveolar wall thickness and increased the volume at closing pressure of the pressure-volume deflation curve, interalveolar septal elastin content, secondary septal crest density, and the mRNA abundance of the leptin receptor (Ob-R) and surfactant protein (SP) B. Neither treatment influenced static lung compliance, maximal lung volume at 40 cmH2O, lung compartment volumes, alveolar surface area, pulmonary glycogen, protein content of the long form signaling Ob-Rb or phosphorylated signal transducers and activators of transcription-3, or mRNA levels of SP-A, C, or D, elastin, vascular endothelial growth factor-A, the vascular endothelial growth factor receptor 2, angiotensin-converting enzyme, peroxisome proliferator-activated receptor γ, or parathyroid hormone-related peptide. Leptin administration in the ovine fetus during late gestation promotes aspects of lung maturation, including up-regulation of SP-B.