DrJorge Bernardino de la Serna

Keywords to MOLECULAR MECHANISMS in INHALATION TOXICOLOGY, PHYSIOPATHOLOGY AND PHARMACOLOGY.
Inhalation Toxicology, Inhalation Pharmacology, Aerosols, Engineered Nanoparticles, Nanomedicine, Toxic Particle and Pathogen-Host interactions, Lung Molecular and Cell Biology, Cell Membrane Biophysics, 4D quantitative Super-resolution Microscopy, Lung-on-a-chip, Immunophysics and Immunoengineering.

ALVEOLAR CAPILLARY BARRIER (ACB) PHYSIOPATHOLOGY

The integrity of the pulmonary respiratory alveolar gas-blood barrier is crucial for effective gas exchange and good health, effective “filtering” of undesirable components, responding to inhaled hazards and developing tolerance mechanisms to attenuate immunopathology. The alveoli are continuously exposed to inhaled micro and nanosized pathogens, which are typically rapidly eliminated with the help of the immune system. Immune responses in the alveoli must be tightly regulated to prevent excessive inflammation and tissue damage. Compromised tissues facilitate immune cell infiltration and extravasation to and from alveolar spaces, generating inflammatory responses. Inappropriate or excessive immune responses cause the development of systemic airway inflammation.

The risk of adverse respiratory effects induced by inhaling nanoparticles (pharmacological, hazardous, or microbial) in the alveoli depends on the exposure lifetime and concentration, where they distribute and accumulate, how they interact (chemistry), and how the immune system clears them.

The alveolar-capillary barrier comprises the alveolar epithelium (AE), interstitial layer and alveolar vascular endothelium (AVE). These juxtaposed layers need to form a leak-proof barrier, where proteins forming cell-cell contacts play a critical role. AE comprises alveolar type 1 and type 2 cells (AT1 and AT2, respectively) and resident alveolar macrophages. During early lung inflammation, elevated levels of immune cells induce cytokine release, which influences the integrity of claudins, the main proteins in the tight junctions of AT1 and AT2 cells. If cell-cell junctions are compromised, AE may develop loose intercellular contacts, increasing permeability, tissue injury and inflammation.

ACB MICROPHYSIOLOGICAL (Organ-on-a-chip) MODELS

In December 2022, in a significant development, the U.S. Food and Drug Administration (FDA) ruled that new medicines no longer require animal testing. This decision followed successful trials of drugs using organoids and organ-on-a-chip technologies (OOC). Microphysiological models, such as OOC and new alternative methods (NAMs), will be pivotal in mechanistic biomedicine and pharmacology research by 2030. This technology will be integral to diagnostic and treatment workflows in clinics, determining the appropriate personalised drug dosage and identifying adverse outcome pathways.

It is not known how the structure and function of the fragile alveolar-capillary barrier and crosstalk between the juxtaposed tissue layers confer the essential integrity and selective permeability to protect us when they are severely immuno-compromised. Our understanding is hampered by the lack of in vitro models and techniques that measure real-time interactions at the alveolar-capillary barrier parallel with physiological and molecular processes to provide holistic structural-functional evidence.

We are developing novel advanced in vitro models that can be used for toxicological and pharmacological testing. We employ cells cultured at the air-liquid interface mimicking the epithelial airways, including lung surfactant and under physiological respiratory mechanics. These models are challenged with nanoparticles to unravel the mechanisms of lung surfactant interaction and cellular uptake. These approaches will help further understand inhaled toxicological nanoparticles' role in compromising our breathing and initiating lung inflammatory processes and airway diseases. The acquired knowledge will be applied in designing novel and better pharmacological drugs.

ACB SENSING AND COMMUNICATION DURING INHALED EXPOSURES

We aim to resolve the molecular basis for cellular communication during environmental exposures and reveal immunologically relevant processes at the alveolar-capillary barrier, such as acute and chronic inflammation and alveolar injuries. We are interested in resolving at the molecular level how airway cell membranes sense and remodel upon exposure. We aim to understand better how plasma and intracellular membranes communicate with their micro/nano-environment, including foreign bio-functional materials, nanoparticles, and micro/nano-organisms. We want to learn how they perceive stimuli and decode signals and how they encode their responses and trafficking mechanisms. We want to know how they harbour supramolecular assemblies and favour particular lipid-protein spatiotemporal arrangements.

We focus on lipid-protein spatiotemporal interactions, distribution, and dynamical functional architectures at the micro- and nano-scale in specialised alveolar cells. To this end, we develop methods and implement applications to quantify the oligomerisation and spatiotemporal dynamics of membrane receptor-cholesterol interactions upon activation, focusing on G protein-coupled receptors (GPCRs). Specifically, we are targeting two GPCRs (GLP1R and S1PR) to resolve how these are activated and decipher the spatiotemporal dynamics and clustering processes that trigger an anti-inflammatory response in the alveolar epithelium. We know cholesterol can be an allosteric modulator in these events, acting from supramolecular cholesterol-rich platforms (often called rafts).

To this end, we implement and adopt innovative techniques such as raster imaging correlation spectroscopy (RICS) and Number and Brightness (N&B) to determine how GPCRs and cholesterol functionally interplay at micro/nanodomains during the activation-triggered process using endogenous ligand and agonist/antagonist binding regulation. Moreover, we apply highly advanced quantitative imaging techniques based on imaging correlation spectroscopy (ICS) to identify molecular localisation, distribution, dynamics, and oligomerisation states during cholesterol-receptor interactions. We use snap-tag ligands and fluorescent proteins to fluorescently report receptors and solvatochromic lipid dyes or bioorthogonal-based cholesterol probes to provide unique insights into membrane lipid lateral packing, cholesterol dynamics and its interaction with receptors.