Paper - Review

DOI: 10.1038/s41577-020-00477-9

Abstract

The community of cells
← that lining our airways
→ 1⃣ play (a collaborative role) ← in the preservation (← of immune homeostasis) 2⃣ provides protection ← from (1⃣ the pathogens 2⃣ pollutants)

The airway epithelium
→ is an (immunologically active barrier surface)
← that 1⃣ senses changes ← in (the airway environment) 2⃣ interacts ← with (resident & recruited immune cells)

Single-cell RNA-sequencing
→ is illuminating → the cellular heterogeneity
→ 1⃣ exists ← in the airway wall 2⃣ has identified → novel cell populations ← with (1⃣ unique molecular signatures 2⃣ trajectories of differentiation 3⃣ diverse functions)

❗: how (our view) ←of the airway epithelial landscape
→ has evolved
→ with the advent of transcriptomic approaches → to cellular phenotyping
→ with a focus → on epithelial interactions ← with 1⃣ the local neuronal 2⃣ immune systems

Introduction

The cell lining
← on the airways
→ constitute → more than just (a barrier)
← between 1⃣ the external environment 2⃣ the underlying mesenchyme

This collection of (specialized epithelial cells)
→ respond → to 1⃣ microbes 2⃣ noxious stimuli
← that overcome → the mucociliary barrier
This collection of (specialized epithelial cells)
→ are → a vital component (← of host defense)
→ 1⃣ interacting ← with (cells of immune system) → to maintain homeostasis 2⃣ facilitating (immune reaction) when necessary

The respiratory epithelium
→ manage responses
→ to the diverse toxins ← that contained within (the inhaled environment)

The epithelial layer
→ incorporates (basal cells)
← in (close proximity) → to 1⃣ secretory cells 2⃣ ciliated cells
→ forming (a tight unit) ← which maintains (a physical barrier)
The epithelial layer
→ is responsive → to (the inhaled environment)
← via (cells & molecules) ← from the immune system

❗: A dynamic cellular structure
→ encompass → (a wide range) ( ←of highly specialized cells)
← which are able → to respond to environmental changes
→ 1⃣ interact with (resident microbial communities) 2⃣ cooperate with (multiple other specialized cellular system)

The respiratory tract
→ is a complex organ system
← 1⃣ the upper respiratory tract 2⃣ the lower respiratory tract 3⃣ the respiratory zone

Specialized epithelial cell populations
→ line → the entire respiratory tract
→ from (the nasal cavity) to the alveoli

Standard immuno-histochemical staining
← for cell type-specific markers
→ has been used → to (characterize & quantify) → epithelial cell populations
← throughout (the human respiratory tract)
∴ Determining → the influence of (anatomical location) ← on (cellular composition)

The airways are lined
← by 1⃣ ciliated cells 2⃣ secretory cells
→ to facilitate mucociliary clearance of (particulate matter & infectious pathogens)

The airway epithelium
→ is an interface ←with the environment
∴ The airway epithelium → is important → to host defense

Immunological activity
→ is necessarily (distinct & shaped)
← by differences ← in 1⃣ environmental conditions 2⃣ resident microbial communities 3⃣ airborne antigens

The epithelial cells
← which lining (the distal alveolar region) ← of the lung
→ are (1⃣ phenotypically 2⃣ functionally) distinct

The advent of (novel sequencing techniques)
→ facilitated → the identification of (novel cell types)
→ reveals (potential functions)

The airway wall
→ represents → a dynamic community (← of epithelial cells)
← which existing in (close association) ← with 1⃣ resident immune cells 2⃣ neuronal cells
∴ 1⃣ maintaining → (mucosal immune homeostasis) 2⃣ facilitating (host defense) ← against inhaled pathogens

Mapping the airway epithelial landscape

scRNA-seq
← single-cell RNA-sequencing
→ un-veiling → a level of (cellular diversity)
← which had not been documented ← using microscopy for phenotyping

scRNA-seq
→ profiles → the transcriptome (← of individual cells)
∴ scRNA-seq → facilitates → an unbiased characterization of (different cell subsets)
← in a hetero-geneous cell population
∴ scRNA-seq → driving → the discovery of un-identified (cell types & states)

❗: computationally interrogate
→ epithelial-immune interactions
← using known ligand-receptor interactions

❗: studying (human epithelial biology)
← using single-cell transciptomics
→ relies ← on the availability of (lung tissue)

Earliest studies → analyzed →
1⃣ Diseased explant parenchymal lung tissue
← from patients ← with IPF (← idiopathic pulmonary fibrosis)
2⃣ Lung lobes
← from donors ← un-suitable for transplant → as control tissue

The acquisition
← of 1⃣ bronchial brushings 2⃣ endobronchial biopsy
→ can be achieved through fibreoptic bronchoscopies
→ carried out ← in the context of routine clinical care

❗: the cellular content
→ differs by the sampling methods used
∴ This will influence → the cell cluster
← obtained by scRNA-seq

Healthy control samples → is more challenging
∵ its necessitates → research volunteers
← undergoing → an invasive procedure

Human lung tissue → is only feasible
→ through close collaboration ← between clinicians 🆚 scientists
→ through (human tissue biobanks)

Logistical practicalities
→ require careful consideration
∵ 1⃣ the hypothermic preservation of tissue 2⃣ the minimization of time delays ← between (fresh sample collection)
→ are critical → to ensuring high-quality scRNA-seq data

A cold ischaemic time of 72 hour
→ result in → an increase in (the presence of mitochondrial reads)
∵ 1⃣ cellular stress & cellular death ← in multiple lung cell types 2⃣ a cell-specific decrease ← in the proportion of (CD4+ & CD8+) cytotoxic T-cells

The enzymatic dissociation (← of tissue)
← with collagenase
→ is widely used → to isolate cells → for scRNA-seq studies

❗: the enzymatic dissociation
→ can induce → the expression of (immediate early genes)
∴ Potentially introducing → (1⃣ an important artifact 2⃣ potential bias)
← in single-cell data sets

Cell
← sharing similar (gene expression profiles)
→ are clustered together
→ can be assigned (an annotation) ← based on known marker genes

A new cluster (← of cells)
→ was identified → that did NOT ❌ map → to any of (the previously known epithelial cell types)
→ based on (its gene expression signature) → "pulmonary ionocyte:

Sub-clustering→ can be used
→ to identify (diverse cell state)
← within a specific cell-type cluster

A single-cell analysis ← of (human nasal epithelial cells)
→ differentiated ← in air-liquid interface cultures
→ demonstrated → a precursor sub-set of FOXJ1-expressing multi-ciliated cells
∴ "deutrosomal cells"

All
← 1⃣ DEUP1 2⃣ FOXN4 3⃣ CDC20B
→ are critical → for centriole amplification & ciliogenesis

Goblet & ciliated cells
← denoted by differential marker gene expression
→ have been described ← in the human airway

Goblet cells
→ could be divided → into two subsets
← according to their uniquely expressed genes

The anatomical origin (← of the cell population)
← within the respiratory tract
→ plays → an important role
← in governing 1⃣ its transcriptional signature 2⃣ its cell state

Spatial variation
← in gene expression profile ← between cells of the same type
→ relate → to the biological function
← required at a specific airway location

Secretory cells
← in the nasal epithelium
→ are enriched → in gene sets
← associated ← with 1⃣ cell motility 2⃣ differentiation 3⃣ sensory perception pathways
Cells ← in the bronchial tree
→ are enriched ← in genes related
→ to 1⃣ innate immunity 2⃣ wound healing response pathways

❗: inconsistence annotation of (secretory cell types)
∵ overlapping (gene expression) signatures

Determinant of (airway epithelial cells state)
→ is revealed
← by the comparison of cells

An analysis of (airway wall biopsies)
← from mild to moderate asthmatics
→ exposed several cell states ← that not found in the healthy airway

Single-cell analysis (← of parenchymal lung tissue)
← from patient of IPF (← a progressive scarring lung disease)
→ have reported → a previously 1⃣ un-identified 2⃣ transcriptionally distinct (cluster of cells)
← in the fibrotic distal lung

These intriguing "aberrant basaloid" cells
→ are co-express 1⃣ genes ← encoding mesenchymal markers 2⃣ genes ← linked to IPF pathogenesis

The pulmonary epithelium
→ represents → a dynamic cellular community

Single-cell transcriptomics
→ captures → a unique snapshot of cells ← at different stages
← in the continuous process of differentiation

A feature
→ was first harnessed
→ to elucidate (epithelial lineage hierarchies)
← at 4 distinct stages of alveolar development ← in mice

The refinement of scRNA-seq bioinformatic approaches
→ has been possible
→ 1⃣ to study cell fate ← in human sample 2⃣ to unveil ← the subtleties of (human airway epithelial cell differentiation)

The transcriptional changes
← that accompany → the differentiation of each cell
→ can be exploited computationally
← by trajectory inference algorithms
← that order cells → according to progress → along their individual trajectories
∴ pseudo-time

A pseduo-time analysis
→ demonstrated
→ 1⃣ goblet cell → could act ← as pre-cursors → to ciliated cells 2⃣ transitory (1⃣ FOXJ1+ 2⃣ MUC5AC+) cells → could be found ← in the context of this differentiation trajectory

Pseudo-temporal ordering of cells
← using a diffusion map approach
→ identified → an intriguing population of (Krt13 expressing cells)
← as an intermediary ← in basal → to club cell differentitaion

This transitional cell
→ was arranged ← in 1⃣ discrete 2⃣ stratified 3⃣ high-turnover structures
→ expressed genes ← associated with 1⃣ squamous epithelial differentiation 2⃣ cell adhesion 3⃣ immuno-modulation

The comprehensive transcriptomic information
← charted at a cellular level ← by scRNA-seq
→ has heightened → our knowledge of the air way epithelial landscape

Genes
← associated with SARS-CoV-2 cell entry
→ were significantly enriched
← in 1⃣ nasal secretory 2⃣ ciliated epithelial cells

Function of rare epithelial cell types

Basal cells
→ are multi-potent stem cell
→ 1⃣ account for a third of all (airway epithelial cells) 2⃣ give rise → to other major sub-populations
← during 1⃣ homeostatic maintenance of the epithelial barrier 2⃣ regeneration in response to injury

Basal cells
→ are anchored → to the basement membrane ← of the conducting airways
→ express 1⃣ KRT5 (← keratin 5) 2⃣ TP63 (← transcription factor tumor protein p63)

1⃣ Lineage tracing 2⃣ Single-cell transcriptomic studies
→ have demonstrated →
→ functionally distinct (basal cell subset) → exist in the airway

These cell states
→ reflect → distinct differentiation stages
← in basal cells

Hillock cells

The advent (← of scRNA-seq)
→ has enabled investigators
→ to examine → cell development trajectories

Montoro et al.
→ defined → a distinct trajectory
← which links ← tracheal basal 🆚 club cells
← via a novel transitional cell ← that uniquely expresses (Krt13 & Krt4)

Krt13+ cells
→ lie ← within contiguous groups of (stratified cells)
← that do not ❌ contain (luminal ciliated cells)

"Hillocks"
→ a structure composed of (luminal Scgb1a1+ Krt13+ cells)
← lying on top of basal cells

Hillocks
→ contain a particularly high number of (cycling cells)
→ expressed markers of (1⃣ cellular adhesion 2⃣ epithelial differentiation 3⃣ barrier function 4⃣ immuno-modulation)

The presence of (a distinct population of Krt13+ Krt4+ cells)
← 1⃣ in the murine trachea 2⃣ in ex vivo differentiated (human basal epithelial cells)
∴ An intermediate population
← between 1⃣ basal stem cells 2⃣ differentiated luminal secretory cells

KRT13
→ is expressed ← in the airway epithelium
← by a sub-population of "cycling" basal cells
← by the expression of genes ← associated with cell proliferation

❓: Hillocks → represent → a truly distinct niche
← rather than ← a metaplastic zone in transition
❓: Determine → 1⃣ their origin 2⃣ their purpose

Tuft cells

The respiratory tract
→ contains → several groups of chemosensory epithelial cells
← that coordinate interactions ← with the external environment

These chemosensory epithelial cells
→ share similarities ← with taste cells
→ are predicted → to evoke (both positive & negative) responses
← from 1⃣ immune cell 2⃣ neuronal cells

These chemosensory epithelial cells
→ have a distinctive morphology
→ are expressed ← in a range of organs

The role of these chemosensory epithelial cells ← in the lung
→ is less certain
→ have been detected ← in 1⃣ the nose 2⃣ trachea 3⃣ proximal airways
→ exists in close contact ← with 1⃣ nerve fibres 2⃣ mediating communication ← between (neuronal pathways 🆚 immune pathways)

Braga et al.
→ could not identify → a unique population of (tuft cell)
← using 1⃣ bronchial brushing 2⃣ endo-broncial biopsy

Tuft cells
→ express → a range of indicative markers
→ e.g. 1⃣ POU2F3 2⃣ TRPM5

scRNA-seq
→ has identified → two terminally differentiated TRpm5+ tuft cells populations
∴ it contributes → to leukotriene synthesis

Tuft cells → are thought
→ to generte cysteinyl leukotrienes
← via the ATP sensor P2Y2

Tuft cell are able
→ to respond to a diverse range of signals
← via the combinatorial expression ← of different receptors

Tuft cells are thought
→ to promote protective respiratory reflexes
→ can also contribute to 1⃣ apnoea 2⃣ local neurogenic inflammation of mucosa

Solitary chemo-sensory cells
→ enhancing → the chemo-responsive protection
← provided by the local neuronal system

Mouse tuft cell
→ express → high level of 1⃣ cholinergic 2⃣ bitter taste signaling transcripts
∵ scRNA-seq analysis

Acetylcholine
→ is released ← from tracheal tuft cell
→ following stimulation with 1⃣ TAS2R ligand denatonium 2⃣ Pseudomonas quinolone quorum-sensing molecules

The detection (← of these quorum-sensing molecules)
← from Gram-negative pathogenic bacteria
→ offers a mechanism
← by which 1⃣ the epithelium triggers capsaicin-sensitive nerve fibers ← releasing 1⃣ CGRP 2⃣ substance P
← which 1⃣ promote innate cascadeds & microvascular leak 2⃣ combat bacterial invasion
∴ Limiting → the population densities of (forming destructive bioflims)

Solitary chemo-sensory cells
→ are found → to be the primary epithelial source
→ facilitating → 1⃣ the activation of group 2 ILC2s 2⃣ the production of IL-13
∴ Solitary chemo-sensory cells → are maintaining → the type 2 environment
← in the upper airways

Ionocyte

Single-cell transcriptomic studies
← of 1⃣ the mouse tracheal epithelium 2⃣ differentiated human tracheal epithelial cells
→ discovered → a novel cluster of cells → "pulmonary ionocytes"
∵ the similarity of their gene expression profiles

Ionocytes
→ express gene
← encoding sub-units of a V-ATPase proton pump
← which regulates 1⃣ ion transport 2⃣ pH

The ionocyte lineage
← in 1⃣ mouse 2⃣ human epithelial cells
→ is "FOX11"
← which belongs → to the forkhead family of (transcription factors)

FOX11
→ regulates the expression of the CFTR gene
→ that encodes → a critical chloride-ion transporter
→ that is (defective & absent) ← cystic fibrosis
∵ Knockout studies

Cystic fibrosis
→ is a life-limiting disease
← characterized by 1⃣ increased mucus viscosity 2⃣ impaired mucociliary clearance 3⃣ chronic infection 4⃣ airway inflammation
∴ Cystic fibrosis leads → to loss of lung function

Basal cells
→ were the principal source (← of 1⃣ ionocytes 2⃣ other rare cell types)
∵ Pulse-seq
← Combining (1⃣ scRNA-seq 2⃣ in vivo lineage tracing)
← over three different time points

Pulmonary neuroendocrine cells

PNECs
→ are solitary cells
← resident within the surface epithelium
← of 1⃣ the trachea 2⃣ bronchi 3⃣ bronchioles

PNECs
→ can also exist
← in neuro-endocrine bodys
← in the intra-pulmonary airways

PNECs function
← as chemo-sensors of the airway
→ respond to change in 1⃣ oxygen 2⃣ stretch 3⃣ chemical stimuli

PNECs
→ are rich source of 1⃣ neuro-peptides 2⃣ neuro-transmitters
← that elicit 1⃣ immune effects 2⃣ physiological effects

PNECs
→ be the earliest (specialized cells type)
← that forms in the lung epithelium ← during development
PNECs
→ be particularly important functionally ← in early life
∵ Neonatal mice (← lacking PNECs) → are protected from allergic inflammation

In vitro analysis
→ predicted → roles for PNECs
→ e.g. 1⃣ oxygen sensing 2⃣ the maintenance of (bronchial & vascular smooth muscle tone) 3⃣ immune responses

A defining characteristic marker (← of PNECs)
→ is the gene "Ascl1"
→ is essential → for their formation

Mice
← that lack Ascl11
→ die at birth
A conditional knockout
← designed to inactive Ascl1 !← in PNEC precursours
→ led to viable mice (← as marked by a lack of CGRP+ cells ← in the airway epithelium)

These Ascl1-mutant mice
→ were normal at baseline
→ were protected ← from 1⃣ severe goblet cell hyperplasia 2⃣ type 2 inflammation
← when exposed to allergens ← during the perinatal period

The expression of the ROBO genes (← the roundabout genes)
→ is vital
→ for 1⃣ the clustering of PNECs → into neuroendocrine bodies 2⃣ limiting → immune cell infiltration 3⃣ preventing → alveolar simplification
← during postnatal lung development

The physiological effects
→ only occurred after birth
∴ The effects of PNEC
→ are dependent ← on the physical exposure to air
← that occurs with the first breath
∴ PNECs are sensor
← of the changing inhaled environment

PNECs
→ were shown → to facilitate (mucus hyper-secretion)
← during allergen exposure
← via 1⃣ neurotrophin 3 regulation of PNEC innervation 2⃣ the secretion of GABA

PNECs
→ represent the only source of GABA
← in 1⃣ primate lungs 2⃣ ex vivo cultures of (human epithelial cells)

GABA production
→ was found → to be absolutely required → for goblet cell hyperplasia
← in 1⃣ both neonatal 2⃣ adult models of allergic inflammation

Abnormalities (← in PNEC numbers)
→ are associated ← with a wide range of (pulmonary diseases)
→ e.g. 1⃣ congenital diaphragmatic hernia 2⃣ small cell lung cancer 3⃣ asthma
∴ A key role → for these cells
← in 1⃣ physiological pathways 2⃣ immune pathways
→ for effective functioning of the lung ← at homeostasis

Immune properties of airway epithelial cells

1⃣ Anti-microbila mediators 2⃣ Airway mucins
← e.g. 1⃣ MUC5AC 2⃣ MUC5B
→ are produced ← by 1⃣ secretory epithelial cells 2⃣ sub-mucosal glands
→ contribute → to the first layer of host defense
← at the airway epithelial surface

SIgA
← Secretory IgA
→ prevents → the adherence of (airborne micro-organisms)
∴ Immune exclusion

Epithelial cells
→ are equipped
← with "pattern recognition receptors"
← e.g. 1⃣ Toll-like receptors 2⃣ cytokine receptors
→ allow them → to respond to signals (← produced by immune cell)

Adjacent airway epithelial cells
→ are linked
← by 1⃣ intra-cellular tight junctions 2⃣ adherens junctions
← 1⃣ regulate → the para-cellular diffusion of (ions & molecules) 2⃣ maintain barrier integrity

These (tight junctions)
→ separate ligands
← which present at the apical epithelial surface
∴ Activation occurs
→ following 1⃣ injury 2⃣ disruption ← of epithelial integrity

Sensing and clearing apoptotic cells

The airway
→ is an environment
← exposed to 1⃣ pollutants 2⃣ pathogens 3⃣ allergens
∴ Apoptosis

The clearances of (apoptotic cells)
→ is carried out
← by 1⃣ professional 2⃣ non-professional phagocytes

Apoptotic epithelial cells
← which labelled with a CypHer5 dye
→ were directly engulfed
→ through the recognition of phosphatidylserine

The inducible deletion of (the small GTPase RAC1)
→ resulted
← in 1⃣ defective apoptotic cell phagocytosis 2⃣ a significant reduction

∴ Airway epithelial cell-mediate phagocytosis (← of apoptotic cells)
← via RAC1 signaling
→ plays a key role
← in taming (the inflammatory response) → to common inhaled allergens

The recognition of (apoptosis cells) ← by airway basal cells
→ plays → a key role
← in determine 1⃣ cell phenotype 2⃣ cell fate
← in the context of inflammation

Basal cell hyperplasia
→ is (an early event) ← in pathogenesis (← of COPD)
← chronic obstructive pulmonary disease

Mouse tracheal basal cells
→ express the AXL receptor
← bound to GAS6

1⃣ Basal cell re-entry → into the cell cycle 2⃣ their proliferation
→ was promoted
← by the presence of AXL
← in a mouse model of (H1N1/PR8 influenza A virus infection)
← 1⃣ is characterized ← by airway infection 2⃣ is ciliated → epithelial cell apoptosis

∴ Basal cell proliferation
← during (airway inflammation)
→ is mediate → though the recognition of (apoptotic cells)
← by the AXL receptor tyrosine kinase

Inflammatory memory

Basal cells
→ 1⃣ are able → to sense & respond → to changes ← in the inflammatory micro-environment 2⃣ have → and intrinsic capacity → for inflammatory memory

CRS
← chronic rhino-sinusitis
→ is a type 2 immune-mediated disease
← characterized by 1⃣ inflammation dysfunction 2⃣ epithelial dysfunction
← 1⃣ with 2⃣ without → polyps

❗: the most striking disease-related changes
← in basal cell
→ 1⃣ differentiating & secretory cell 2⃣ glandular cell populations

Notable basal cell expansion
← at the expense of (epithelial cell diversity)
← in the polyp samples

Basal cells
→ up-regulated → 1⃣ IL-4-responsive 2⃣ IL-13-responsive genes
→ displayed → a failure → to differentiate

The up-regulation of (transcription factors)
← 1⃣ KLF5 2⃣ ATF3
→ to maintain → un-differentiated (cell states)
→ corresponding → to enriched motifs ← in sorted polyp basal cells
∵ 1⃣ Bulk RNA-sequencing 2⃣ ATAC-seq

∴ Intrinsic changes ← at an epigenetic level
→ may be responsible → for the differences observed ← in polyp basal cell state
→ may be governed ← by the local type 2 inflammatory milieu

1⃣ IL-4 2⃣ IL-13
→ induced more than 10 times
← in non-polyp basal cell > in polyp basal cell
∵ Bulk RNA-sequencing

CTNNB1
←Wnt pathway activator
→ was expressed ← in polyp basal cells
∴ A memory of (in vivo exposure)
→ to type 2 cytokines
← by polyp basal cells

Circadian rhythm

Neuro-hormonal control (← of the circadian rhythm)
← by (the suprachiasmatic nucleus)
← in 1⃣ the brain 2⃣ peripheral tissues 3⃣ their cells
→ possess → an intrinsic circadian clock
← responsible → for rhythmic immune oscillations

1⃣ CLOCK 2⃣ PER2
← the clock gene products
→ were expressed ← in CCSP+ club cells

Circadian oscillations
→ could be reset ← in response to gluco-corticoids
→ were lost ← with the selective ablation of (club cells)

A notable time-of-day variation ← in inflammatory response
← e.g. the BAL neutrophil count
→ was observed ← in response → to an aerosolized lipo-polysaccharide challenge

Driving → this pulmonary inflammatory response
→ was CXCL5
← the neutrophil chemo-attractant

REV-ERBα
→ is a nuclear receptor
→ that sits ← in (a negative feedback loop downstream) of (core transcriptional activators) of (the molecular clock)
← e.g. 1⃣ CLOCK 2⃣ BMAL1

REV-ERBα
→ plays → a key role
← in the circadian modulation of (innate immune response)
← in 1⃣ myeloid cells 2⃣ airway epithelial cells

This inflammatory response
→ was mediated
← through increased expression of CXCL5
← at 1⃣ the transcript 2⃣ the protein level

The dual deletion of (1⃣ REV-ERBα 2⃣ REV-ERBβ)
→ resulted in a further increase ← in (neutrophilic inflammation)

∴ REV-ERB proteins
→ play → a homeostatic anti-inflammatory function

Immune cell interactions at the epithelial surface

scRNA-seq
→ facilitates → the mapping of (1⃣ the diverse innate 2⃣ the adaptive immune cell communities)
← of the airway niche

Airway epithelial cells
→ act ← in concert with resident
→ recruited immune cells → to regulated pulmonary immunity

1⃣ Human sputum 2⃣ BAL
→ facilitate → the detailed characterization of (immune populations)
← within the airway
1⃣ Bronchial brushing samples 2⃣ Biopsies
→ capture → both 1⃣ the epithelial cell 2⃣ the immune cell components

∴ Computational advances
→ are leveraged → to unveil → the complex epithelial-immune crosstalk
← occurs in 1⃣ the airway space 2⃣ in alveolar space

Macrophages
→ are the most abundant immune cells
← which located in the airway lumen
→ are found readily ← in 1⃣ human sputum samples 2⃣ airway brushings 3⃣ BAL

Resident airway macrophages
→ are perinatally derived ← from fetal monocytes
→ are self-maintained locally throughout life
∵ Murine studies

Most tissue-resident airway macrophages
← in the donor lung
→ were replaced ← by airway macrophages
← from the circulating monocyte pool of the recipient

Dominant airway immune populations
← from resident airway macrophages
→ to recruited 1⃣ monocytes 2⃣ neutrophils
∴ 1⃣ Pro-inflammatory phenotypes 2⃣ Gene expression changes
→ suggestive of impaired phagocytosis
∵ A single-cell transcriptomic study

Bi-directional interactions
← between 1⃣ resident airway macrophages 2⃣ epithelial cells
→ ensure the maintenance of (a homeostatic state) of (immune tolerance)
→ to 1⃣ harmless stimuli 2⃣ appropriate protective responses

Macrophages
← which CD11c-expressing alveolar
→ formed direct connection ← with alveolar epithelial cells
← by connexin 43-containing gap junctions
→ to limit → the inflammatory response to LPS

❓: how dysregulated immuno-regulatory interactions
← at this environmental interface
→ contribute to human disease

BAL
→ was studied ← in human subjects (← with atopic asthma)
← before 🆚 after → sub-segmental broncho-provocation ← with an identified allergen
→ to explore → the epithelial-immune axis

CSF1
← colony-stimulating factor 1
→ was elevated ← in BAL samples
← after aero-allergen challenge
→ was shown → to be secreted
← by airway epithelial cells

Epithelial cell-derived CSF1
→ increased → alveolar dendritic cells
← expressing the CSFR1 receptor

Epithelial cell 🆚 Dendritic cells interactions
→ enhance 1⃣ antigen presentation 2⃣ augment adaptive allergic airway responses

A robust immune response
→ to respiratory pathogens
→ is also contingent → on an effective adaptive T-cell response

T-RM cells
← tissue-resident memory T-cells
→ poised to generate → a rapid immune response
→ to sub-sequently encountered pathogens

1⃣ CD4+ 2⃣ CD8+ T-RM cells
← which expressing CD69
→ were transcriptionally distinct → to circulating CD69- effector memory T-cells
→ expressed (adhesion molecules) → that promote retention ← within the lung mucosal tissue

Lung airway CD8+ T-RM cells
→ were continually re-plenished
← by interstitial CD8+ T-RM cells
∵ parabiosis experiments

Donor & recipient T-cells
→ were localized
← around the airways ← in biopsy samples
→ were infiltrating T-cells → from the recipient acquired a T-RM phenotype

Crosstalk
← between 1⃣ T-RM cells 2⃣ airway epithelial cells
→ can shape (the immune response)
→ to environmental antigens

The persistence of (CD4+ T-RM cells)
← around the airways
→ was associated ←with a rapid response → to allergen re-challenge
∴ Airway hyper-responsiveness → a cardinal feature of asthma

A bio-informatics tools
→ interrogate cell-cell interaction
← in the asthmatic airway wall
→ un-veiled increased (predicted crosstalk)
← between 1⃣ T help 2 cells 2⃣ epithelial cells
← which known to play → an important role ← in the pathogenesis

The specific properties ← of (the airway micro-environment)
→ can drive → (adaptive gene expression changes)
← in immune cells

CD8+ airway T-RM cells
→ displayed 1⃣ a distinct transcriptomic 2⃣ a epigenetic profile
← enriched for genes associated ← with 1⃣ the integrated stress response 2⃣ amino acid starvation
← which led → to a gradual loss of these cells

MAIT cells
← mucosal-associated invariant T-cells
→ contribute → approx. 4% of the total T-cells
← in human airway wall biopsies

A semi-invariant αβ TCR
→ allows MAIT cells
→ to recognize → metabolites of riboflavin biosynthesis

MAIT cells
→ generate → a rapid pro-inflammatory cytokine response
→ 1⃣ to provide protection ← against respiratory pathogens 2⃣ to display → a tissue repair transcriptomic signature
∴ Other important function ← in 1⃣ barrier integrity 2⃣ healing post-infection

Mediators
← produced by resident immune cells
→ can hinder the epithelial cell
← response to pathogens ← at the airway mucosal surface

1⃣ Type I (IFNα & IFNβ) 2⃣ Type III (IFNλ) interferons
→ are key mediators ← in the host anti-viral response
→ have recently been shown → to compromise (epithelial repair) following infection

1⃣ Airway epithelial proliferation 2⃣ regeneration
→ was reduced
← in a TP53-dependent manner
← by prolonged exposure to IFNλ

The epithelial interferon response
→ is also directly influenced
← by the type of invading pathogen

A delayed type I interferon response
→ drives → the accumulation of 1⃣ inflammatory monocytes 2⃣ inflammatory macrophage
→ leads → to severe immuno-pathology ← in mice infected with SARS-CoV

A robust expression ← of 1⃣ gene encoding chemokines 2⃣ pro-inflammatory cytokines
← which is responsible → for many of the clinical manifestations of the disease

Complex interaction
→ exist ← between 1⃣ epithelial cells 2⃣ innate & adaptive immune cells
→ to maintain host defense

Airway neuroimmune interactions

The general location ← of (airway epithelial cells)
→ enables them
→ to sense & react → to environmental changes
← via the secretion of a range of mediators
← which facilitate interaction ← with 1⃣ immune cells 2⃣ stromal cells

The airways
→ are richly innervated ← with sensory neurons
→ facilitating the execution of these avoidance reflexes

Extensive crosstalk
← between 1⃣ neuronal & immune systems 2⃣ specialized chemosensory pulmonary epithelial cells
→ is → for the regulation of (tissue homeostasis)

This integrated landscape
→ is also able → to coordinate responses
→ 1⃣ to invading → pathogens & noxious particles 2⃣ to monitor → environmental changes 3⃣ to react → to physical events

A mechanosensory ion channel
← expressed by myeloid cells ← in mice
→ was 1⃣ to sense environmental cyclical hydrostatic pressure change 2⃣ to generate → a pro-inflammatory response

The lung
→ contain → a range of different neural cells
← that react → to 1⃣ noxious 2⃣ potentially harmful stimuli

Cholinergic neurons
→ regulate 1⃣ airway tone 2⃣ airway smooth muscle contraction 3⃣ mucus secretion 4⃣ vasodilation
← via the interaction ← with muscarinic acetylcholine receptors

Activated lung neurons
→ release → stimulatory peptides
← that contribute → to the ensuing (inflammatory reaction)

A bronchoscophic radio-frequency ablation therapy
→ is used → to durably disrupt parasympathetic pulmonary nerves
∵ it decreases 1⃣ airway resistance 2⃣ mucus hyper-secretion 3⃣ inflammation

The combination of (1⃣ specialized chemosensory cells 2⃣ direct neuronal interaction)
→ may also represent → an alternative way
← in which respond → to inhaled 1⃣ respiratory pathogens 2⃣ airborne pollutants 3⃣ allergens

The activation of (epithelial TRPV4)
→ triggers → protective responses
→ to bacterial LPS

The specific deletion of (TRPV1+ neurons)
→ resulted ← in enhanced (immune protection)
→ to lethal pneumonia

1⃣ TRPV1 2⃣ Nav1.8+ nociceptor sensory neurons
→ were shown → to influence bacterial dissemination
← via the suppression of 1⃣ neutrophil number 2⃣ surveillance activities 3⃣ the regulation of resident γδT cell numbers

Epithelial-neuronal interaction
→ facilitate (lung inflammation)
← via the secretion of 1⃣ neuropeptides 2⃣ neurotransmitters

Many immune cells
→ express receptors
← which facilitate communication ← with the epithelium

❗: Which (neuronal & immune system)
→ integrate → to promote → a range of type 2 cytokine response
← that facilitate 1⃣ anti-microbial 2⃣ inflammatory 3⃣ tissue protective type 2 repair responses
∵ ILC2s → express a range of receptors → for 1⃣ neuropeptides 2⃣ neurotransmitters

ILC2s
→ interact directly ← with neuron
← via 1⃣ neuropeptides 2⃣ neurotransmitters 3⃣ neurotrophic factors

ILC2
← expression of VIPR2
→ enables the cells → to respond → to neuronal-derived VIP
∴ ILC2 creates a feedback loop ← whereby IL-5 stimulates nociceptors → to produce more VIP

NMU
← which secreted by thoracic dorsal root ganglia
→ interact ← with NMUR1 expressed by lung ILC2
→ regulated ← by IL-25

Mice
← which lacking PNECs
→ have a reduced expression ← of both (1⃣ CGRP 2⃣ GABA)
← after allergen exposure

CGRP
→ modulates → type 2 cytokine production
∴ CGRP constrains → the magnitude of the type 2 response

Concluding remarks and future directions

The cellular communities
← of the airway epithelium
→ are 1⃣ diverse 2⃣ dynamic 3⃣ yet functionally cohesive

1⃣ Epithelial 2⃣ stromal 3⃣ immune cell
→ can be modulated
← by their local tissue micro-environment

Mapping cell location
← in a specific anatomical tissue micro-environment
→ will enhance our understanding of
← 1⃣ cell molecular phenotype 2⃣ cell function

A multi-omic approach
← utilizing 1⃣ single-cell transcriptomics 2⃣ mass spectrometry-based proteomics
→ was used → to chart 1⃣ the molecular alteration 2⃣ the cellular alterations

The combination
← with 1⃣ comprehensive demographic 2⃣ clinical information
→ will allow → to link changes ← in disease phenotype

The functional validation ← of (transcriptomic data)
→ requires → robust in vitro culture systems

---