Supplementary Appendix

This appendix has been provided by the authors to give readers additional information about their work.

Supplement to: Thompson BT, Chambers RC, Liu KD. Acute respiratory distress syndrome. N Engl J Med 2017;
377:562-72. DOI: 10.1056/NEJMra1608077
 Acute Respiratory Distress Syndrome

Electronic Supplement

Table of Contents

1) Expanded Review of ARDS Pathogenesis 2-4

2) Approaches to ARDS Subclassification: the search for 4-7

treatment responsive subphenotypes

3) Abbreviations 7-8

4) Bibliography 8-10

1
1) Expanded Review of ARDS Pathogenesis

In this section we present an expanded discussion of the pathogenesis of ARDS from the main

manuscript and Figure 2. Abbreviations are defined in section 3 of this supplement.

The initial injury response is referred to as the exudative phase of ARDS and begins within 24

hours of the inciting direct or indirect insult as outlined in Table 2 of the review. This phase is

characterized by innate immune cell mediated damage of the alveolar endothelial and epithelial

barriers and the accumulation of protein-rich edema fluid within the interstitium and alveolus as

noted in Figure 2, panel B. Resident alveolar macrophages (Amφ) sense the presence of

microbial components (PAMPs such as LPS) or tissue injury (DAMPs such as HMGB1 and

mitochondrial DNA) via the pattern recognition receptors (PRRs; e.g. TLR).1 PRR signalling

leads to the NFκB-dependent polarization of Amφ into highly inflammatory M1 (or M1-like) mφ

and the initiation of the exudative phase. Complement activation and the release of potent pro-

inflammatory mediators (e.g. TNF, IL6, IL17), as well as multiple chemokines (e.g. IL8, CCL2,

CCL7) amplifies the inflammatory response by promoting the accumulation of neutrophils and

monocytes to sites of tissue injury via their cognate chemokine receptors (e.g. CXCR1/2 and

CCR2).

Although ARDS can occur in the absence of neutrophils, excessive neutrophilic inflammation is

a major contributor to lung injury in ARDS. Activated neutrophils contribute to injury by releasing

pre-stored inflammatory mediators, reactive oxygen species (ROS) and proteinases (e,g.

neutrophil elastase, myeloperoxidase, MMPs), as well as via the formation of neutrophil

extracellular traps (NETs) and highly injurious histones which lead to the activation of the NOD-

like receptor P3 (NLRP3) inflammasome and the release of IL-1β and IL-18 (reviewed in 2). The

resultant injury leads to loss of basement membrane integrity, epithelial-endothelial barrier

2
disruption and culminates in interstitial and intra-alveolar flooding. The TNF-mediated

expression of tissue factor by the injured and activated endothelium and epithelium leads to

dysregulated intra-vascular and intra-alveolar coagulation, platelet aggregation and

microthrombi formation, as well as hyaline membrane formation along denuded basement

membranes. Direct inhibition of surfactant function and lack of further production impede

alveolar patency. The resultant alveolar flooding and collapse lead to severely compromised

gas diffusion and hypoxemia.

The neutrophil chemokine network in the inflamed lung is highly micro-compartmentalized and

involves a cascade of chemokines, including the archetypal neutrophil chemokine, IL-8/CXCL8,

as well as CXCL10, CCL2, and CCL7 3. Recent experimental evidence further supports a role

for innate lymphoid cells and adaptive immune cells in mediating neutrophil recruitment and

promoting epithelial permeability via an αβTH17/IL-17A axis.4

Our understanding of the molecular mechanisms leading to endothelial activation, adhesion

molecule expression, microvascular vascular injury, and barrier disruption in ARDS has also

evolved with key roles identified for multiple mediators (TNF, IL-6, LPS, ROS, histamine), as

well as mechanical stretch via activation of the endothelial actin-myosin contractile apparatus.

There has been much recent interest in angiopoietin-2 (Ang-2), which is produced by activated

endothelial cells and competes with Ang1 for tie2 binding and thereby destabilizes vascular

junctional formation. Widespread damage to the alveolar epithelium also results in the loss of

alveolar ion channels and impairs the generation of osmotic forces required for alveolar fluid

clearance and thereby further contributes to alveolar flooding.

The repair programs initiated during the second or proliferative phase of ARDS are essential

for host survival (Figure 3, panel A). The formation of a provisional fibrin/fibronectin-rich matrix

along the denuded basement membranes is conducive to the proliferation and accumulation of

3
fibroblasts, myofibroblasts and locally generated pluripotent mesenchymal progenitor cells to

allow for repair. Alveolar epithelial regeneration is mediated by alveolar epithelial cells (AEC)II

and lineage-negative progenitor cell differentiation into AECI cells and subsequent spreading

along the denuded basement membrane. The re-establishment of tight and adherens junctions

leads to restoration of epithelial barrier function and surfactant production by AECII. The re-

expression of alveolar ion and aquaporin channels on AEC I and AECII (EnaC, Na+K+ ATPase,

CFTR and AQ5) facilitates the resorption of alveolar edema fluid.5 Endothelial cell and

endothelial progenitor proliferation restores endothelial barrier function.6 In experimental models

of ARDS, reparative or M2-like alveolar macrophages orchestrate the termination and resolution

of inflammation via the release of resolvins and lipoxins and promote epithelial and endothelial

repair processes via the release of growth factors (e.g. KGF, HGF, GM-CSF and VEGF). The

engulfment of apoptotic neutrophils (efferocytosis) shifts macrophages towards an M2, anti-

inflammatory phenotype and is enhanced by the release of TGFβ by regulatory T lymphocytes.

The release of matrix metalloproteinases (MMPs) further degrades the chemotactic gradient

and prevents further inflammatory cell migration. The resorption of the provisional matrix by

MMPs and lysosomal degradation pathways is a critical final event in restoring alveolar

architecture and function.

The final fibrotic phase (Figure 3, panel B) of ARDS does not occur in all patients but has

been linked to prolonged mechanical ventilation and increased mortality. Even in the low-tidal

volume era, ventilator-induced lung injury may continue to be a major contributor to the

development of lung fibrosis.7 The underlying mechanisms remain poorly understood but in

experimental models, the extent of damage to the alveolar basement membrane is a critical

determinant of whether alveolar repair is successful, or conversely sets the scene for the

development of fibrosis. The fibrogenic response is mediated via the dramatic expansion and

differentiation of resident fibroblasts into highly synthetic myofibroblasts in response to a micro-

4
environment characterized by the expression of multiple pro-fibrotic mediators (e.g. PDGF,

TGF-β, IGF-1 etc). Experimental models of ventilator-associated lung injury have also

highlighted potential roles for epithelial-mesenchymal transition (EMT) and the recruitment of

circulating bone marrow-derived fibrocytes 7, although the contribution of these cell types to the

fibrogenic response in the context of human ARDS remains to be established.

Multiple mediators have been implicated in the fibrogenic response, with current evidence

pointing to a major role for the integrin-dependent activation of latent TGFβ1 via coagulation-

dependent PAR-1 signaling. A role for the mechanotransduced activation of TGF-β1 has also

recently been demonstrated and may provide a plausible mechanistic explanation for the link

between prolonged mechanical ventilation and the development of fibrosis.8

2) Approaches to ARDS Subclassification: the search for treatment responsive

subphenotypes

Risk Factors: Direct versus Indirect Lung Injury

Direct lung injury from bacterial/viral pneumonia or aspiration of gastric contents is hypothesized

to begin with epithelial injury, whereas indirect lung injury from sepsis and pancreatitis may

begin with endothelial injury and systemic inflammation. Animal models show greater alveolar

destruction and hyaline membrane deposition, greater infiltration and neutrophil apoptosis, and

more severely impaired lung edema clearance with direct injury. Patients with direct injury have

differing biomarker profiles, and genetic risk factors.9 Pediatric patients with direct lung injury

may benefit from exogenous surfactant, though adults do not, and some patients (e.g., those

with pneumonia and associated septic shock) may have both forms of injury (reviewed in 10).

5
Timing relative to insult or risk factor

In the setting of trauma-associated ARDS, latent class analysis identified 3 subgroups of

patients based on the timing of ARDS relative to the initial insult. Early-onset ARDS was

associated with higher Abbreviated Injury Scale scores and more severe hypotension, as well

as with higher levels of plasma RAGE and Ang-2.11 Discovery genetic approaches also identify

Ang-2, a protein that increases vascular permeability, as a potential ARDS mediator.12

Baseline Physiology: Mild/Moderate/Severe by PaO2/FiO2

Patients with more severe ARDS have, on average, greater lung weights, higher rates of DAD

and pneumonia on biopsy or post-mortem, and are more likely to die from refractory hypoxemia

per se, as opposed multiorgan failure, the most common cause of death in patients with ARDS

overall.13 Higher levels of PEEP, proning and neuromuscular blockade appear to have mortality

benefit in subsets of patients with moderate-severe ARDS, suggesting that restricting clinical

trials to more severe ARDS subsets may enrich for those who are more likely to have a

treatment response to lung targeted therapies, a form of predictive enrichment.14

Baseline Physiology: High Dead Space Fraction and Acute Cor Pulmonale

A higher dead space fraction is associated with higher mortality.15 Such patients require a high

minute ventilation to maintain a normal PaCO2 levels even when CO2 production is normal.

One explanation is impairment of the lung vasculature leading to areas of the lung that are

ventilated but not perfused. Microthrombi, vascular remodeling, and pathologic

vasoconcsriction may also lead to acute cor pulmonale (ACP).16 If all four components of the

ACP risk score (pneumonia as the cause of ARDS, driving pressure > 18 cm H20, PaO 2/FiO2 <

150, and PaCO2 ≥ 48 cm H2O) are present, over 70% of patients have ACP on

transesophageal echocardiography.17 High dead space and high risk for ACP may serve as

6
selection criteria for more focused trials of anticoagulant, vasodilator, or other vascular targeted

therapies.

Markers for steroid responsiveness (PCP III)

Elevated levels of procollagen peptide III (PCP III) in bronchoalveolar lavage (BAL) reflect the

fibroproliferative phase of ARDS and may predict a favorable response to corticosteroid

therapy.18 A large RCT of PCP III directed corticosteroid for ARDS is underway.

Hyperinflammatory subtype by Latent Class Analysis

Using over 30 clinical variables and protein biomarkers from three ARDS Network trials, two

clusters of patients were identified using latent class analysis. ~30% of patients in each trial fit a

cluster with higher plasma proinflammatory biomarkers levels, lower protein C and bicarbonate

levels, and a higher proportion with sepsis as the cause of ARDS.19,20 The hyperinflammatory

subphenotype has higher mortality that improves with higher PEEP and conservative fluid

management strategies with opposite effects in the other subtype. Differential responses to

treatment by subphenotype suggests this approach identifies subtypes with different biological

mechanisms of disease.

Mediation Analysis for Treatment Targets: platelets

A genetic variant (SNP) strongly associated with platelet count is also associated with ARDS

risk and death. A portion of the genetic association between the SNP and ARDS risk and death

is mediated through changes in platelet count.21,22 These data suggest a causal role for

platelets and ARDS pathogenesis and support platelets as a treatment target for ARDS 23.

7
Proteomics and Metabolomics

High dimensional proteomic analysis of BAL fluid showed decreased expression of coagulation,

iron homeostasis, and immune activation proteins with increased actin cytoskeleton, collagen

metabolism, and glycolysis proteins in non-survivors of ARDS. Large scale metabolomics show

increased expression of lactate, citrate, and creatine both plasma and BAL of patients with

ARDS with decreased metabolites of guanosine and urate metabolism in BAL. Exhaled breath

condensate identified three volatile compounds (octane, acetaldehyde, and 3-methylene) during

ARDS but not ventilated controls. These preliminary findings, coupled with mediation analyses
24
offer the potential to find treatment targets for ARDS (reviewed in ).

3) Abbreviations:

AECI: type I alveolar epithelial cell; AECII: type II alveolar epithelial cell; Aφ: alveolar

macrophage; AQ5: aquaporin 5; CFTR: cystic fibrosis transmembrane conductance regulator;

CCL-2,-7: C-C chemokine ligand -2,-7; CCR2: C-C chemokine receptor 2; CXCR1/2: C-X-C

chemokine receptor 1/2; DAMP: damage-associated molecular pattern; ENaC: epithelial

sodium channel; HMGB1: High mobility group box 1 protein; IL-6, -8, -17, -18: interleukin-6, -8, -

17, -18; NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells; NET: neutrophil

extracellular trap; PAMP: pathogen associated molecular pattern; DAMP: danger associated

molecular pattern; PRR: pattern recognition receptor; PDGF: platelet derived growth factor;

ROS: reactive oxygen species; Na+K+ ATPase: sodium potassium ATPase; TLR: Toll-like

receptor; TGF-β: transforming growth factor beta; PEEP = Positive end-expiratory pressure;

RAGE = Receptor for advanced glycation end-products; Ang-2 = Angiopoetin 2; DAD = Diffuse

Alveolar Damage

8
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