University of Groningen Developmental and pathological roles of BMP/follistatin-like 1 in the lung Tania, Navessa

University of Groningen

Developmental and pathological roles of BMP/follistatin-like 1 in the lung

Tania, Navessa

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1

Regulation of pulmonary inflammation by

mesenchymal cells

Hatem Alkhouri,

Wilfred J. Poppinga, Navessa P. Tania, Alaina Ammit,

Michael Schuliga

Regulation of pulmonary inflammation by mesenchymal cells

Hatem Alkhouri

_{, Wilfred J. Poppinga}

_{, Navessa P. Tania}

_{, Alaina Ammit}

_andMichael

Schuliga

_{Respiratory Research Group, Faculty of Pharmacy, University of Sydney, Sydney, New}

South Wales, Australia

_{Department of Molecular Pharmacology, University of Groningen, Groningen, The}

Netherlands

_{Groningen Research Institute of Asthma and COPD (GRIAC), University of Groningen,}

Groningen, University Medical Centre Groningen, The Netherlands

_{Deptartment of Pharmacology and Therapeutics, University of Melbourne, Parkville,}

Victoria, Australia

_{Lung Health Research Centre, University of Melbourne, Parkville, Victoria, Australia}

6

Abstract

Pulmonary inflammation and tissue remodelling are common elements of chronic

respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD),

idiopathic pulmonary fibrosis (IPF), and pulmonary hypertension (PH). In disease,

pulmonary mesenchymal cells not only contribute to tissue remodelling, but also

have an important role in pulmonary inflammation. This review will describe the

immunomodulatory functions of pulmonary mesenchymal cells, such as airway smooth

muscle (ASM) cells and lung fibroblasts, in chronic respiratory disease. An important

theme of the review is that pulmonary mesenchymal cells not only respond to

inflammatory mediators, but produce their own mediators, whether pro-inflammatory

or pro-resolving, which influence the quantity and quality of the lung immune response.

The notion that defective pro-inflammatory or pro-resolving signalling in these cells

potentially contributes to disease progression is also discussed. Finally, the concept

of specifically targeting pulmonary mesenchymal cell immunomodulatory function to

improve therapeutic control of chronic respiratory disease is considered.

1. Introduction

Worldwide, more than 250 million people suffer from a debilitating or lethal chronic

respiratory disease,

_{such as asthma, chronic obstructive pulmonary disease (COPD),}

idiopathic pulmonary fibrosis (IPF) or pulmonary hypertension (PH). Asthma,

characterized by airway inflammation, remodelling and hyper-reactivity, is one of the

most prevalent chronic respiratory diseases, causing ~1/4 of a million deaths per year

globally.

_{COPD, comprised of irreversible breakdown of lung tissue (emphysema) and}

airway wall remodelling, contributes to ~3 million deaths per year, and is increasing

in incidence.

_{IPF, albeit less common than asthma or COPD, is a lethal interstitial}

lung disease characterised by a relentlessly progressive and invasive form of lung

parenchymal fibrosis.

_{Secondary PH, a comorbidity caused primarily by hypoxia in lung}

disease, features increased pulmonary vascular resistance.

_{There remains no effective}

treatment for severeasthma (5-10% of asthmatics), COPD and IPF.

The consistent presence of inflammatory cells in the lungs of patients unequivocally

establishes pulmonary inflammation as an important component of chronic respiratory

disease. The lung inflammatory profiles of patients vary depending on the disease and

severity, and change upon exacerbation.

_{Airway inflammation in asthma is associated}

with an increase in mast cells, eosinophils and CD4

_{T-helper-2 (Th2) lymphocytes.}

However, for asthmatics with fixed airway obstruction, the inflammation is more

neutrophilic with greater CD8

_{T-helper-1 (Th1) cell involvement, akin to COPD, which}

is also characterised by fixed airway obstruction.

_{Whilst IPF has a predominant Th2}

cell profile, the ratio of CD8+ to CD4

_{lymphocytes increases with disease severity.}

_Like

COPD, neutrophils and macrophages are also present in lung tissue of patients with IPF.

In PH, perivascular infiltration of dendritic cells, macrophages, mast cells, T-lymphocytes

(CD4

_{and CD8}

_{) and B-lymphocytes occurs.}

_{In chronic respiratory disease, infiltrating}

inflammatory cells produce an array of inflammatory mediators which act by autocrine

and paracrine mechanisms to not only regulate inflammatory cell function, but also

pulmonary mesenchymal cells in tissue remodelling.

In chronic respiratory disease, there is an important relationship between

inflammation and tissue remodelling. The latter describes the structural changes in

lung tissue which may contribute to respiratory dysfunction. Pulmonary mesenchymal

cells are structural cells with a well-recognised role in tissue remodelling processes

in disease. In asthma and COPD, airway smooth muscle (ASM) cell hyperplasia and

hypertrophy cause ASM enlargement, whereas airway fibroblasts contribute to sub-epithelial fibrosis in the airway wall.

_{In IPF, lung fibroblasts lung fibroblasts}

_{have an}

integral role in the progressive fibrosis which begins in the lung interstitium and invades

alveoli spaces.

_{In PH, pulmonary vascular smooth muscle cells have a prominent}

role in the medial enlargement of blood vessels, which in effect reduces lumen size,

increasing vascular resistance.

_{Abnormalities of the extracellular matrix (ECM) are a}

key feature of tissue remodelling in lung disease.

_{Mesenchymal cells, by the synthesis}

and deposition of collagens I and III and other ECM components (e.g. fibronectin),

expand the volume of the ECM in the sub-epithelial layer of the airway wall, within ASM

6

bundles or in the lung interstitium.

_{Aside from important biomechanical contributions}

in tissue remodelling, pulmonary mesenchymal cells are also potent producers of an

array of inflammatory mediators, including cytokines, chemokines and cell adhesion

molecules (CAMs).

_{These inflammatory mediators, as well as the ECM produced by}

pulmonary mesenchymal cells, influence the type and quantity of inflammatory cells

that infiltrate airway and lung tissue in chronic respiratory disease. Furthermore, the

potential importance of inflammatory responses regulated by pulmonary mesenchymal

cells in tissue remodelling is becoming increasingly recognised. In this review, the

immunomodulatory functions of pulmonary mesenchymal cells and their potential

roles in the progression of chronic respiratory disease will be described.

2. Immunomodulatory function of pulmonary mesenchymal cells

This section will provide an overview of the types of immunomodulatory functions of

pulmonary mesenchymal cells, as summarized in

Figure 1.

Figure 1. Immunomodulatory functions of pulmonary mesenchymal cells. The solid black

arrows designate the pro-inflammatory mediators produced directly or indirectly by pulmonary

mesenchymal cells which contribute to pulmonary inflammation in disease. The black

hatched arrows represent sources of pro-inflammatory mediators which regulate pulmonary

mesenchymal cell function, including the production and expression of pro-inflammatory

mediators. The types and phenotype of the pulmonary mesenchymal and inflammatory cells

varies for disease and disease severity. Abbreviations are defined in the text.

2.1. Pro-inflammatory mediators

Pulmonary mesenchymal cells coordinate inflammatory responses by producing pro-inflammatory mediators which lead to inflammatory cell recruitment and activation.

The production of pro-inflammatory mediators by ASM cells, particularly in the context

of asthma, has been extensively studied and the subject of many reviews, including

one recent review.

_{Table 1 provides an overview of the broad range of cytokines,}

chemokines and CAMs, which have been shown to be expressed by pulmonary

mesenchymal cells, primarily in in vitro cell culture studies. The ECM produced by

these cells also influences inflammatory cell recruitment. Versican and hyaluronan

for instance are ECM components produced by lung fibroblasts which regulate T-cell

trafficking and functioning in inflamed lung tissue.

_{Pro-inflammatory mediator}

expression in pulmonary mesenchymal cells is stimulated primarily by cytokines and

growth factors produced by inflammatory cells and the epithelium.

_{The regulation}

of immunomodulatory function of these cells also involves pro-resolving mediators

(section 2.2), the innate immune system (section 2.3), the plasminogen activation

system (section 2.4) and the coagulation system (section 2.5).

2.2. Pro-resolving mediators

Pulmonary mesenchymal cells may be targets for or produce mediators which have a

role in resolving inflammation. Most pro-resolving mediators with anti-inflammatory

activity, including the resolvins, protectins and lipoxins, are derived from dietary omega-3

polyunsaturated fatty acids.

_{Administration of pro-resolving lipid mediators, including}

protectin D1, resolvin D1 and resolvin E1, are protective in models of lung injury and

disease.

_{Endogenous protectin D1 is increased in the airways in response to allergen}

challenge, but less so for asthmatics than non-asthmatics.

_{Such observations}

suggest that dys-regulated production of pro-resolving lipid mediators may contribute

to chronic respiratory disease.

Whilst inflammatory cells are a major source of pro-resolving lipid mediators,

_{pulmonary mesenchymal cells are a target. In cultures}

of human lung fibroblasts, resolvin D1 inhibits cigarette smoke extract- and

IL-1β-induced cytokine release

_{and endotoxin-induced COX-2 expression and prostaglandin}

E

(PGE

) production.

_{Current knowledge about the release of pro-resolving mediators}

by pulmonary mesenchymal cells is limited, aside from the production of annexin A1,

an anti-inflammatory protein.

_{Annexin A1, like resolvin D1, is a ligand for the lipoxin A}

receptor, ALX/FPR2. Annexin A1 expression and release is increased in lung fibroblasts

following treatment with glucocorticoids.

_{Furthermore, the silencing of annexin A1}

augments TNFα-induced IL-6 release from lung fibroblasts,

_{suggesting that annexin}

A1 production may be an important immunomodulatory function of pulmonary

mesenchymal cells.

2.3. Toll like receptors (TLRs)

Toll-like receptors (TLRs) activate the innate immune system in response to infection

and tissue injury. TLR ligands are: (i) derived from pathogens, including bacterial cell-surface lipopolysaccharides (LPS) and the double stranded RNA of viruses; or (ii) formed

6

Table 1. Immunomodulatory proteins expressed by airway smooth muscle (ASM) cells

and lung fibroblasts.

Type Protein Pulmonary mesenchymal cell Cytokines IL-1 ASM23_{, lung fibroblasts}19

IL-4 Lung fibroblasts24

IL-6 ASM,25 _{lung fibroblasts}24

IL-10 ASM23 IL-11 ASM25 IL-13 Lung fibroblasts26 GM-CSF ASM,27 _{lung fibroblasts}19,28 LIF ASM29 OX40 ligand ASM30

Chemokines CXCL1 (Gro-α) ASM,31 _{lung fibroblasts}32

CXCL5 (ENA-78) Lung fibroblasts32

CXCL6 (GCP-2) ASM23

CXCL8 (IL-8) ASM,33 _{lung fibroblasts}19,26

CXCL9 (MIG) ASM34

CXCL10 (IP-10) ASM,35 _{lung fibroblasts}36

CXCL11(ITAC) ASM34

CXCL12 (SDF-1α) ASM,34 _{lung fibroblasts}19

CCL2 (MCP-1) ASM,37 _{lung fibroblasts}19

CCL3 (MIP-1α) ASM38

CCL5 (RANTES) ASM,39 _{lung fibroblasts}19,26

CCL4 (MIP-1β) ASM23

CCL7 (MCP-3) ASM,38 _{lung fibroblasts}40

CCL8 (MCP-2) ASM37

CCL11 (Eotaxin) ASM,41 _{lung fibroblasts}42

CCL16 (MTN-1) ASM23 CCL17 (TARC) ASM43 CCL19 (MIP-3) ASM44 CCL20 (MIP-3α) ASM45 CX3CL1 (Fractalkine) ASM46 SCF ASM47

CAMs ICAM-1 ASM,48 _{lung fibroblasts}49

VCAM-1 ASM,19,48 _{lung fibroblasts}49

CD40 ASM19,50 CD44 ASM51 CD90 (Thy-1) Lung fibroblasts52

Abbreviations: CD, cluster of differentiation; ENA, epithelial-derived neutrophil activating; GM-CSF, granulocyte macrophage-colony stimulating factor; ICAM, intracellular adhesion molecule;

IL, interleukin; LIF, leukaemia inhibitory factor; SCF, Stem cell factor; VCAM, vascular cell adhesion

molecule.

endogenously, such as fibrinogen

_{and annexin A2.}

_{The binding of TLR ligands to their}

receptors leads to the activation of nuclear factor NF-κB and/or interferon regulatory

transcription factor 3/7, which stimulates the gene expression of inflammatory

mediators. The dysregulation of TLR signalling may contribute to the development of

chronic respiratory disease. The activation of TLR4 by fibrinogen cleavage products of

coagulation proteases possibly contributes to asthma pathophysiology.

_Pulmonary

mesenchymal cells express TLR2,

_TLR3,

_TLR4,

_{and TLR9}

_{and their activation}

stimulates IL-6, IL-8 and eotaxin production.

_{ASM cells release the stress-response}

protein, annexin A2, which stimulates IL-6 production in both macrophages

_{and ASM}

cells

_{via TLR4. In lung fibroblasts, TLR3 activation stimulates the production of RANTES,}

IP-10, IL-8, type 1 IFN, TGF-β, IL-4 and IL-13,

_{and TLR4 regulates proliferation.}

2.4. The plasminogen activation system

In interstitial lung tissue, the conversion of plasminogen to plasmin (“activation”), a pro-inflammatory serine protease,

_{contributes to disease.}

_{Plasminogen, a plasma protein,}

is relevant in lung pathology as vascular leak leads to its extravasation into inflamed

lung tissue. Both lung fibroblasts and ASM cells activate extracellular plasminogen with

subsequent effects on IL-6 and IL-8 production and cell proliferation.

_{These effects}

occur at low µg/mL concentrations of plasminogen, substantially lower than that

detected in plasma. At higher concentrations of plasminogen, increased PGE

synthesis

and/or apoptosis are observed.

_{For ASM cells, plasminogen activation is mediated}

by the urokinase plasminogen activator (uPA), in a manner accelerated by the annexin

A2 hetero-tetramer (AIIt),

_{an extracellular protein complex comprised of annexin A2}

and S100A10 (p11). The AIIt also serves as a signal transducer for plasmin in mediating

its pro-inflammatory effects on ASM cells

_{and macrophages.}

_{Whilst currently little is}

known about the role of annexin A2 in respiratory disease, it is becoming increasingly

recognised for its importance in cancer.

_{Both uPA and annexin A2 may be novel drug}

targets in the treatment of chronic respiratory disease

_{(section 5.4).}

2.5. The coagulation system

The coagulation system also contributes to pulmonary inflammation in disease.

_Like

plasminogen, the inactive zymogens of coagulation proteases enter inflamed lung tissue

as a consequence of vascular leak, in a process accompanied by platelet aggregation

and activation of the coagulation cascade.

_{Through the actions of thrombin, the main}

activator of the coagulation system, TLR4-activating fibrinogen cleavage products are

generated. Interestingly, plasmin is also involved in the formation of fibrinogen cleavage

products,

_{suggesting that the convergence of both the coagulation and plasminogen}

activation systems may play an important role in pulmonary inflammation in disease.

Thrombin and factor Xa (FXa), another coagulant, also activate PAR receptors, including

those on ASM cells and lung fibroblasts,

_{to elicit pro-inflammatory and remodelling}

activities.

_{The targeting of thrombin or FXa reduces pulmonary inflammation and}

tissue remodelling in murine models of lung injury and disease.

6

3. Pulmonary mesenchymal cells in chronic respiratory disease

3.1. Asthma

In asthma, allergen-induced airway inflammation contributes to airway

hyper-responsiveness (AHR), a process that involves spasmodic ASM contraction. Inflammation

has direct and indirect roles in AHR, causing vascular leakage, mucus hyper-secretion,

epithelial shedding, ASM thickening and sub-epithelial fibrosis.

_{Pro-inflammatory}

mediators produced by ASM cells and airway fibroblasts, including IL-8, IP-10,

MIP-1α, RANTES and eotaxin, contribute to the recruitment of mast cells, lymphocytes,

eosinophils and neutrophils in asthma.

_{ASM abnormalities in asthmatics may}

contribute to an increased hyper-secretory phenotype. Cytokine-induced production

of IL-8,

_IP-10,

_ITAC,

_eotaxin

_{and MIP-3α}

_{is greater in cultures of ASM cells}

obtained from asthmatic donors than non-asthmatics donors. Furthermore, ASM cells

of asthmatics produce relatively more collagen, fibronectin and fibulin-1,

_ECM

proteins which may facilitate inflammatory cell adhesion and activation. Increased

calcium handling, caused by abnormal sarco/endoplasmic reticulum calcium ATPase

(SERCA) pump function and expression, increases eotaxin expression in ASM cells of

asthmatics.

_{Furthermore, JNK signalling and STAT-1 activation is diminished,}

whilst p65 NF-κB activation is higher

_{in ASM cells of asthmatic than non-asthmatic}

donors. Additionally, TNF-α induced p38 mitogen-activated protein kinase (MAPK)

signalling is greater in ASM cells of donors with severe asthma, than other asthma or

control groups.

_{ASM cells of asthmatics lack expression of the full length transcription}

factor CCAAT/enhancer binding protein (C/EBPα).

_{As a consequence, C/EBPβ binding}

to chemokine promoters increases, causing cytokine hyper-secretion.

_{This may be}

due to lack of an important anti-inflammatory protein, MAPK phosphatase 1 (MKP-1),

a critical MAPK deactivator that is explored in greater depth in section 5.3. Reduced

expression of MKP-1 was responsible for over-activation of the p38 MAPK pathway and

corticosteroid insensitivity of alveolar macrophages in severe asthma compared with

non-severe asthma.

Although airway fibroblast secretory function is an area of active research,

asthma-

associated changes in airway fibroblast inflammatory mediator production is under-explored. Whilst IL-1b-induced GM-CSF and IL-8 production is increased more in the

airway fibroblasts of asthmatics than non-asthmatics,

_{the mechanism behind this}

differential cytokine production remains unknown. Interestingly, airway fibroblasts

of asthmatics in culture express lower levels of IL-13 Rα2 (a decoy receptor for IL-13

signalling) at baseline than airway fibroblasts of controls,

possibly augmenting IL-13-induced inflammation in asthma.

3.2. COPD

COPD, characterized by shortness of breath, cough and mucus hyper-secretion, is

caused primarily by tobacco exposure. Genetics/epigenetics

_{and the pulmonary}

to ASM-related pathology, such as AHR, being far more pronounced in asthma. In COPD,

the number of airway fibroblasts with a more contractile phenotype (myofibroblasts)

is greater,

_{likely caused by increases in the expression and activity of rho-associated}

coiled-coil protein kinase 1 (ROCK1).

_{Such increases will reduce airway elasticity,}

as will versican, the production of which is increased in airway fibroblasts of COPD

patients.

_{Airway fibroblasts of COPD patients also express higher levels of IL-6 and}

IL-8 than controls.

_{Intriguingly, airway fibroblasts from COPD patients, and not from}

control subjects, produce prostacyclins in response to TGF-β.

Prostacyclins have anti-inflammatory effects in pulmonary fibrosis and PH.

3.3. IPF

Interstitial lung diseases (ILDs) are characterized by an abnormality in the interstitium,

the area in the lung parenchyma between the capillaries and alveolar spaces. In IPF,

a lethal form of ILD, the abnormality is a relentlessly progressive form of fibrosis that

causes irreversible damage of lung structure and function. Whilst an increased number

of inflammatory cells in the lungs of patients with IPF suggests a role of inflammation,

an abnormal wound-repair response of epithelial/fibroblast origin is thought to be

an underlying cause.

_{Lung fibroblasts have a pivotal role in IPF, proliferating and}

differentiating into collagen producing, contractile myofibroblasts to form fibroblastic

foci. The expression of fibroblast growth factor (FGF9) is increased in IPF fibrotic foci in

situ and lung fibroblasts of IPF patients in vitro in response to TGF-β1.

By contrast, IFN-inducible expression of STAT1 and IP-10 is repressed in lung fibroblasts of IPF patients.

Human lung fibroblasts from IPF patients show constitutive activation of STAT3,

_which

mediates oncostatin M induced fibroblast chemotaxis.

_{Oncostatin M is secreted by}

inflammatory cells, such as macrophage and dendritic cells upon bacterial infection.

Oncostatin M is a potent mediator of pulmonary inflammation,

_{and is involved in the}

induction of pulmonary eosinophilia and goblet cell hyperplasia in mice,

being up-regulated in the lung of IPF patients. Furthermore, in lung fibroblasts, eotaxin expression

is induced by oncostatin M, suggesting that lung fibroblasts play an important role in

oncostatin M-induced inflammation in IPF.

3.4. Pulmonary hypertension

PH, whether primary or secondary to an accompanying chronic respiratory disease

is characterized by vasoconstriction, in situ thrombosis and pulmonary vascular

remodelling. Hypoxia, chronic inflammation and shear stress contribute to PH

pathology.

_{In proximal pulmonary vessels that were previously muscularized,}

medial thickening is caused by the hypertrophy, hyperplasia and ECM production of

resident pulmonary vascular smooth muscle cells. In previously non-muscular

pre-capillary arterioles, the pulmonary vascular smooth muscle cells that contribute to

medial thickening are derived from intermediate cells in blood vessels or adventitial

fibroblasts, which differentiate into pulmonary vascular smooth muscle cells. In PH, the

vascular adventitia has an important role in regulating and contributing to perivascular

inflammation.

_{Pulmonary adventitial fibroblasts, through the production and release}

6

of pro-inflammatory mediators, induce the infiltration and activation of monocytes

and macrophages. Epigenetic alterations in pulmonary adventitial fibroblasts from

chronically hypoxic hypertensive calves are linked to a heightened pro-inflammatory

phenotype with the expression of IL-1β, IL-6, MCP-1, CXCL12, RANTES, CCR7, CXCR4,

GM-CSF and VCAM-1 being increased.

_{In severe PH, the epigenetic reprogramming}

of human pulmonary adventitial fibroblasts to a pro-inflammatory phenotype is

associated with the decreased expression of miR-124, which regulates Notch1/PTEN/

FOXO3/p21Cip1 and p27Kip1 signalling.

_{Interestingly, aberrant PTEN phosphatase}

activity may also contribute to the pro-inflammatory phenotype of pulmonary vascular

smooth muscle cells in PH. PTEN, which inhibits Akt/PI3kinase signalling, regulates

a number of cell processes including inflammation.

_{Selective deletion of the PTEN}

gene in pulmonary vascular smooth muscle cells increases macrophage infiltration and

vascular remodelling in a murine model of PH.

4. Interactions between pulmonary mesenchymal cells and inflammatory cells

4.1. Mast cell-airway smooth muscle cell interactions

Mast cell-ASM cell interactions have an important role in asthma pathophysiology.

_The

number of mast cells within the ASM layer of asthmatics is higher than non-asthmatics,

correlating with disease severity.

_{In asthma, the mast cells residing in the ASM}

layer are predominantly mast cell

,

_{being smaller and less granular}

_{compared to}

the mast cells found elsewhere in the airway wall, or within the ASM layer of non-asthmatics. Mast cell recruitment to the ASM layer requires mast cell expression of

chemokine receptors and ASM cell production of chemokines. Lung mast cells express

a wide range of chemokine receptors, including CCR3, CXCR1, 2, 3 and 4, with CXCR3

being the most highly expressed on mast cells within the ASM layer in asthma.

Important chemokines produced by ASM cells involved in mast cell recruitment include:

IL-8 (binds CXCR1);

_{IP-10 (binds CXCR3);}

_{SDF-1α (binds CXCR4);}

_{RANTES (binds}

CCR1, 3 and 5);

_{and eotaxin (binds CCR3).}

_{These chemokines, in conjunction with}

SCF and TGF-β, are involved in the movement of mast cells to the ASM layer.

_ASM

cells of asthmatics produce higher levels of IP-10 than ASM cells of non-asthmatics

following treatment with Th1 cytokines.

_{Under Th2 inflammatory conditions, mast cell}

chemotaxis involves IL-8 and eotaxin.

_{Interestingly, the ASM cells of non-asthmatics}

release factor(s) that inhibit mast cell chemotaxis under either Th1 or Th2 inflammatory

conditions.

_{CXCL1 is an inhibitory factor of mast cell migration, and is produced less}

in ASM cells of asthmatics than non-asthmatics.

_{Upon ASM-mast cell contact, ASM}

cells induce mast cell proliferation and maintain mast cell survival.

_{This interaction}

is mediated by membrane-bound SCF expressed on ASM cells and soluble IL-6 and

CADM1 produced by mast cells.

_{In addition, numerous mast cell produced mediators}

directly affect ASM cell function, a topic that has been extensively reviewed.

These mediators cause exaggerated bronchoconstriction and also modulate ASM cell

4.2. Monocyte/macrophage-fibroblast interactions

In chronic respiratory disease, blood circulating monocytes infiltrate lung tissue to

differentiate into macrophages or dendritic cells. The phagocytic and antigen presenting

functions of these cells are important in innate and adaptive immunity respectively.

Fibroblasts are ideally suited to regulate monocyte trafficking, differentiation and

functioning because of their synthetic capacity and sentinel-like positioning in interstitial

spaces. Monocytes stimulate GM-CSF production by lung fibroblasts in a manner

involving physical contact between the two cell types.

_{Lung fibroblast production of}

GM-CSF, which regulates monocyte/macrophage function, is in turn increased by TNF-α

and/or IL-1β, cytokines produced by activated macrophages. In mouse models of PH,

pulmonary adventitial fibroblasts produce soluble mediators, including GM-CSF, which

influence monocyte/macrophage cell adhesion, infiltration and cytokine production.

Airway fibroblasts from patients with COPD express higher levels of the integrin

αvβ8, which activates TGF-β, in turn stimulating CCL2 and CCL20 production in airway

fibroblasts by an autocrine manner.

_{In a murine model of COPD, αvβ8-regulated CCL2}

and CCL20 production stimulates dendritic cell migration to boost an adaptive immune

response.

_{Alternatively activated (M2) macrophages, induced by Th2 cytokines (e.g.}

IL-4 and IL-13), are increasingly being recognised for their role in chronic respiratory

disease. M2 activated macrophages are the pre-dominant macrophage phenotype

present in the lungs of IPF patients

_{and are detected in higher numbers in the lung}

of COPD patients who continue smoking than those who stop.

_{The M2 macrophages}

have an impaired role in innate immunity, but produce a myriad of pro-inflammatory

and pro-fibrogenic mediators such as TGF-β, IL-13, CCL2, CCL17, CCL18 and CCL22.

Alveolar macrophages from non-IPF donors produce more CCL18 when either treated

with Th2 cytokines, co-cultured with lung fibroblasts or exposed to native collagen.

The latter effect of collagen occurs in a manner mediated by the β

-integrin.

_{As CCL18}

stimulates collagen production in lung fibroblasts, an axis between M2 macrophages

and lung fibroblasts, involving a CCL18-driven positive feedback loop, may perpetuate

fibrosis in IPF.

5. Novel strategies to target pulmonary mesenchymal cell immunomodulatory

function

5.1. cAMP elevating agents

There is still a need to find new therapies for chronic respiratory diseases for which,

anti-inflammatory glucocorticoids alone are ineffective.

_{Roflumilast, an oral}

phosphodiesterase (PDE4) inhibitor, is an anti-inflammatory drug for COPD, but has side

effects including nausea. Interestingly, both PDE4 inhibitors and β

-adrenergic receptor

agonists cause a rise in intracellular second messenger cyclic AMP (cAMP), but are used

pharmacologically for different targets, one inflammation, the other bronchoconstriction

(in asthma and COPD). In cultures of normal human lung fibroblasts, roflumilast, and the

β

-agonist, indacaterol, act synergistically to attenuate inflammatory cytokine secretion

and differentiation into a pro-fibrotic phenotype.

_{The levels of PGE}

, an endogenous

lipid mediator that increases cAMP production, are higher in lung

_{and lung fibroblasts}

6

from COPD patients.

_{However, in COPD, PGE}

effects on the cAMP pathway are

reduced due to an increase in PDE4 activity.

_{As increased PDE4 activity also reduces}

β

-agonist effectiveness, these findings imply the potential benefit of combining PDE4

inhibitors with cyclic AMP elevating agonists. Interestingly, the addition of plasmin(ogen)

to lung fibroblasts from IPF patients overcomes a similar PGE

resistance, by rearranging

the intracellular compartmentalization of the cAMP pathway.

_{This rearrangement}

is mediated by an increased expression of the A-kinase anchoring protein AKAP9, a

scaffolding protein for protein kinase A (PKA), which amplifies the downstream pathway

of PGE

-cAMP-PKA.

_{Thus the anti-inflammatory and anti-fibrotic properties of PGE}

2

on pulmonary mesenchymal cells may potentially be restored in chronic respiratory

diseases by approaches that rearrange cAMP compartmentalization.

5.2. TGF- β

pathways

Aberrant TGF-β

signalling, important in regulating pulmonary mesenchymal cells

function, contributes to pulmonary inflammation and remodelling in disease.

Inhibiting specific aspects of TGF-β

signalling may be an effective strategy to treat

chronic respiratory disease.

_{The TGF-β}

1

superfamily member, activin A, is linked

with the progression of PH, stimulating pulmonary vascular smooth muscle cell

proliferation.

_{Administration of follistatin, an endogenous inhibitor of activin A}

attenuates inflammation and remodelling in a murine model of pulmonary fibrosis

and asthma.

_TGF-β

-inducible connective tissue growth factor (CTGF) is implicated in

the pathogenesis of IPF. Inhibition of CTGF reduces collagen promoter activity and its

expression in bleomycin-induced mice lung fibroblasts, suggesting CTGF neutralization

may be an option for the treatment of IPF.

_{HS 6-O-sulfotransferases 1 (HS6ST1) is}

up-regulated in lung fibroblasts of IPF patients,

_{and its silencing reduces TGF-β}

1

activation and subsequent collagen I and α-smooth muscle actin expression. Such data

suggests that HS6ST1 inhibition could potentially reduce TGF-β

-mediated lung fibrosis.

Interestingly, the IL-6 antagonist Sant7 attenuates TGF-β

-induced proliferation of lung

fibroblasts obtained from ILD patients,

_{suggesting that targeting IL-6 may selectively}

block an important TGF-β

-mediated fibrotic response. Finally, inhibition of GSK-3, a

mediator of TGF-β

-induced pulmonary mesenchymal cell differentiation, may also be a

potential molecular target for chronic lung diseases.

5.3. MKP-1

In recent years, the important anti-inflammatory role played by the MAPK deactivator

MKP-1 in regulating inflammation in asthma has emerged. Upregulation of MKP-1 is

one of the ways in which common anti-asthma medicines, such as β

-agonists and

glucocorticoids, mediate their anti-inflammatory effects.

_{MKP-1 is a critical}

negative feedback controller, limiting the extent and duration of pro-inflammatory

MAPK-driven cellular signalling pathways in pulmonary mesenchymal cells such as

severe asthma.

_{The concept of enhancing MKP-1 expression and/or activity to}

control inflammation is currently under investigation,

_{as is the potential use of p38}

MAPK inhibitors to improve corticosteroid-mediated therapeutic control of chronic

respiratory disease, especially in severe asthma.

_{Further studies are warranted.}

5.4. Urokinase & Annexin A2

Urokinase and annexin A2 production by pulmonary mesenchymal cells is potentially

important in chronic respiratory disease (section 2.4). Both uPA and annexin A2 are

becoming increasingly recognised as important pathological mediators, particularly

in cancer, and their targeting by either pharmacological or antibody-based therapies

reduces tumour growth and/or metastasis in a number of pre-clinical cancer models.

Furthermore, uPA inhibitors are well tolerated in humans and have provided promising

results in recent phase I and II trials for cancer.

_{Both uPA and annexin A2 gene-deletion}

reduces pulmonary inflammation in various murine models,

_{and uPA antibodies}

reduce inflammation and oedema in a mouse model of acute lung injury.

_However,

further pre-clinical characterization of these inhibitors as therapy for chronic respiratory

disease is required.

6. Conclusion

In disease, pulmonary mesenchymal cells not only respond to inflammatory mediators,

but also contribute to inflammation by producing chemokines, cytokines, CAMs

and ECM matrix which recruit and activate inflammatory cells. The dysregulation of

pulmonary mesenchymal cell immunomodulatory function is likely to contribute to the

pathogenesis of lung disease. The specific targeting of aberrant immunomodulatory

functioning in pulmonary mesenchymal cells may be a strategy to treat lung disorders

such as severe asthma, COPD, IPF and PH, for which there are no current effective

therapies.

6

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