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\begin{document}

\title{Biologics and Airway Remodeling in Severe Asthma}



\author[1]{Gilda Varricchi}%
\author[2]{Sebastian Ferri}%
\author[2]{Jack Pepys}%
\author[1]{Remo Poto}%
\author[1]{Giuseppe Spadaro}%
\author[2]{Nappi Emanuele}%
\author[2]{Giovanni Paoletti}%
\author[3]{Christian Virchow}%
\author[2]{Enrico Heffler}%
\author[2]{Giorgio Walter Canonica}%
\affil[1]{Universita degli Studi di Napoli Federico II Dipartimento di Scienze Mediche Traslazionali}%
\affil[2]{Humanitas University}%
\affil[3]{Universitatsmedizin Rostock Abteilung Pneumologie und Interdisziplinare Internistische Intensivmedizin}%


\vspace{-1em}



  
  \date{May 19, 2024}


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\selectlanguage{english}
\begin{abstract}
Asthma is a chronic inflammatory airway disease resulting in airflow
obstruction, which in part can become irreversible to conventional
therapies, defining the concept of airway remodeling. The introduction
of biologics in severe asthma has led in some patients to the complete
normalization of previously considered irreversible airflow obstruction.
This highlights the need to distinguish a ``fixed'' bronchial
obstruction due to structural changes unresponsive to current therapies,
from a ``reversible'' one as demonstrated by lung function normalization
during biological therapies not previously obtained even with high dose
systemic glucocorticoids. The mechanisms by which exposure to
environmental factors initiates the inflammatory responses that trigger
airway remodeling are still incompletely understood. Alarmins represent
tissue-derived cytokines that initiate immunologic events leading to
inflammatory airway remodeling. Biological therapies can improve airflow
obstruction by addressing these airway inflammatory changes. In
addition, biologics might prevent and possibly even revert ``fixed''
remodeling due to structural changes. Hence, it appears clinically
important to separate the therapeutic effects (early and late) of
biologics as a new paradigm to evaluate the effects of these drugs and
future treatments on airway remodeling in severe asthma.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Asthma is a heterogeneous chronic inflammatory disease of the
respiratory system that affects approximately 10\% of
adults\textsuperscript{1}. A typical feature of asthma is a variable
airflow limitation associated with symptoms such as dyspnoea, cough,
wheezing and chest tightness\textsuperscript{2}. The heterogenicity of
the immunologic disorder is reflected in different phenotypes that
differ in etiology, pathogenic mechanisms, symptoms, and
severity\textsuperscript{3}. Based on airway inflammation, asthma has
been subdivided into type 2-high (T2-high) and -low (T2-low), although
the latter form is rare in clinical practice\textsuperscript{3-5}. A
similar distinction is made between eosinophilic and non-eosinophilic
asthma (GINA 2021) \textsuperscript{6}. Severe asthma is defined as
asthma that is not well controlled despite the administration of
high-dose drug therapy\textsuperscript{7}. Up to 10\% of asthma patients
have severe asthma, with a reduced quality of life, and increased risk
of exacerbations, hospitalizations, and death\textsuperscript{8, 9}.

In T2-high asthma, immunologic stimuli (e.g., allergens, viral and
bacterial superantigens) activate primary effector cells of allergic
disorders (i.e., mast cells, basophils) through the engagement of
specific IgE to release a plethora of interleukins (ILs) (e.g., IL-3,
IL-4, IL-5, IL-13) \textsuperscript{10, 11}. Eosinophils and their
mediators contribute to the pathogenesis of allergic asthma and play
pivotal roles in eosinophilic asthma. T2-low asthma is heterogeneous,
incompletely defined and understood and presumably includes different
phenotypes characterized by the involvement of mast cells, macrophages,
neutrophils and/or a mixture of these immune cells
\textsuperscript{3,5}. Bronchial epithelial cell-derived alarmins (e.g.,
TSLP, IL-33 and IL-25) are upstream cytokines that initiate immunologic
events culminating in airway remodeling \textsuperscript{12-14}. The
latter is a complex process requiring a timely expression of
fibrogenic\textsuperscript{15} and angiogenic factors causing profound
structural alterations of the bronchial walls and blood
vessels\textsuperscript{16,} \textsuperscript{17}. These alterations
contribute to the reduction of airway caliber and stiffening, resulting
clinically in airflow limitations and respiratory
symptoms\textsuperscript{18}.

\textbf{Airway remodeling}

Airway remodeling can affect both large and small
airways\textsuperscript{19} and is characterized by structural changes
including goblet cell hyperplasia, subepithelial matrix protein
deposition and fibrosis, overexpression of angiogenic factors, and
hyperplasia/hypertrophy of airway smooth muscle (ASM)
cells\textsuperscript{15-17, 20, 21}. Increased deposition of
extracellular matrix (ECM) proteins in the reticular basement membrane
(RBM), lamina propria, and submucosa is a characteristic of asthmatic
airways and contributes to the airway wall thickening and airflow
obstruction\textsuperscript{22, 23}. Collagen fibers, fibronectin and
tenascin are the most abundant elements of the ECM in the asthmatic
lung\textsuperscript{24-27}. Aberrant accumulation of ECM proteins leads
to alterations in tissue structure and function, contributing to airway
remodeling in asthma\textsuperscript{28-30}. ASM hypertrophy/hyperplasia
(e.g., increased ASM mass) are features of asthmatic airway
remodeling\textsuperscript{18, 3132, 33}. ASM cells in asthmatic
individuals also produce increased amounts of collagen and
fibronectin\textsuperscript{34, 35}. The increase in the ASM mass is
responsible for bronchial obstruction\textsuperscript{36}, loss of
function\textsuperscript{20} and greater susceptibility to external
triggers\textsuperscript{37-39}.

Angiogenesis is fundamental to providing the blood vessels to maintain
tissue homeostasis \textsuperscript{40}, whereas inflammatory
angiogenesis is a critical factor in the development of a disease
process\textsuperscript{41-43}. Blood vessel density and vascular area
are increased in patients with asthma\textsuperscript{16, 17}. Another
feature of asthma is goblet cell hyperplasia, mucin overproduction and
mucus hypersecretion\textsuperscript{44}. Figure 1 schematically
illustrates the fundamental characteristics of the normal bronchial
airway and the main features of airway remodeling in asthma.

A plethora of cytokines {[}transforming growth factor \selectlanguage{greek}β \selectlanguage{english}(TGF-\selectlanguage{greek}β\selectlanguage{english}),
platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),
epidermal growth factor (EGF){]}), vascular endothelial growth factors
(VEGFs){]} and chemokines (e.g., CXCL2, CXCL3, IL-8/CXCL8) contribute
directly and indirectly to airway remodeling in
asthma\textsuperscript{45-47}. TGF-\selectlanguage{greek}β, \selectlanguage{english}produced by macrophages and
eosinophils, is a main mediator responsible for airway remodeling by
inducing epithelial-mesenchymal transition (EMT)\textsuperscript{48}.

IL-4 activates ASM cells, causing an increase in actin and collagen
synthesis as well as TGF-\selectlanguage{greek}β \selectlanguage{english}release by the bronchial
epithelium\textsuperscript{37, 49}. IL-5, on the other hand promotes
subepithelial and peri-bronchial fibrosis through the recruitment and
activation of eosinophils, a major source of TGF-\selectlanguage{greek}β\selectlanguage{english}\textsuperscript{50,
51}. IL-13 induces the release of TGF-\selectlanguage{greek}β \selectlanguage{english}which increases goblet cell
hyperplasia and thus mucus hypersecretion\textsuperscript{37, 52-54}.

Human eosinophil granules are armed with cytotoxic major basic protein
(MBP), eosinophil peroxidase (EPX), eosinophil cationic protein (ECP),
eosinophil derived neurotoxin (EDN), and galactin-10 (also known as
Charcot-Leyden protein)\textsuperscript{55}. ECP and MBP induce the
release of preformed (histamine and tryptase) and \emph{de
novo}synthesized mediators (prostaglandin D\textsubscript{2}:
PGD\textsubscript{2}) from human mast cells\textsuperscript{56}.
Activated human eosinophils secrete LTC\textsubscript{4} and a wide
array of type 2 cytokines (i.e., IL-5, IL-4, IL-13) and
TGF-\selectlanguage{greek}β\selectlanguage{english}\textsuperscript{45, 57}. Altogether, current data indicate that
eosinophils play a globally pathogenic role in airway remodeling.

Macrophages are the predominant immune cells in human lung
parenchyma\textsuperscript{58} and are involved in immune responses as
well as tissue remodeling\textsuperscript{59}. Human lung macrophages
(HLMs) contribute to airway remodeling through the release of TGF-\selectlanguage{greek}β,
\selectlanguage{english}matrix metalloproteinases (MMPs), angiogenic (VEGF-A, ANGPT2) and
lymphangiogenic factors (i.e., VEGF-C)\textsuperscript{60, 61}. Human
lung mast cells (HLMCs) are important lung-resident immune cells
involved in asthmatic airway remodeling \textsuperscript{62}. IgE- and
non-IgE-mediated activation of HLMCs induces the release of several
pro-fibrotic cytokines (e.g., IL-13 and TNF-\selectlanguage{greek}α\selectlanguage{english}), as well as inflammatory
mediators (e.g., PGD\textsubscript{2} and tryptase)\textsuperscript{63}.
Tryptase induces fibroblast, endothelial and epithelial cell
proliferation, further fueling airway remodeling in asthmatic
individuals\textsuperscript{64}. Neutrophils have also been shown to
produce MMP-9\textsuperscript{65, 66}, angiogenic
factors\textsuperscript{40} and neutrophil extracellular traps
(NETs)\textsuperscript{67} and can be associated with severe
asthma\textsuperscript{67}.

The airway epithelium is a key component of the innate immune system and
the initiator of airway remodeling in asthma\textsuperscript{14}. A
plethora of environmental insults (e.g., allergens, cytokines, microbial
proteins, smoke extracts, chemical and physical insults) damage and/or
activate epithelial cells to release several cytokines, including
TSLP\textsuperscript{13, 68}, IL-33\textsuperscript{14, 69},
IL-25/IL-17E\textsuperscript{12}, TGF-\selectlanguage{greek}β \selectlanguage{english}and granulocyte-macrophage
colony-stimulating factor (GM-CSF), which can recruit dendritic cells
(DCs), mast cells and other immune cells\textsuperscript{70}.

Thymic stromal lymphopoietin (TSLP), constitutively expressed by human
bronchial epithelial cells\textsuperscript{71-74}, can be rapidly
released as a result of cell injury in response to a variety of
inflammatory stimuli \textsuperscript{71, 75-79}. TSLP is also released
by DCs\textsuperscript{80}, mast cells\textsuperscript{81},
HLMs\textsuperscript{60}, and fibroblasts\textsuperscript{82, 83}. There
are two isoforms of TLSP: the short (sf) and the long (lf) isoforms. The
latter is expressed at a low or undetectable level at steady state, but
its expression increases during inflammation \textsuperscript{60, 84}.
For instance, house dust mites induce lfTSLP but not sfTSLP in human
bronchial epithelial cells\textsuperscript{85}. TSLP immunostaining is
increased in the airway epithelium in asthmatic
patients\textsuperscript{74} and its concentrations are increased in the
broncho-alveolar lavage (BAL) fluid of asthmatics \textsuperscript{73}.
Moreover, bronchial allergen challenge of asthmatics increases the
expression of TSLP\textsuperscript{+} cells in the epithelium and
submucosa \textsuperscript{72}. TSLP is overexpressed in the airways of
severe asthma patients\textsuperscript{73} and exerts its effects by
binding to a high-affinity heterodimeric receptor complex composed of
TSLPR and IL-7R\selectlanguage{greek}α\selectlanguage{english}\textsuperscript{13}.

TSLP plays a role in airway remodeling\textsuperscript{86} by promoting
the differentiation of Th2 cells and innate lymphoid cells
(ILCs)\textsuperscript{13} and the induction of epithelial-mesenchymal
transition (EMT) in airway epithelial cells\textsuperscript{87}. Human
lung fibroblasts are also a significant source of
TSLP\textsuperscript{83, 88}. Through an autocrine mechanism, TSLP can
activate human lung fibroblasts\textsuperscript{89} to release type I
collagen \textsuperscript{90} and promote the proliferation of ASM
cells\textsuperscript{91}. TSLP has also been shown to cause goblet cell
hyperplasia and mucus production\textsuperscript{92-94}. TSLP activates
human eosinophils \textsuperscript{13}, mast cells \textsuperscript{81,
95} and HLMs\textsuperscript{60}. The multiple activating properties of
TSLP on a plethora of immune and structural cells indicate that this
cytokine plays a role in T2-high and T2-low asthma.

IL-33, an IL-1 superfamily alarmin released by airway epithelial cells
and endothelial cells\textsuperscript{96}, activates the ST2 receptor on
several cells of the innate and the adaptive
immunity\textsuperscript{96}. Epithelial cell-derived IL-33 induces type
2 cytokines (i.e., IL-5 and IL-13) in human mast cells
\textsuperscript{97}, collagen and fibronectin release from airway
fibroblasts\textsuperscript{98, 99}. Collectively, IL-33 and IL-33/ST2
signaling pathways might be involved in both airway inflammation and
asthma remodeling through the activation of several immune and
structural cells.

IL-25, also known as IL-17E, is a unique cytokine of the IL-17 family
produced by airway epithelial cells\textsuperscript{12}. Airborne
allergens, ATP, and viral infections upregulate IL-25 and its receptor
IL-17RB in airway epithelial cells and submucosa\textsuperscript{100,
101}. IL-25 modulates EMT of alveolar epithelial cells and local tissue
remodeling\textsuperscript{102} and upregulates cytokine expression in
lung fibroblasts\textsuperscript{103}. IL-25 drives lung fibrosis in
several mouse models\textsuperscript{102, 104, 105}.

Finally, IgE itself could play a role in airway remodeling by
stimulating the production of interleukins\textsuperscript{106}. Several
investigators have reported that human monomeric IgE, in the absence of
cross-linking, can induce the release of cytokines (e.g., IL-4) and
chemokines (e.g., CXCL8) from mast cells\textsuperscript{107, 108}.
Moreover, Roth \emph{et al} . have shown that \emph{in vitro} incubation
of serum containing IgE obtained from allergic asthmatics caused ASM
proliferation and marked production of type I
collagen\textsuperscript{109}.

A recent study in a mouse model of asthma demonstrated dynamic changes
in the respiratory microbiota at different stages of the disease. In
particular, \emph{Staphylococcus} and \emph{Cupriavidus} were more
abundant during airway remodeling\textsuperscript{110}. Additional
studies are urgently needed to investigate whether the dysbiosis of
airway microbiota could also play a role in the progression from
allergic inflammation to airway remodeling in humans.

Airway remodeling can sometimes cause irreversible airflow limitation
with consequent poor symptom control and lack of response to
treatment\textsuperscript{20, 111, 112}.

\textbf{Targeting airway remodeling}

Experimental and human studies have demonstrated that glucocorticoids
and certain anti-inflammatory drugs can reduce inflammation and
morbidity in mild to moderate asthma. However, evidences of beneficial
effects of glucocorticoids on airway remodeling have not been
demonstrated, with several reports showing contradictory
results\textsuperscript{17, 113, 114}. The introduction of several
biological immunotherapies (e.g., anti-IgE, anti-IL-5/IL-5R\selectlanguage{greek}α,
\selectlanguage{english}anti-IL-4R\selectlanguage{greek}α, \selectlanguage{english}anti-TSLP)\textsuperscript{2, 115} has favored the
development of a personalized medicine approach for the treatment of
patients with severe asthma \textsuperscript{116-119}. There is some
evidence that these biologics can improve not only clinical symptoms but
also certain features of airway remodeling and functional decline of
FEV\textsubscript{1}. Therefore, it will be important to evaluate the
effects of biological immunotherapies in randomized clinical trials
(RCTs) and real-life settings\textsuperscript{120, 121}.

\textbf{Omalizumab}

Omalizumab is a humanized IgG1/\selectlanguage{greek}κ \selectlanguage{english}monoclonal antibody (mAb) that binds to
the Fc fragment of IgE\textsuperscript{122}. This mAb inhibits binding
of IgE to the high-affinity IgE (Fc\selectlanguage{greek}ε\selectlanguage{english}RI) receptor on human mast cells and
basophils\textsuperscript{123, 124}. Several studies have documented the
efficacy of omalizumab in improving allergic asthma and reducing
symptoms and exacerbations\textsuperscript{125-127} also in children and
pregnant women\textsuperscript{128-130}. Although omalizumab did not
cause FEV\textsubscript{1} improvement in RCTs\textsuperscript{131,
132}, there is some evidence that this mAb can improve
FEV\textsubscript{1} in real-life settings\textsuperscript{133, 134} and
can reduce the thickness of the basement membrane \textsuperscript{135,
136} and fibronectin deposits in asthmatic airways
\textsuperscript{137}in addition to preventing exacerbation-induced
inflammatory alterations in the airways. IgE-containing serum from
asthmatic patients stimulated\emph{in vitro} mesenchymal cell
proliferation and accumulation of collagen and fibronectin. Both
proliferation and matrix deposition were prevented by preincubation of
the cells with omalizumab\textsuperscript{109}.

\textbf{Mepolizumab}

Mepolizumab is an IgG1-\selectlanguage{greek}κ \selectlanguage{english}anti-IL-5 mAb approved as add-on treatment for
severe eosinophilic asthma (SEA)\textsuperscript{138, 139}. IL-5 is the
most important growth, differentiation, and activation factor of human
eosinophils\textsuperscript{140}. This cytokine acts on eosinophils by
binding to the specific IL-5 receptor (IL-5R), which consists of an IL-5
receptor \selectlanguage{greek}α \selectlanguage{english}(IL-5R\selectlanguage{greek}α\selectlanguage{english}) subunit and the common receptor \selectlanguage{greek}β \selectlanguage{english}subunit
(\selectlanguage{greek}β\selectlanguage{english}c)\textsuperscript{141}. IL-5, together with IL-3 and GM-CSF, is
crucial for the maturation of human eosinophils in the bone marrow
\textsuperscript{141, 142}. IL-5 is mainly produced by type-2 ILC2s, Th2
cells, mast cells, invariant NKT cells, and eosinophils themselves
\textsuperscript{140}. Human eosinophils can also be activated by
IL-33\textsuperscript{96} and TSLP\textsuperscript{13}.

The efficacy and safety of mepolizumab have been demonstrated in several
RCTs \textsuperscript{143-146}. The DREAM study, conducted with
mepolizumab at 3 different doses administered intravenously (\emph{i.v}
.), showed the clinical efficacy but there were no statistically
significant changes in FEV\textsubscript{1,} although there was an
improvement, compared to baseline, in FEV\textsubscript{1} in the
mepolizumab group \emph{versus}placebo \textsuperscript{143}. In the
MENSA study, the FEV\textsubscript{1} increase was rapid, starting from
the first administration, and persisted over time\textsuperscript{146}.
Two additional studies showed a rapid and long-lasting improvement in
pre-bronchodilator FEV\textsubscript{1} in the mepolizumab group
compared to placebo\textsuperscript{147, 148}. There is some evidence
that mepolizumab can also improve FEV\textsubscript{1} in real-life
settings\textsuperscript{149-151}.

Interestingly, some patients first enrolled in the COSMOS study and
subsequently in the long-term COSMEX study, with a suspension of more
than 12 weeks of mepolizumab between the two, reported a transient
worsening of their symptoms and FEV\textsubscript{1,} which rapidly
improved upon reintroduction of mepolizumab\textsuperscript{145}. A
clinical trial (NCT03797404) is evaluating the effects on airway
remodeling during mepolizumab treatment.

In a pioneering study, Flood-Page and collaborators examined the
bronchial biopsies of mild atopic asthmatic patients obtained before and
after treatment with three mepolizumab infusions\textsuperscript{57}.
They demonstrated that the thickness and density of tenascin in the RBM,
the airway TGF-\selectlanguage{greek}β\selectlanguage{english}1\textsuperscript{+} eosinophils and the BAL
concentrations of TGF-\selectlanguage{greek}β\selectlanguage{english}1 were increased in mild asthmatic patients
compared to normal subjects. As expected, mepolizumab reduced bronchial
eosinophil numbers but also TGF-\selectlanguage{greek}β\selectlanguage{english}1\textsuperscript{+} eosinophils,
thickness and tenascin immunoreactivity and the concentration of TGF-\selectlanguage{greek}β\selectlanguage{english}1
in BAL fluid\textsuperscript{57}.

\textbf{Reslizumab}

Reslizumab is a humanized IgG4-\selectlanguage{greek}κ \selectlanguage{english}anti-IL-5 mAb developed by drafting
technology from a rat mAb with high affinity against human
IL-5\textsuperscript{152}. Reslizumab binds to a small region
corresponding to amino acids 89-92 of IL-5, which are critical for
binding to IL-5R\selectlanguage{greek}α\selectlanguage{english}\textsuperscript{153}. Reslizumab administration
results in clinical improvement in asthma and an increase in
FEV\textsubscript{1} in patients with eosinophilic counts \textgreater{}
400 cells/\selectlanguage{greek}µ\selectlanguage{english}L compared to placebo\textsuperscript{153-158}. Apparently
contrasting results have been reported on the effects of reslizumab on
the improvement of FEV\textsubscript{1} compared to placebo. Although
several studies reported that reslizumab significantly improved
FEV\textsubscript{1} compared to placebo\textsuperscript{154, 157},
other investigators found that reslizumab had no improvement in
FEV\textsubscript{1} compared to those receiving placebo
\textsuperscript{154}. A real-life study recently confirmed the efficacy
of reslizumab treatment in reducing the number of exacerbations and
increasing FEV\textsubscript{1} 6 months after the beginning of
treatment\textsuperscript{159}.

\textbf{Benralizumab}

Benralizumab is a humanized, afucosylated IgG1-\selectlanguage{greek}κ \selectlanguage{english}mAb that targets the \selectlanguage{greek}α
\selectlanguage{english}subunit of the IL-5 receptor (IL-5R\selectlanguage{greek}α\selectlanguage{english}) and it binds with high affinity to
the main Fc receptor for IgG expressed by natural killer (NK) cells,
macrophages and neutrophils, which results in eosinophil
apoptosis\emph{via} antibody-dependent cell-mediated
cytotoxicity\textsuperscript{160}. Several studies have demonstrated
that benralizumab administration leads to subjective and functional
clinical improvement in patients with SEA. RCTs, reported an improvement
in FEV\textsubscript{1} at 12 weeks from the beginning of treatment
\textsuperscript{161-163}. A post-hoc analysis conducted on SIROCCO and
CALIMA data further documented that benralizumab promoted functional
improvement even in patients with fixed obstruction, an alteration found
in approximately 16\% of patients with severe
asthma\textsuperscript{164}. A recent study extended the previous
results by showing that benralizumab caused a rapid (4 weeks)
improvement in FEV\textsubscript{1}, which increased after 12 weeks and
persisted throughout the period (24 weeks) of observation
\textsuperscript{163}.

Cachi and collaborators evaluated the effects of benralizumab on airway
remodeling examining biopsies from patients with
SEA\textsuperscript{165}. Benralizumab reduced the number of eosinophils
in the bronchial lamina propria and ASM mass compared to placebo. In the
benralizumab group, there were no significant changes in the number of
myofibroblasts compared to the control group. The effects of
benralizumab on ASM mass were attributed to an indirect effect mediated
by the depletion of local TGF-\selectlanguage{greek}β\selectlanguage{english}1\textsuperscript{+} eosinophils in the
bronchial lamina propria.

Pelaia and collaborators emphasized the clinical efficacy of
benralizumab in terms of lung function in real life after the first
administration of this mAb\textsuperscript{166}. Padilla-Galo\emph{et
al.} reported an improvement in FEV\textsubscript{1} after three months
of treatment with benralizumab, which lasted up to six months
\textsuperscript{167}. Similar results were obtained in a retrospective
study\textsuperscript{168}. The authors ascribed the rapid improvement
of lung function to the early effects of the mAb on peripheral blood and
bronchial eosinophils. Although these observations were obtained in
small cohorts of patients, they emphasize that the improvement caused by
benralizumab on lung function observed in real life is more evident
compared to certain RCTs\textsuperscript{92, 161, 162}. Finally,
clinical trials (NCT04365205, NCT03953300) are evaluating the effects of
benralizumab on airway remodeling.

\textbf{Dupilumab}

The Th2-like cytokines IL-4 and IL-13 and the heterodimeric IL-4
receptor (IL-4R) complexes that they activate play a key pathogenic role
in asthma \textsuperscript{169}. Dupilumab is a human IgG4 mAb that
targets the IL-4 receptor \selectlanguage{greek}α \selectlanguage{english}chain (IL-4R\selectlanguage{greek}α\selectlanguage{english}), common to both IL-4R
complexes: type 1 (IL-4R\selectlanguage{greek}α/γ\selectlanguage{english}c; IL-4 specific) and type 2
(IL-4R\selectlanguage{greek}α/\selectlanguage{english}IL-13R\selectlanguage{greek}α\selectlanguage{english}1: IL-4 and IL-13 specific)\textsuperscript{170}.

In several RCTs, dupilumab reduced the annualized rate of asthma
exacerbations in patients with moderate-to-severe uncontrolled asthma
compared to placebo\textsuperscript{171-173}. Dupilumab in patients with
severe asthma caused rapid (2 weeks) and long-lasting improvement in
FEV\textsubscript{1} \emph{versus}placebo\textsuperscript{172, 173}.
Furthermore, the trend curves of FEV\textsubscript{1} post
bronchodilation showed a loss of function in the placebo group and no
decrease in FEV\textsubscript{1} over time in the dupilumab group. The
latter findings suggest that dupilumab might exert a positive effect on
structural airway remodeling. Moreover, in a mouse model of asthma, dual
IL-4/IL-13 blockade with dupilumab prevented eosinophil infiltration
into lung tissue without affecting circulating eosinophils
\textsuperscript{174}. A real-life retrospective study demonstrated that
4 weeks of treatment were necessary to achieve a significant improvement
in FEV\textsubscript{1} compared to baseline\textsuperscript{175}.

It should be noted that blood eosinophilia was reported in 4 to 25\% of
patients in the dupilumab group compared to 0.6\% in the placebo
group\textsuperscript{173, 176}. This paradoxical effect has also been
reported in patients treated with dupilumab for moderate-to-severe
atopic dermatitis \textsuperscript{177}.

The VESTIGE study (NCT04400318), evaluating dupilumab effects on lung
function and structural airway changes using functional respiratory
imaging (FRI), is ongoing.

Additionally, indirect evidence of a combined anti-inflammatory effect
as well as on tissue and structural cells can be derived from the
observation that all of the above mentioned anti-Th2 cytokine or
receptor antibodies have been shown to reduce nasal polyposis in
patients with chronic rhinosinusitis\textsuperscript{178-181}.

\textbf{Tezepelumab}

Tezepelumab is a human mAb, which binds with high affinity to TSLP, an
epithelial cell-derived cytokine implicated in the pathogenesis of
different phenotypes of asthma\textsuperscript{13}. TSLP, a pleiotropic
cytokine overexpressed in the airway epithelium of
asthmatics\textsuperscript{74}, exerts its effects by binding to a
high-affinity heterodimeric receptor complex composed of TSLPR and
IL-7R\selectlanguage{greek}α \selectlanguage{english}\textsuperscript{13}. TSLP concentrations are increased in BAL
fluid of asthmatics\textsuperscript{73} and bronchial allergen challenge
increases TSLP expression in the asthmatic epithelium and submucosa.
Importantly, serum concentrations of TSLP are increased during asthma
exacerbations\textsuperscript{182}. Finally, TSLP induces the release of
angiogenic and lymphangiogenic factors from HLMs\textsuperscript{60}.
TSLP can promote airway remodeling \emph{via} the activation of human
lung fibroblasts\textsuperscript{89}.

The FDA has recently approved tezepelumab for the treatment of severe
asthma with no phenotype or biomarker limitations. Tezepelumab is the
first of a new class of biologics that antagonize an alarmin (e.g.,
TSLP), which plays a pivotal role in the pathogenesis of
asthma\textsuperscript{13, 68}. The phase II PATHWAY study showed that
three different doses (70 mg, 210 mg, or 280 mg s.c. every 4 weeks) of
tezepelumab reduced the number of annual exacerbation rates regardless
of blood eosinophil count, with a significant increase in
prebronchodilator FEV\textsubscript{1} at 52 weeks from the start of
treatment compared to the placebo group\textsuperscript{183}. These
results were extended in the phase III NAVIGATOR study in which
tezepelumab (210 mg s.c. every 4 weeks) reduced asthma exacerbations at
week 52 and significantly improved FEV\textsubscript{1} regardless of
peripheral blood eosinophils in adolescent and adult patients with
severe uncontrolled asthma\textsuperscript{184} although there was a
trend toward a better improvement with higher eosinophil counts in
subgroup analysis.

Studies conducted in different animal models using TSLP antibodies have
demonstrated that TSLP blockade reduces airway inflammation, TGF-\selectlanguage{greek}β\selectlanguage{english}1
levels, hyperreactivity and airway remodeling\textsuperscript{185-188}.
The phase II CASCADE study evaluated the effects of tezepelumab on
airway remodeling by performing bronchoscopic biopsies in
moderate-to-severe asthma patients \textsuperscript{189}. Tezepelumab
caused a greater reduction from baseline to the end of treatment in
airway submucosal eosinophils compared to placebo. There were no other
significant changes either at the level of other immune cells
(neutrophils, mast cells, and T cells) and at the structural level
(e.g., RBM thickness, epithelial integrity). Interestingly, tezepelumab
administration was associated with lower hyperresponsiveness to mannitol
inhalation compared to placebo. The latter finding was confirmed in an
independent study \textsuperscript{190}. These preliminary results on
the effects of tezepelumab on airway remodeling are of translational
interest for several reasons. There is overwhelming evidence that
fibroblasts are a source of TSLP\textsuperscript{82, 83} and there is
evidence that a functional TSLP signaling axis plays a role in fibrotic
lung disease\textsuperscript{83}. Figure 2 schematically illustrates the
mechanisms of action of different biologics and their immunological and
cellular targets in the context of airway remodeling.

\textbf{Future perspectives}

\textbf{Astegolimab}

Astegolimab is an IgG2 mAb that blocks IL-33 signaling by targeting ST2,
the IL-33 receptor \textsuperscript{96}. The phase 2b ZENYATTA
study\textsuperscript{191} evaluated the safety and efficacy of
astegolimab in patients with severe asthma. Astegolimab was safe, well
tolerated, and effective in reducing the annualized asthma exacerbations
at week 54. Astegolimab did not show a significant benefit compared to
placebo in the absolute change in FEV\textsubscript{1} at week 54.

\textbf{Itepekimab}

Itepekimab is a human IgG4 anti-IL-33 mAb. A phase 2 trial compared the
safety and efficacy of itepekimab (300 mg s.c. every 2 weeks), dupilumab
(300 mg. s.c. every 2 weeks), itepekimab plus dupilumab, or placebo in
patients with moderate-to-severe asthma\textsuperscript{192}. The
primary endpoint, loss of asthma control, was similar in the itepekimab
(22\%), combination (27\%) and dupilumab (19\%) groups and lower than in
the placebo (41\%) group. Pre-bronchodilator FEV\textsubscript{1}
increased with itepekimab and dupilumab monotherapies but not with
combination therapy. Ipetekimab improved asthma control and quality of
life compared to placebo and reduced peripheral blood eosinophils. The
latter results are consistent with a role for IL-33 in the pathogenesis
of asthma exacerbations and airflow limitations in asthma. Further
investigations are needed to investigate whether blockade of the
IL-33/ST2 axis can modify airway remodeling in patients with asthma.

\textbf{Imaging of airway remodeling}

The direct assessment of human airway remodeling can be
performed\emph{in vivo} on bronchial biopsies in asthmatic
patients\textsuperscript{57, 189, 193}. Bronchial biopsies identify
useful information on bronchial and pulmonary alterations, such as
thickening of the bronchial tissue, infiltration and density of
inflammatory cells, aberrant accumulation of elements of ECM, and ASM
hyperplasia and hypertrophy \textsuperscript{15}. However, this
procedure has several limitations that should be pointed out. First, it
is not part of the routine clinical evaluation of asthmatic patients.
Second, it does not easily facilitate serial biopsies in the same
patient. Third, the results can be significantly influenced by the
operator and the techniques used to assess tissue remodeling.

During the last decade, high-resolution computed tomography (HRCT) and
nuclear magnetic resonance (NHR) are gaining a place as non-invasive
techniques to examine different aspects of airway remodeling in asthma.
Hoshino found increased airway wall thickening in asthmatics assessed by
HRTC \textsuperscript{194}. Endobronchial ultrasound (EBUS) was higher
in asthma patients than healthy controls \textsuperscript{195}. Hartley
\emph{et al.} found that the proximal airway wall area was increased in
asthmatics compared to controls\textsuperscript{196}. The loss of the
peripheral pulmonary vasculature, also termed pruning, was associated
with asthma severity\textsuperscript{197}. Recently, Eddy and co-workers
demonstrated that the total number of CT-visible airways was correlated
to asthma severity\textsuperscript{198}.

Preliminary results derive from imaging studies that have evaluated the
effects of biological therapies on airway remodeling using HRCT.
Hoshino\emph{et al.} reported that 16-week of treatment with omalizumab
reduced the airway wall thickness and the number of sputum
eosinophils\textsuperscript{136}. In another study, 48-week treatment
with omalizumab reduced the airway wall area corrected for body surface,
but no changes in percentage wall area, without changes in the luminal
area\textsuperscript{199}. In two studies conducted by Haldar \emph{et
al.}\textsuperscript{200, 201} on airway remodeling by HRCT, it was
found that the biological treatment determined a greater variation in
pre/post treatment luminal area when compared to the placebo group. A
recent study evaluated the impact of one-year mepolizumab therapy on
airway remodeling through EBUS and HRTC. Improved airway remodeling
(e.g., reduction in bronchial wall thickness) was better noticeable in
invasive EBUS than in non-invasive HRCT\textsuperscript{202}. In the
phase 2 CASCADE study, tezepelumab increased the CT scan-determined
lumen area across airway generations\textsuperscript{189}.

Hyperpolarized helium-3 MRI of the lung has demonstrated regional
heterogeneity of lobar ventilation in asthma\textsuperscript{203}, which
is correlated with asthma severity\textsuperscript{204}. MRI ventilation
defects (VDP) are correlated to sputum eosinophilia in severe
asthma\textsuperscript{205} and are a predictor of
exacerbation\textsuperscript{206}. Collectively, studies with CT and MRI
in asthma have shown some structural and functional changes in airways
and pulmonary vasculature associated with more severe disease but are
unable to differentiate between reversible and potentially irreversible
changes.

Finally, fractional exhaled nitric oxide (FeNO) has been proposed to
assess airway structure variations in asthma patients, especially in the
distal airway. FeNO was associated with bronchial wall thickening in the
third to the sixth generation of bronchial trees\textsuperscript{207}.

\textbf{Conclusions and perspectives}

Immunotherapy with mAbs targeting IgE, several cytokines or their
receptors has revolutionized the treatment landscape for patients with
severe asthma. mAbs that block IgE (omalizumab), the IL-5/IL-5R\selectlanguage{greek}α \selectlanguage{english}axis
(mepolizumab, reslizumab, benralizumab), IL-4R\selectlanguage{greek}α \selectlanguage{english}(dupilumab), or TSLP
(tezepelumab) can produce durable responses in the majority of patients
with severe asthma.

Airway remodeling is a cardinal feature of bronchial asthma and is
responsible for structural alterations of the airways and lung
parenchyma determining airway hyperresponsiveness and the development of
fixed airflow obstruction \textsuperscript{15}. Damaged epithelial
barrier \textsuperscript{208,} \textsuperscript{209}, subepithelial
matrix proteins and collagen deposition \textsuperscript{22,23,}
\textsuperscript{27}, infiltration and activation of inflammatory cells
\textsuperscript{57,} \textsuperscript{66,193}, goblet cell hyperplasia
\textsuperscript{44}, overexpression of inflammatory angiogenesis
\textsuperscript{16,17} and hyperplasia and hypertrophy of ASM
cells\textsuperscript{18,} \textsuperscript{31} are major features of
airway remodeling in asthma \textsuperscript{111,}
\textsuperscript{210}.

At present, we have incomplete knowledge on the short- and long-term
effects of biological therapies on airway remodeling in asthma. However,
biological therapies targeting IgE, IL-5/IL-5R\selectlanguage{greek}α, \selectlanguage{english}IL-4R\selectlanguage{greek}α, \selectlanguage{english}TSLP and
IL-33/ST2 can improve not only clinical symptoms but also certain
features (e.g., FEV\textsubscript{1}, FeNO) of airway remodeling in
asthma \textsuperscript{173,} \textsuperscript{183,211-214}. These
findings allow us to speculate that biologics could promote the
resolution of allergic inflammation. Late phases of airway remodeling
are associated with infiltration/activation of both pro-fibrotic immune
cells (e.g., mast cells, eosinophils and macrophages) and structural
cells (e.g., fibroblasts, myofibroblasts, ASM cells and endothelial
cells). Biologics may have late effects on immune and structural cells
in addition to early effects on airway inflammation. The effects of each
biologic on specific features of airway remodeling are summarized in
Table 1.

Biologics are presently used for the treatment of patients with severe
asthma who are likely to have a prolonged history of persistent and/or
repetitive immunological insults. Repetitive or prolonged injury can
lead to a pathological state of fibrosis associated with reduced lung
function. Experimental studies indicate that there is a limited-time
window in which stopping inflammation avoids fibrosis. Beyond this
time-window, tissue remodeling inevitably occurs even if inflammation is
resolved. In this circuit, macrophages, the most abundant immune cells
in the human lung, can transition between different states, including
pro-inflammatory, anti-inflammatory, and pro-fibrotic states. Although
there are no clinical and experimental data, we would like to
hypothesize that perhaps early treatment of mild/moderate asthma with
biologics might represent an innovative strategy to limit the
irreversible airway remodeling of severe asthma.

There are several limitations in studying \emph{in vivo} the effects of
biological therapies on airway tissue remodeling in asthmatic patients.
Bronchial asthma is a highly heterogeneous disorder and different forms
of airway remodeling could likely underlie several asthma
pheno-/endotypes \textsuperscript{3}. The quantitative identification of
immune cells in the airway epithelium and submucosa should also take in
consideration the functional heterogeneity of
eosinophils\textsuperscript{215}, macrophages \textsuperscript{60,193,}
\textsuperscript{216}, mast cells\textsuperscript{41,}
\textsuperscript{217-220}, neutrophils\textsuperscript{221,}
\textsuperscript{222}, and basophils\textsuperscript{40,}
\textsuperscript{223}. Potential airway remodeling biomarkers (e.g.,
galectin 3 and YKL40) are under investigation. Blood detection of these
molecules, compared to invasive methods, could be useful in the future
to evaluate bronchial remodeling\textsuperscript{224}.

In conclusion, a deeper understanding of the immunological mechanisms of
the formation of different forms of airway tissue remodeling in various
asthma phenotypes is needed. The use of single-cell transcriptomics will
be of paramount importance to chart the cellular landscape and specific
signaling networks of upper and lower airways in healthy and asthmatic
subjects \textsuperscript{193}. The results emerging from these studies
could help in the generation of new reliable diagnostic biomarkers and
targeted therapeutic approaches to improve asthma treatment.

\textbf{Figure Legends}

\textbf{Figure 1} Schematic representation of normal airway (left side)
and asthmatic airway remodeling (right side). In healthy subjects,
ciliated epithelial cells function as both a physical barrier and an
immunological organ that has evolved to defend living organisms against
pathogens and physical insults, as well to maintain tissue homeostasis.
Fibroblasts and macrophages contribute to tissue homeostasis in the
submucosa. In severe asthma, several triggers (i.e., allergens,
cytokines, cigarette smoke, microbial products, and physical insults)
damage bronchial epithelial cells that rapidly release several alarmins
(i.e., TSLP, IL-33, IL-25/IL-17E) that are constitutively expressed.
These alarmin cytokines are first reactor and rapidly initiate innate
and adaptive immune responses in asthma. Asthma is associated with
chronic inflammation and remodeling of the immune and stromal
compartments of the airway wall\textsuperscript{208, 209}. Analysis of
the immune and stromal cell populations in the bronchial biopsies of
asthmatic patients\textsuperscript{193} revealed structural changes
including mucus cell hyperplasia with a marked increase in goblet cell
numbers, which are rare in healthy airways. Subepithelial matrix protein
(collagen, fibronectin, tenascin) deposition and
fibrosis\textsuperscript{22, 23, 27, 29-31, 44, 57}, increased blood
vessel density and overexpression of angiogenic
factors\textsuperscript{16, 17}, and hyperplasia and hypertrophy of
airway smooth muscle (ASM) cells are features of airway remodeling in
asthma. The goblet cell transcriptional phenotype is altered in asthma,
with upregulation of proinflammatory and remodeling
genes\textsuperscript{193}. Mast cell numbers expressing high levels of
tryptase genes and prostaglandin D synthase are increased in asthma.
These are intraepithelial cells and tend to accumulate in asthmatic
airway epithelium\textsuperscript{225} and increase with disease
severity \textsuperscript{226}. Luminal and tissue macrophages are
increased in asthma and contribute to airway remodeling by releasing a
plethora of inflammatory and angiogenic factors \textsuperscript{58,
60}. Eosinophilia and neutrophil numbers are increased in different
asthma phenotypes\textsuperscript{57, 65, 66, 165, 189}. Pathogenic
effector CD4\textsuperscript{+} T cells are enriched in asthmatic
airways\textsuperscript{193}. Airway remodeling is also characterized by
complex interactions between inflammatory and structural cells
\textsuperscript{209}\emph{via} direct physical interactions and
secreted proteins and small molecules. A wealth of growth factor
signaling pathways including FGF, EGFR, TGF, PDGF, and VEGFs participate
in the cell-cell interactions between structural and immune cells.

\textbf{Figure 2} Here the mAbs effective in severe asthma are listed
and their known immunological mechanisms are summarized. The targets of
approved add-on biologic treatments of severe asthma include IgE
(omalizumab), IL-5 (mepolizumab and reslizumab), IL-5 receptor
(benralizumab), IL-4/IL-13 receptor complex (dupilumab) and anti-TSLP
(Tezepelumab).

\textbf{Abbreviations:}

AHR, airway hyperresponsiveness; ANGPT, angiopoietin; ASM, airway smooth
muscle; BAL, brocho-alveolar lavage; \selectlanguage{greek}β\selectlanguage{english}c, common receptor \selectlanguage{greek}β \selectlanguage{english}subunit;
COPD, chronic obstructive pulmonary disease; DC, dendritic cell; EBUS,
endobronchial ultrasound; ECM, extracellular matrix; ECP, eosinophil
cationic protein; EDN, eosinophil derived neurotoxin; EGF, epidermal
growth factor; EMT, epithelial-mesenchymal transition; EPX, eosinophil
peroxidase; Fc\selectlanguage{greek}ε\selectlanguage{english}RI, high affinity IgE; FeNO, fractional exhaled nitric
oxide; FDA, food and drug administration; FGF, fibroblast growth factor;
GINA, global initiative for asthma; GM-CSF, granulocyte-macrophage
colony-stimulating factor; HLM, human lung macrophage; HLMC, human lung
mast cell; HRCT, high-resolution computed tomography; IL, interleukins;
IL-5R\selectlanguage{greek}α, \selectlanguage{english}IL-5 receptor \selectlanguage{greek}α; \selectlanguage{english}ILC, innate lymphoid cell; i.v., intravenously;
lfTSLP, long form TSLP; mAb, monoclonal antibody; MBP, major basic
protein; MMP, matrix metalloproteinase; NET, neutrophils extracellular
trap; NHR, nuclear magnetic resonance; NK cell, natural killer cell; NKT
cell, natural killer T cell; PDGF, platelet-derived growth factor;
PGD\textsubscript{2}, prostaglandin D\textsubscript{2}; RCTs, randomized
clinical trials; sfTSLP, short form TSLP; TGF-\selectlanguage{greek}β, \selectlanguage{english}transforming growth
factor \selectlanguage{greek}β; \selectlanguage{english}TSLP, thymic stromal lymphopoietin; T2-high, type 2-high;
T2-low, type 2-low; VDP, ventilation defect; VEGF, vascular endothelial
growth factors.

\textbf{Author Contributions}

G.V., G.W.C, E.H. and J.C.W. drafted the manuscript; S.F., J.P., R.P.,
G.S., E.N. and G.P. edited the manuscript. All authors have read and
agreed to the published version of the manuscript.

\textbf{Funding}

This work was supported in part by grants from the CISI-Lab Project
(University of Naples Federico II), TIMING Project and Campania
Bioscience (Regione Campania).

\textbf{Acknowledgments}

We would like to thank Dr. Gjada Criscuolo for critical reading of the
manuscript.

\textbf{Conflicts of Interest}

\begin{itemize}
\tightlist
\item
  E.H. received grants and personal fees from: AstraZeneca, Sanofi,
  Regeneron, Novartis, GSK, Circassia, Nestl\selectlanguage{ngerman}è Purina, Stallergenes-Greer
  outside the submitted work.
\item
  G.V., S.F., J.P., R.P., G.S., have not potential conflicts of interest
  to declare.
\item
  JCV is a full time employee of the University of Rostock as a full
  time professor and chair of the departments of Pneumology and
  intensive care medicine and has lectured for and received honoraria
  from: AstraZeneca, Avontec, Bayer, Bencard, Bionorica,
  Boehringer-Ingelheim, Chiesi, Essex/Schering-Plough, GSK,
  Janssen-Cilag, Leti, MEDA, Merck, MSD, Mundipharma, Novartis,
  Nycomed/Altana, Pfizer, Revotar, Sandoz-Hexal, Stallergens, TEVA,
  UCB/Schwarz-Pharma, Zydus/Cadila has participated in advisory boards
  for Avontec, Boehringer-Ingelheim, Chiesi, Essex/Schering-Plough, GSK,
  Janssen-Cilag, MEDA, MSD, Mundipharma, Novartis, Regeneron, Revotar,
  Roche, Sanofi-Aventis, Sandoz-Hexal, TEVA, UCB/Schwarz-Pharma
\end{itemize}

and has received research grants from the Deutsche
Forschungsgesellschaft, Land Mecklenburg-Vorpommern, GSK, MSD

GWC honoraria for lectures, presentations, speakers bureaus AstraZeneca
GSK-Novartis-Sanofi-Stallergenes Greer-Hal
Allergy-Menarini-Chiesi-Mylan-Valeas-Faes.

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