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\begin{document}

\title{PATHOGENESIS OF NON-TYPEABLE HAEMOPHILUS INFLUENZAE INFECTIONS IN
CHRONIC SUPPURATIVE LUNG DISEASE}



\author[1]{Grigorios  Chatziparasidis}%
\author[2]{Ami Kantar}%
\author[3]{Keith Grimwood}%
\affil[1]{IASO Hospital}%
\affil[2]{Instituti Ospedalieri Bergamaschi}%
\affil[3]{Griffith University School of Medicine and Dentistry}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The respiratory tract antimicrobial defense system is a multilayered
defense mechanism that relies upon mucociliary clearance and components
of both the innate and adaptive immune systems to protect the lungs from
inhaled or aspirated microorganisms. One of these potential pathogens,
non-typeable \emph{Haemophilus influenzae} ( \emph{NTHi}), adopts
several, multifaceted redundant strategies to successfully colonize the
lower airways and establish a persistent infection. \emph{NTHi} can
impair mucociliary clearance, express multiple multi-functional adhesins
for various cell types within the respiratory tract and evade host
defenses by surviving within and between cells, forming biofilms,
increasing antigenic drift, secreting proteases and antioxidants, and by
host-pathogen cross-talk, impair macrophage and neutrophil function.
\emph{NTHi} is recognized as an important pathogen in several chronic
lower respiratory disorders, such as protracted bacterial bronchitis,
bronchiectasis, cystic fibrosis and primary ciliary dyskinesia. The
persistence of \emph{NTHi} in human airways, including its capacity to
form biofilms, results in chronic infection and inflammation, which can
ultimately injure airway wall structures. The complex nature of the
molecular pathogenetic mechanisms employed by \emph{NTHi} is
incompletely understood but improved understanding of its pathobiology
will be important for developing effective therapies and vaccines,
especially given the marked genetic heterogeneity of \emph{NTHi} and its
possession of phase-variable genes. Currently, no vaccine candidates are
ready for large phase III clinical trials.%
\end{abstract}%



\sloppy


\textbf{PATHOGENESIS OF NON-TYPEABLE \emph{HAEMOPHILUS
INFLUENZAE}INFECTIONS IN CHRONIC SUPPURATIVE LUNG DISEASE}

Grigorios Chatziparasidis MD,PhD\textsuperscript{1,2}, Ahmad Kantar MD,
PhD\textsuperscript{3,4} and Keith Grimwood MD,
FRACP\textsuperscript{5,6}

\textsuperscript{1}Paediatric Respiratory Unit, IASO Hospital, Larissa,
Thessaly, Greece.

\textsuperscript{2} Faculty of Nursing, Thessaly University, Larissa,
Greece.

\textsuperscript{3}Pediatric Asthma and Cough Centre, Instituti
Ospedalieri Bergamaschi, Bergamo, Italy.

\textsuperscript{4}Vita-Salute San Raffaele University, Milan, Italy

\textsuperscript{5}School of Medicine and Dentistry, and Menzies Health
Institute Queensland, Griffith University, Gold Coast, Queensland,
Australia.

\textsuperscript{6}Departments of Infectious Disease and Paediatrics,
Gold Coast Health, Southport, Queensland, Australia.

\textbf{Corresponding author:}

Grigorios Chatziparasidis

Pediatric Respiratory Unit, IASO Hospital, Larissa

41110 Thessaly, Greece

\emph{gchatziparasidis@gmail.com}

\textbf{Key words} : Nontypeable \emph{Haemophilus
influenzae,}respiratory infection, lung damage.

\textbf{Grants} : KG is supported by the Australian National Health and
Medical Research Council (NHMRC) Centre for Research Excellence in
paediatric bronchiectasis grant 1170958.

\textbf{Running head:} \emph{H. influenzae} and chronic suppurative lung
disease

\textbf{ABBREVIATIONS}

AECs ciliated airway epithelial cells

AI-2 autoinducer-2

AMPs antimicrobial peptides

ASL airway surface liquid

BAL bronchoalveolar lavage

C4bP C4b binding protein

CEACAM-1 carcinoembryonic antigen-related cell adhesion molecule

CF cystic fibrosis

CFTR cystic fibrosis transmembrane conductance regulator

COPD chronic obstructive pulmonary disorder

CSLD chronic suppurative lung disease

ECM extracellular matrix

eDNA extracellular deoxyribonucleic acid

FISH fluorescent in-situ hybridization

Hap \emph{Haemophilus influenzae} adhesion protein

Hia \emph{Haemophilus influenzae} adhesion

HMW high-molecular weight

ICAM-1 intracellular adhesion molecule-1

Ig immunoglobulin

IL interleukin

LOS lipo-oligosaccharide

MAC membrane attack complex

MCC mucociliary clearance

METs macrophage extracellular traps

NETs neutrophil extracellular traps

NLRP3 NLR family pyrin domain containing 3

nNO nasal nitric oxide

NTHi nontypeable \emph{Haemophilus influenzae}

OMP outer membrane protein

PAMPs pathogen associated molecular patterns

PBB protracted bacterial bronchitis

PCD primary ciliary dyskinesia

PHiD-CV \emph{Haemophilus influenzae} protein D conjugate vaccine

QSS quorum sensing system

ROS reactive oxygen species

SOD superoxide dismutase

TLR toll-like receptor

\textbf{ABSTRACT}

The~respiratory tract antimicrobial defense system~is a multilayered
defense mechanism that relies upon mucociliary clearance and components
of both the~innate and~adaptive immune systems to protect the lungs from
inhaled or aspirated microorganisms. One of these potential pathogens,
non-typeable \emph{Haemophilus influenzae} (\emph{NTHi} ), adopts
several, multifaceted redundant strategies to successfully colonize the
lower airways and establish a persistent infection. \emph{NTHi} can
impair mucociliary clearance, express multiple multi-functional adhesins
for various cell types within the respiratory tract and evade host
defenses by surviving within and between cells, forming biofilms,
increasing antigenic drift, secreting proteases and antioxidants, and by
host-pathogen cross-talk, impair macrophage and neutrophil
function.\emph{NTHi} is recognized as an important pathogen in several
chronic lower respiratory disorders, such as protracted bacterial
bronchitis, bronchiectasis, cystic fibrosis and primary ciliary
dyskinesia. The persistence of \emph{NTHi} in human airways, including
its capacity to form biofilms, results in chronic infection and
inflammation, which can ultimately injure airway wall structures. The
complex nature of the molecular pathogenetic mechanisms employed by
\emph{NTHi} is incompletely understood but improved understanding of its
pathobiology will be important for developing effective therapies and
vaccines, especially given the marked genetic heterogeneity of
\emph{NTHi} and its possession of phase-variable genes. Currently, no
vaccine candidates are ready for large phase III clinical trials.



\textbf{INTRODUCTION}

\emph{Haemophilus influenzae} are facultative, anaerobic, Gram-negative
coccobacilli requiring two accessory growth factors, X (hemin) and V
(nicotinamide adenine dinucleotide, NAD). They~are restricted human
pathogens comprising both encapsulated (a--f) and unencapsulated or
non-typeable (non-typeable \emph{H. influenzae} {[}\emph{NTHi} {]})
strains. Encapsulated serotypes cause invasive disease, while\emph{NTHi}
strains are genetically diverse and have adapted to colonize the
nasopharynx and cause local mucosal infections. \emph{NTHi} are acquired
by either person-to-person transmission from respiratory droplets or
from direct contact with respiratory secretions. Nasopharyngeal
colonization by \emph{NTHi} begins during infancy, where one-third are
colonized by 1-year of age and nearly one-half by age
2-years.Colonization rates are higher in Indigenous children, those with
older siblings, amongst childcare attendees, and during viral
respiratory infection\emph{NTHi} carriage is dynamic, with frequent
strain turnover; the same child can acquire a new strain with loss of
the old ones or carry multiple strains simultaneously, some of which are
shared with primary caregivers, suggesting that transmission is
occurring between them.

In children, \emph{NTHi} colonizing the nasopharynx can cause otitis
media, sinusitis, and conjunctivitis, while in preterm and young
infants, and those with serious underlying comorbidities, they may
occasionally invade leading to sepsis and bacteremic pneumonia. As an
upper airway colonizer, \emph{NTHi} can also access the lungs by
inhalation or via micro-aspiration episodes and direct mucosal
dispersion. In healthy children, these incursions are transient, as
microbes are removed by mucociliary clearance (MCC) and innate immune
defenses. However, \emph{NTHi} are able to colonize the lower airways of
children with chronic suppurative lung disease (CSLD) where impaired
MCC, airway wall injury, and regional microenvironments favor microbial
growth. This review describes the lower airway defenses, and
how\emph{NTHi} evades these defenses to establish infection. In so
doing,\emph{NTHi} contributes to the pathogenesis of CSLD, an umbrella
term used here to encompass protracted bacterial bronchitis (PBB),
bronchiectasis, cystic fibrosis (CF) and primary ciliary dyskinesia
(PCD). The role of \emph{NTHi} in asthma is beyond the scope of this
review and is discussed elsewhere.\textsuperscript{17}

\textbf{LOWER AIRWAY DEFENSES}

The~lung has layered defenses involving~innate and~adaptive immune
protection against infection. Airway epithelia, through their barrier
and MCC functions, production of antimicrobial peptides (AMPs),
inflammatory mediators, and ability to transport immunoglobulin (Ig) A
and IgM antibodies into the airway lumen, play a central role.

MCC is the main innate defense mechanism and involves mucus production
and its proximal transport by ciliary beating. The apical surfaces of
ciliated airway epithelial cells (AECs) are bathed by airway surface
liquid (ASL) comprised of an upper gel-like mucus layer composed
principally of MUC5AC and MUC5B mucins that entrap inhaled
microorganisms, and beneath it the periciliary fluid allowing rapid
ciliary beating. The mucus layer also secretes potent antimicrobial
molecules, such as lysozyme, defensins, IgA, and IgG. Respiratory cilia
are hair-like projections from the apical membranes of AECs that, by
beating synchronously, propel airway mucus and entrapped microorganisms
towards the oropharynx, where they are either expectorated or
swallowed.\textsuperscript{21} Respiratory cilia may also have
chemosensory, signal transduction, and cellular growth regulatory
functions. They express members of the bitter taste family of receptors
to direct innate immune defenses responding to foreign antigens.

Microorganisms penetrating the mucus layer reach a second line of
defense that includes~AMPs secreted by AECs that are activated after
sensing microbes by pattern recognition receptors, such as Toll-like
receptors (TLR). AMPs, like lysozyme and lactoferrin, are also expressed
constitutively into the ASL and are now supplemented locally by
defensins and other AMPs, cytokines, and chemokines from recruited
phagocytes and activated AECs. AMPs selectively target vital microbial
structures, taking advantage of structural and biochemical differences
between the host and the microbes. Microorganisms resistant to AMPs are
killed by~reactive oxygen species (ROS) produced by~neutrophils and
alveolar macrophages.

The third line of defense is~adaptive immunity mediated by B-lymphocytes
(humoral immunity) and T-lymphocytes (cellular immunity) where clonal
rearrangement of antigen receptor genes generates long-term
antigen-specific memory. The importance of adaptive immunity is
underlined by congenital and acquired disorders of adaptive immunity,
such as agammaglobulinemia, common variable immunodeficiency, and human
immunodeficiency virus infections, which are all risk factors for
recurrent pneumonia and bronchiectasis. Establishment of a new pathogen
in the lower airways requires them to evade these defenses, to compete
with other resident microbes, and to adapt to the nutrient availability
and physicochemical properties of the local microenvironment. Here we
describe how \emph{NTHi} adapt to survive in the lungs of children with
CSLD, where they exploit impaired local defenses and contribute to
airway wall injury.

\textbf{HOW \emph{NTHi} ESTABLISHES LOWER AIRWAY INFECTION}

Following either inhalation of respiratory droplets or micro-aspiration
and mucosal dispersion from the upper to lower airways,
\emph{NTHi}employs several strategies to establish infection:
perturbation of MCC, adherence to AECs, evading immune defenses, forming
biofilms, and scavenging iron and other essential nutrients, allowing
them to persist and survive in the lungs (Figure-1).

\textbf{Perturbation of MCC} \emph{NTHi} colonization is facilitated by
impaired MCC. Such impairment may be congenital, as in CF or PCD, or
acquired following exposure to respiratory viruses, chronic
inflammation, cigarette smoke, indoor air pollution or \emph{NTHi}
itself. In CF, mutations in the CF transmembrane conductance regulator
(CFTR) chloride channel compromise MCC by decreasing chloride and
bicarbonate secretion into the ASL, thereby reducing its volume and pH,
increasing mucus viscosity, and impeding ciliary function. While in PCD,
mutations in motile ciliopathy-associated genes result in AECs with
dyskinetic or static cilia.

Respiratory viruses, notably rhinoviruses, respiratory syncytial virus
and influenza, downregulate genes critically involved in cilia formation
through an ill-defined mechanism.Moreover, viruses can lead to shedding
of ciliated AECs or dysregulated cilial function, further reducing MCC
and increasing the risk of secondary bacterial infection. Rhinovirus
challenge studies in adults with chronic obstructive pulmonary disease
(COPD) induced bacterial co-infection in 60\% of subjects compared with
10\% of healthy, non-smoking controls. \emph{NTHi} was the principal
secondary bacterial pathogen, with bacterial load peaking at 2-weeks and
persisting for at least 6-weeks after the rhinovirus infection. Possible
mechanisms for secondary \emph{NTHi} infections are that rhinoviruses
disrupt AEC barrier function by damaging tight inter-epithelial cell
junctions, permitting \emph{NTHi} to enter paracellular
sites.Rhinoviruses and\emph{NTHi} also share a common cellular receptor
and rhinoviruses inhibit macrophage interleukin (IL)-1 responses to
\emph{NTHi} and diminish IL-8 responses via TLR-2 dependent degradation
of IRAK-1.

CSLD in children is characterized by chronic airway inflammation, which
may also adversely impact upon MCC. Chronic inflammation in asthma is
associated with decreased cilial motility, disorientated beating
direction, and ultrastructural damage. In COPD, the intraflagellar
transport of structural proteins in the respiratory cilia is
dysregulated; consequently, cilia length is shortened and less capable
of propelling the overlying mucus.Furthermore, in adults with COPD,
squamous cells replace pseudostratified epithelia, resulting in a
significant reduction in ciliated cuboidal cell numbers. Cigarette smoke
exposure also has detrimental effects upon ciliogenesis and cilia
function,decreasing CFTR activity, and it is associated with decreased
ciliary beat frequency. Nevertheless, it is difficult to disentangle the
relationships between inflammation, infection, resident lung microbiota,
and aerotoxicants, and their individual impact upon MCC.

Experimental models indicate \emph{NTHi} may also interfere with MCC
directly. \emph{NTHi} adheres initially to the mucus layer via its outer
membrane proteins (OMP), such as P2 and P5, following which isolates
expressing the highly conserved surface lipoprotein, protein-D, and
lipo-oligosaccharide (LOS) inhibit cilial function. Activation of the
host protein, protein kinase C epsilon, mediates these effects, but this
is an inconsistent finding, and penetrating paracellular foci is also
thought to be an important strategy for escaping the mucociliary
apparatus.

\textbf{Adherence to AECs}

Having bypassed MCC defenses, \emph{NTHi} employs multiple
surface-expressed multifunctional adhesion proteins, such as proteins E
and F, that bind to nonciliated AECs and extracellular matrix (ECM)
proteins, in particular laminin and vitronectin. Adherence is enhanced
when there is structural damage to the airway mucosa from viral
infections or chronic inflammation exposing ECM proteins. The trimeric
autotransporters, Hi adhesin (Hia), Hi adhesion protein (Hap), and
high--molecular weight (HMW) proteins 1 and 2, play an important role in
colonization, even though only Hap is highly conserved in
\emph{NTHi}strains. Moreover, the type IV pili on \emph{NTHi} cell
surfaces mediate adherence to AECs by interacting with the intracellular
adhesion molecule-1 (ICAM-1), which also serves as a cellular receptor
for rhinoviruses that may help explain their frequent co-infections. The
ICAM-1 receptors on the AEC surface increase in numbers in COPD
patients, in smokers, and exhibit a positive feedback loop
following\emph{NTHi} exposure. Additionally, P1 fimbriae bind to the
carcinoembryonic antigen-related cell adhesion molecule (CEACAM-1),
while P5 fimbriae adhere to both CEACAM-1 and ICAM-1 on AEC surfaces,
respectively.\textsuperscript{59,60} As well as binding to mucins, other
OMPs adhere to exposed ECM proteins.\textsuperscript{61} Indeed, the
interaction between the ECM exposed within the COPD airway environment
and Hap facilitates \emph{NTHi} attachment to AECs and this interaction
may also operate in the lungs of children with CSLD.

\textbf{Evasion of immune defenses}

Having overcome the first layer of host lung defenses, \emph{NTHi} must
also evade innate and adaptive immune responses. Pulmonary phagocytic
cells (alveolar macrophages and neutrophils) constantly sense the local
microenvironment for pathogen associated molecular patterns (PAMPs),
which are molecules with highly conserved motifs associated with human
pathogens. PAMPs bind to pattern recognition receptors, such as TLRs,
which activate intracellular pathways to secrete chemotaxins, including
IL-8 to recruit and upregulate neutrophils to the site of infection, and
to initiate intracellular killing of phagocytosed
pathogens.\textsuperscript{64} When phagocytes encounter
\emph{NTHi}there is a sustained release of ROS, both within
intracellular phagolysosomes and into the extracellular environment. The
generation of ROS is also associated with neutrophil and macrophage
extracellular traps (NETs and METs, respectively), which are composed of
a mesh of DNA, histones and bactericidal proteins to neutralize invading
pathogens. The production of ROS represents the primary mechanism
adopted by these cells to eliminate phagocytosed pathogens.
Nevertheless, \emph{NTHi} can neutralize the effects of oxidative stress
by producing antioxidants, including catalase,
peroxiredoxin-glutaredoxin and superoxide dismutase (SOD), which in
addition to forming biofilms is one of the mechanisms it uses to resist
NET.

Furthermore, submucosal plasma cells produce dimeric IgA molecules,
which bind to an epithelial cell membrane protein, the secretory
component, and are transported through AECs to be released as secretory
IgA into mucosal secretions.\textsuperscript{67} IgA aids lung defenses
by preventing microbial adherence, neutralizing toxins and boosting
adaptive immunity. There are two types of IgA (IgA1 and IgA2) of which
IgA1 accounts for more than 90\% of the total IgA. However,
\emph{NTHi}produces an IgA1 protease, IgaA, encoded by the \emph{IgaA}
gene as an extracellular endopeptidase that cleaves IgA1 at the hinge
region, disabling its antibacterial functions and aiding adherence to
and invasion of AECs. Approximately half the \emph{NTHi} strains also
possess a second IgA1 protease gene, \emph{IgaB,} that encodes IgaB,
which seems necessary for intracellular survival.

The human complement system is also part of the innate defenses against
pathogenic bacteria. It has multiple functions and when activated
complement mediates the inflammatory response and elimination of
pathogens and enhances adaptive immunity. The complement system leads to
C3b deposition on the bacterial cell surface with formation of the
membrane attack complex (MAC) and bacterial lysis. There are three
complement activation pathways, which can all be inhibited by\emph{NTHi}
. The classical and lectin pathways are blocked by the C4b binding
protein (C4bP), whereas factor H is believed to be main inhibitor of the
alternative pathway. C4bP and factor H are captured by OMP P5 and
protect\emph{NTHi} against opsonization and phagocytosis, while also
preventing MAC formation. Vitronectin, a potent negative regulator of
the terminal pathway can also be bound by \emph{NTHI} proteins E and F,
further inhibiting the formation of the MAC by \emph{NTHi} . Moreover,
LOS covering the outer cell wall of \emph{NTHi} can block bactericidal
antibodies accessing cell surface structures and activating the
classical complement pathway.\textsuperscript{73} Finally,
\emph{NTHi}may benefit from other co-pathogens, such as \emph{Moraxella
catarrhalis,} and their inhibition of the complement
system.\textsuperscript{74}

\textbf{Persistence and survival of \emph{NTHi} in the lungs}

After adhering successfully to AECs, \emph{NTHi} must still obtain
nutrients and continue evading host
defenses.\textsuperscript{,}\emph{NTHi} have absolute growth
requirements for iron, but are unable to synthesize heme, which is
essential for their survival. Consequently,\emph{NTHi} has multiple core
and accessory genes producing proteins expressed on the cell surface to
acquire iron from free heme and hemoglobin sequestrators, such as
hemopexin and haptoglobin.\textsuperscript{77} However, the exact
mechanisms remain poorly understood because of the number, diversity and
functional redundancy of the encoding genes. Recent studies identified
that some of these genes are phase-variably expressed and rapidly
acquire point mutations during persistent infection, resulting in
accumulated amino acid changes in surface exposed regions of iron
acquisition proteins, suggesting these are survival strategies following
immune selection pressures.\textsuperscript{79} Other OMPs, such as P2,
also undergo antigenic drift with key amino acid sequence changes
occurring in key epitopes, which enable \emph{NTHi} to persist despite
the presence of potentially protective antibodies.

\emph{NTHi} utilize phase variation to facilitate adaptation and
survival within multiple niches throughout the respiratory tract. Phase
variation is the random and reversible switching of gene expression,
which permits reversible loss or gain of surface structures, such as
adhesins (HMW1 and HMW2), LOS and iron-acquisition proteins (heme
receptors and hemagglutinating pili).\textsuperscript{81} Phase-variable
genes have changes in the length of simple sequence repeats and when
located in the open reading frame of a gene this variation in length
determines if the encoded protein is either expressed (ON) or not (OFF).
Phase-variable DNA methyltransferases also control the expression of
multiple genes involved in colonization and survival within different
ecological niches and such systems are termed phasevarions to describe
phase-variable regulons.\textsuperscript{81} Taken together, these
random changes in gene expression enable \emph{NTHi} to generate
phenotypic variants, which are better suited to the local lung
environment or more able to evade host defenses.

Widely regarded as an extra-cellular pathogen, \emph{NTHi} can access
and survive in the paracellular spaces between AECs due to the adherence
and invasion protein, HMW1,\textsuperscript{85} and following
downregulation of cadherin and claudin proteins responsible for
epithelial cell-to-cell interactions and epithelial tight junction
integrity, respectively.\textsuperscript{86} As a further survival
strategy, \emph{NTHi} can also invade mononuclear cells and AECs by
utilizing multiple virulence factors, including IgaA protease, LOS via
the platelet-activating factor receptor leading to cytoskeletal
remodeling, several adhesins, including protein D, Hap, or P1 fimbrae
binding to the CEACAM-1 receptor and the phase-variable adhesin HMW1 and
protein E binding to vitronectin.\textsuperscript{69,85,87} Under iron
restricted conditions within the lungs, \emph{NTHi} enter AECs by
macropinocytosis to form bacterial communities within the
cytoplasm.\textsuperscript{88} Once inside the cell, \emph{NTHi} must
adapt to the low pH and increased oxidative state within the phagosome
of macrophages or the limited nutrient availability within the AEC
cytoplasm. Relatively little is known of how \emph{NTHi} survives and
traffics within this environment. Transcriptional factors, such as the
ferric uptake regulator cassette which regulates the expression of
multiple genes controlling the uptake and utilization of iron, and IgaB
that cleaves lysosomal-associated membrane protein-1, appear to be
important, and \emph{NTHi} can survive for at least 72-hours inside
AECs.

Furthermore, lung explant cultures from COPD and CF patients undergoing
transplantation for end-stage lung disease revealed that despite
negative sputum and lung tissue cultures, \emph{NTHi} was detected
widely throughout the lung parenchyma and lower airway tissue sections
by immunoperoxidase staining and polymerase chain reaction assays. While
most NTHi were detected in clusters in extracellular and paracellular
locations, in CF patients they were found within macrophages too. The
discrepancy between culture and non-culture results was attributed to
pre-operative antibiotics and the inhibitory effects
of\emph{Pseudomonas} aeruginosa upon
\emph{NTHi}growth.\textsuperscript{91} However, paracellular and
intracellular locations of \emph{NTHi} and biofilms may also be
important.

\textbf{Biofilm aggregates}

\emph{NTHi} can form biofilms (Figure-2), which are aggregated bacterial
communities encased in an ECM of polysaccharides, proteins and
extracellular DNA (eDNA) that adhere to a surface.\textsuperscript{92}As
biofilms mature, the resident bacteria undergo a series of behavioral
changes, including differential gene expression and increased ECM
production, in response to increasing cell density and environmental
stresses from decreasing oxygen tension and nutrient
availability.\textsuperscript{93} Crucial bacterial factors for biofilm
formation include LOS, pili, eDNA, and a functioning quorum sensing
system (QSS). Bacteria utilize QSS for inter-cellular communication
where cells collectively regulate their gene expression in response to
cell density as sensed by the concentration of small soluble autoinducer
signal molecules produced and secreted by
bacteria.\textsuperscript{94}In \emph{NTHi} , biofilm development and
dispersal are mediated by autoinducer-2 (AI-2), which is controlled by
the LuxS/RbsB system regulating LOS and pili expression, including PilA,
P2 and P6. DNA-NET-like structures captured by DNA-binding proteins from
the DNABII family form a mesh network within biofilms where they help
make up important structural components.\textsuperscript{95}

The ECM of biofilms helps protect \emph{NTHi} from environmental
stresses, including acting as a physiochemical barrier to cellular and
innate host defenses and to antimicrobial agents. For example, in
addition to stimulating biofilm formation, eDNA binds the AMP, human
beta-defensin-3, while peroxiredoxin-glutaredoxin and catalase protect
against neutrophil-induced oxidative stress, and IgA proteases cleaving
IgA are found on the ECM.\textsuperscript{96} There are several
potential mechanisms within biofilms capable of mediating antimicrobial
resistance. These include the (i) ECM limiting antibiotic access to
bacteria within the biofilm, (ii) negatively charged eDNA sequestering
positively charged antibiotics, (iii) hypoxic and nutrient restricted
environment deep within biofilms transforming bacteria into a
semi-dormant, metabolically inert state rendering antibiotics relying
upon active cell growth and division ineffective, and (iv) horizontal
gene transfer of antibiotic resistance genes that result in deactivating
enzymes, altered membrane permeability and cellular targets, and
upregulated multidrug efflux pumps.\textsuperscript{97} Nevertheless,
despite much in-vitro and experimental model work on biofilms, evidence
for their presence in patients with CSLD is relatively limited. They
have however, been detected in the sputum of adults with CF and
COPD,\textsuperscript{98,99} and in bronchoalveolar lavage (BAL) fluid
from children with PBB and
bronchiectasis.\textsuperscript{100}Importantly, by using fluorescent
in-situ hybridization (FISH) staining in a small subset of BAL samples,
researchers were able to demonstrate the polymicrobial nature of the
biofilms, which included in addition to\emph{NTHi} other recognised
respiratory pathogens (\emph{Streptococcus pneumoniae, M. catarrhalis} )
and upper airway commensals, such as\emph{Prevotella}
species.\textsuperscript{100}

\emph{NTHi}\textbf{-induced host damage}

Incursion by \emph{NTHi} into the lower airways induces both innate and
adaptive immune responses. \emph{NTHi} infection stimulate TLRs and NLR
family pyrin domain containing 3 (NLRP3) inflammasomes stimulate AECs to
release AMPs and proinflammatory mediators, including tumor necrosis
factor-alpha, IL-6 and IL-8, that recruit and activate macrophages and
neutrophils leading to increased ROS production, and NETs formation, the
latter designed to immobilize and destroy pathogens. In this
hyperinflammatory environment, high concentrations of proteolytic
enzymes, such as neutrophil elastase, and ROS are released by activated
and dying neutrophils, with the latter leading to local increases in
free radicals. Despite these host defensive measures, \emph{NTHi} are
resistant to NETs and survive oxidative stress by forming biofilms and
possessing antioxidants, while also capitalizing on the ROS-induced
tissue damage as a nutrient source to establish and sustain their
presence in the airways. The tissue damage results from excessive
oxidative stress secondary to \emph{NTHi}persistence and an imbalance
between oxidants and antioxidants, leading to oxidation-induced damage
of proteins, lipids, and DNA in the airways. These changes, labelled by
some as NETosis, contribute to a pathway leading to impaired lung
function and destruction of local airway wall architecture as
demonstrated by airway hyperresponsiveness, mucus hypersecretion, AEC
shedding, vascular exudation, and, finally, airway remodeling. Moreover,
the damaged mucosa facilitates \emph{NTHi}persistence because of further
disruption to MCC and the higher affinity of \emph{NHTi} for damaged
tissues, which sustains the airway inflammatory response and promotes a
vicious cycle of infection, inflammation and impaired bacterial
clearance. (Figure-3).

\emph{NTHi} \textbf{and chronic suppurative lung diseases}

\emph{NTHi} is a normal upper airway commensal, but when it becomes
established in the lower airways it elicits a strong, but ineffective
inflammatory response.\textsuperscript{26} As it is an important
pathogen in patients with COPD, much of our understanding of the
clinical implications of chronic \emph{NTHi} infection is from this
patient group. Indeed, in COPD it accounts for nearly half of all
isolates and is responsible for over 30\% of disease exacerbations.
Moreover, the severity of COPD exacerbations is associated
with\emph{NTHi} airway colonization. However, large knowledge gaps
remain to explain why \emph{NTHi} only affects a subset of COPD patients
and what host-pathogen and microbial (eg. bacteria-bacteria and
virus-bacteria) interactions cause this
susceptibility.\textsuperscript{112} There is accumulating evidence that
the biofilm-phase of \emph{NTHi} may be important and knowing more about
the mechanisms involved may promote targeted treatment of this chronic
disease. Recent studies question the ability of antibiotics to eradicate
lower airway \emph{NTHi} infection in COPD patients since antibiotics do
not prevent reinfection, nor do they alter the disease
course\textsuperscript{76}. Consequently, there has been substantial
interest in creating an effective \emph{NTHi}vaccine.

\emph{NTHi} is the major pathogen in bronchiectasis and is accompanied
by airway neutrophilic infiltration and inflammatory markers, such as
IL-8 and neutrophil elastase. Alveolar macrophages from PBB and
bronchiectasis patients have reduced capacity to phagocytose
\emph{NTHi}and apoptotic neutrophils, allowing the latter to become
necrotic and release ROS and proteolytic enzymes into the
airways.\textsuperscript{115} NETs, which in bronchiectasis are a key
marker of disease severity,\textsuperscript{116} may induce IL-17
secreting lymphocytes (Th17 and NK cells) in bronchial submucosa to
further drive neutrophil recruitment and mucus hypersecretion
perpetuating the cycle of inflammation.\textsuperscript{117} Compared to
healthy controls, peripheral blood mononuclear cells from bronchiectasis
patients also have an attenuated \selectlanguage{greek}γ-\selectlanguage{english}interferon response to \emph{NTHi} ,
but not to mitogens, suggesting a specific impairment in cell mediated
immunity,\textsuperscript{118} which improves after receiving the highly
conserved \emph{NTHi} surface protein, protein D, incorporated into the
pneumococcal \emph{Haemophilus influenzae} protein D conjugate vaccine
(PHiD-CV).

In CF patients \emph{NTHi} are most often detected in children and
adolescents and can be the predominant pathogen cultured in BAL fluid
from young children.\textsuperscript{14} Nevertheless, their role as
pathogens has been questioned.\textsuperscript{119} However, studies
from the 1990s revealed that high \emph{NTHi} loads
(\emph{\textgreater{}} 10\textsuperscript{5} colony-forming units) in
BAL cultures were associated with significantly higher neutrophil and
IL-8 levels than when no pathogens were
present.\textsuperscript{120,121} A North American CF registry-based
study also found that isolating\emph{NTHi} from respiratory secretions
was associated with accelerated decline in lung function in the
transition from adolescence to adulthood.\textsuperscript{122} Recently,
\emph{NTHi} isolates from CF patients inoculated into a murine lung
model induced chronic neutrophilic inflammation driven by Th17 cells and
IL-17 cytokines. Taken together, these studies suggest a pathogenic role
for \emph{NTHi}in CF patients.

In PCD, nasal nitric oxide (nNO) levels are dramatically reduced for
uncertain reasons. Recent in-vitro data suggest low levels of nNO make
it easier for \emph{NTHi} to adhere to primary AECs from PCD patients
and form biofilms, while exogenous nitric oxide with azithromycin
enhanced bacterial killing in biofilms compared to azithromycin alone.

PBB and bronchiectasis are on a continuum, and in children they have
similar lower airway pathogen profiles, core microbiota, neutrophilic
airway inflammation and impaired cellular immune responses to\emph{NTHi}
.\textsuperscript{40,128-130} A recent 5-year follow-up study of 194
children with PBB reported recurrent PBB and \emph{NTHi} in BAL cultures
predicted a future diagnosis of bronchiectasis, emphasizing the
importance of \emph{NTHi} in disease progression and outcome.

\textbf{Summary}

\emph{NTHi} is a highly adapted human commensal and opportunistic lower
airway pathogen possessing multi-function virulence factors exhibiting
efficient adherence, immune evasion and nutrient scavenging properties.
These allow it to invade cells and form biofilms in order to survive
within the lungs of children with CSLD. The molecular pathogenesis
of\emph{NTHi} is not fully understood and large knowledge gaps exist
over its mechanistic role in progressive lung disease. Identifying
critical cellular pathways and, including host-pathogen cross-talk and
interactions with other lung microbes, are crucial for developing novel
therapeutic agents and vaccine candidates
(Box).\textsuperscript{132}NTHi vaccines are an active area of research,
but \emph{NTHi} strains have both marked genetic heterogeneity from
recombination with other\emph{NTHi} and phase-variability, and no
vaccine candidate is ready for large field trials. Two recent,
relatively small vaccine trials, one in children with CSLD targeting
\emph{NTHi} protein D in the PHiD-CV vaccine and another in adults with
COPD targeting three\emph{NTHi} surface proteins, failed to meet their
primary outcome of reducing exacerbations, despite both demonstrating
good immunogenicity highlighting that further mechanistic studies are
needed involving\emph{NTHi} and patients with
CSLD.\textsuperscript{133,134}

\textbf{FIGURE LEGENDS}

\textbf{Figure-1} . The main strategies used by
nontypeable\emph{Haemophilus influenzae} to establish a persistent
infection in the lower airways of patients with chronic suppurative lung
disease.

\textbf{Figure-2.} \emph{NTHi} biofilm life cycle. (A) \emph{NTHi} in
planktonic form escapes local airway defenses and irreversibly attaches
to receptors on airway cells. (B) After evading local niches,\emph{NTHi}
aggregates and starts the production of extracellular polymeric
substance matrix which favors \emph{NTHi} survival in the form of
biofilm. (C) Biofilm properties enhance growth and survival
of\emph{NTHi} and other bacteria. (D) Maturation of biofilm is followed
by\emph{NTHi} dispersal in planktonic forms to subsequently develop
biofilms in secondary niches. \emph{NTHi} : nontypeable
\emph{Haemophilus influenzae} . \emph{Created by biorender.com}

\textbf{Figure-3} . Schematic representation of \emph{NTHi} -induced
host damage. AMs: alveolar macrophages, AMPs: antimicrobial peptides,
Aps: antioxidant peptides, IL: interleukin, NE: neutrophil elastase,
NKs: natural killer cells, NPs: neutrophils, \emph{NTHi} :
nontyeable\emph{Haemophilus} influenzae, ROS: reactive oxygen species,
Th: T helper cells. \emph{Created by biorender.com}

\textbf{REFERENCES}

\textbf{Box: Future research questions on the pathogenesis and role
of\emph{NTHi} infections in pediatric chronic suppurative lung disease}

Can animal models replicate the complexity of \emph{NTHi} infection in
CSLD?

What are the important interactions between \emph{NTHi} and the host
microbiome?

What role do cilia play in \emph{NTHi} host-pathogen interactions?

How long do \emph{NHTi} survive within cells and in intercellular
spaces?

How long do \emph{NTHi} strains survive in the lungs? Are they replaced
or constantly changing as a result of intraspecies transformation and
recombination of genes from the supragenome?

What are the critical functions of host-pathogen cross-talk that
allow\emph{NTHi} to evade or manipulate host defences?

What part does NETosis have in the pathogenesis of CSLD?

What is the role of \emph{NTHi} biofilms in progressing CSLD and how
might this be overcome by increasing susceptibility to host defences and
antimicrobial agents?

What are the regulatory mechanisms controlling a relatively small number
of multi-functional proteins that allow \emph{NTHi} to adapt to
environmental changes?

What is the nature, timing and frequency of antigenic, epigenetic and
phase variation in lung \emph{NTHi} isolates?

What are the critical phase-variable genes for establishing
\emph{NTHi}lung infection and how they might be important targets for
vaccine candidates?

Can \emph{NTHi} virulence be manipulated by small molecules targeting
the quorum sensing system?

What are the mechanisms of antimicrobial resistance in biofilms and
where is the reservoir of antibiotic resistant genes?

What impacts do CFTR modulators have upon \emph{NTHi} infection in CF
patients?

What are the host mechanisms of protective immunity?

Will multi-valent vaccines containing highly conserved surface antigens,
including antigens under phase-variable control expressed during
critical periods of establishing infection overcome the challenges
of\emph{NTHi} genetic heterogeneity and protect against
\emph{NTHi}infection?

CF: cystic fibrosis, CFTR: cystic fibrosis transmembrane conductance
regulator, CSLD: chronic suppurative lung disease, \emph{NTHi} ,
nontypeable \emph{Haemophilus influenzae.}

\textbf{Figure-1} . The main strategies used by
nontypeable\emph{Haemophilus influenzae} to establish a persistent
infection in the lower airways of patients with chronic suppurative lung
disease.

\textbf{Figure-2.} \emph{NTHi}biofilm life cycle. (A) \emph{NTHi} in
planktonic form escapes local airway defenses and irreversibly attaches
to receptors on airway cells. (B) After evading local niches,
\emph{NTHi} aggregates and starts the production of extracellular
polymeric substance matrix which favors\emph{NTHi} survival in the form
of biofilm. (C) Biofilm properties enhance growth and survival of
\emph{NTHi} and other bacteria. (D) Maturation of biofilm is followed by
\emph{NTHi} dispersal in planktonic forms to subsequently develop
biofilms in secondary niches. \emph{NTHi} : nontypeable
\emph{Haemophilus influenzae} . \emph{Created by biorender.com}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure-3} . Schematic representation of \emph{NTHi} -induced
host damage. AMs: alveolar macrophages, AMPs: antimicrobial peptides,
Aps: antioxidant peptides, IL: interleukin, NE: neutrophil elastase,
NKs: natural killer cells, NPs: neutrophils, \emph{NTHi} :
non-typable\emph{Haemophilus} influenzae, ROS: reactive oxygen species,
Th: T helper cells. \emph{Created by biorender.com}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\selectlanguage{english}
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