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\begin{document}

\title{Endotype clustering in chronic rhinosinusitis based on the chemokine
expression pattern}



\author[1]{Ulrike Förster-Ruhrmann}%
\author[1]{Greta Pierchalla}%
\author[2]{Agnieszka Szczepek}%
\author[1]{Joachim Fluhr}%
\author[1]{Metin Artuc}%
\author[1]{Torsten Zuberbier}%
\author[3]{Claus Bachert}%
\author[1]{Heidi Olze}%
\affil[1]{Charité Universitätsmedizin Berlin}%
\affil[2]{Charite Universitatsmedizin Berlin}%
\affil[3]{University Hospital Ghent}%


\vspace{-1em}



  
  \date{June 05, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Chronic rhinosinusitis with and without nasal polyps
(CRSwNP/ CRSsNP) is an inflammatory disease affecting the nasal and
sinus mucosal lining. Here, to further characterize this heterogeneous
disease, we performed an extended endotyping of CRS using the nasal
tissue from CRS-patients and a new approach of expression profiling of
chemokines related to Th2-type cytokine IL-5. Methods: In this
case-control study, we included 66 patients with CRS (CRSwNP n=26;
CRSsNP n=40) diagnosed according to the EPOS 2020 criteria. The control
group (n=25) consisted of CRS-free patients scheduled for inferior
turbinate surgery. The concentration of following chemokines and
cytokines was determined in tissue samples obtained during routine
surgery from all subjects: TARC/ CCL17, PARC/ CCL18, eotaxin/ CCL11,
MCP-3/ CCL7, MIP-1\selectlanguage{greek}α/ \selectlanguage{english}CCL3, IP-10/ CXCL10, ENA-78/ CCL5, and IL-5. The
analysis was performed by partition-based clustering. Results: In CRS
tissues, the concentration of eotaxin, TARC, total IgE, IL-5, and ECP
was significantly higher than in control (p\textless{}0.005). The
analysis identified seven clusters. Cluster-1 was IL-5- and inflammatory
chemokines-negative (11\% CRSwNP). Cluster-2 had low IL-5 concentration
and elevated MCP-3/CCL7 (100\% CRSsNP). Clusters -3 and -4 expressed
IP-10/CXCL10 (type-1-dominated), TARC/CCL17 and eotaxin/CCL11 (both
type-2-dominated) (CRSwNP 13-31\%). Clusters 5-7 had high concentration
of IL-5, TARC/ CCL17, PARC/ CCL18, and eotaxin/CCL11 (type-2-dominated),
NP 71-100\%, asthma 19-50\%, N-ERD 29\%. Conclusions: Our chemokine
expression-based extended analysis identified distinct CRS endotype
clusters, possibly impacting future diagnosis, monitoring, and
biologics-based treatment of CRS.%
\end{abstract}%



\sloppy




\textbf{Introduction}

Chronic rhinosinusitis (CRS) impairs quality of life as well as work
productivity (1) and has a high socioeconomic burden (1). In the Global
Allergy and Asthma European Network (GA\textsuperscript{2}LEN) European
multicentre study involving 56 000 adult subjects, a prevalence rate of
CRS of 10.9\% was determined with a range from 6.9 to 27.1\% in Europe
(2). The CRS comprised heterogeneous diseases and was differentiated by
the European Position Paper on Rhinosinusitis and Nasal Polyps (EPOS
2020) in phenotypes with and without nasal polyps (CRSwNP/ CRSsNP) (3).
Subtypes are non-steroidal anti-inflammatory drugs (NSAIDs)-exacerbated
respiratory disease (N-ERD), cystic fibrosis, and the allergic fungal
rhinosinusitis (3). CRS is associated with asthma (4). Evidence-based
therapies consist of nasal corticosteroids in CRSsNP and nasal/oral
corticosteroids in CRSwNP (3). In cases without improvement under
conservative treatment, surgery of nasal sinuses is recommended
according to the EPOS 2020 criteria (3). Despite the evidence-based
therapy options, an acceptable level of disease control is still not
reached in a high proportion of patients (5). There is a particularly
high risk of disease recurrence following nasal sinus surgery for
patients with CRSwNP (5, 6). New therapy concepts developed for severe
CRSwNP consist of modified nasal sinus surgeries as reboot operations
that aim to entirely remove all nasal sinus mucosa and allow healthy
reepithelialization from the preserved nasal mucosa (7). Biological
treatment options are gaining evidence as an efficient therapy (8-10) in
CRSwNP. The CRS can be characterized by a wide range of factors, such as
histopathological findings, specific inflammatory and T-cell patterns,
tissue remodeling parameters, the concentration of eicosanoids, and IgE
production (11). The current phenotyping in CRSwNP and CRSsNP might not
sufficiently reflect pathophysiological processes within the CRS
disease. Therefore, elucidation of the pathomechanisms underlying CRS,
with the aim to develop specific and personalized therapy strategies,
might improve specific therapy options. First results of the
GA\textsuperscript{2}LEN Sinusitis Cohort study in the framework of the
European FP6 research initiative have already been published by Tomassen
et al. (12). This multicenter case-controlled study focused on the
analysis of immune markers of nasal sinus tissues from CRS patients.
This second cluster analysis followed the first analysis, and the CRS
patients were assigned to groups CRSsNP, CRSwNP, and asthma comorbidity.
The study included parameters regarding tissue remodeling,
proinflammatory cytokines, cytokines of type 1-, type 2 inflammation and
Th17 lymphocyte pattern, eosinophil and neutrophil activation markers as
well as Staphylococcus aureus enterotoxin-specific IgE (SE-IgE). Based
on the detected IL-5 concentrations, CRS was divided into ten clusters
with low/ undetectable IL-5 and moderate or high concentrations of IL-5.
The group of IL-5-negative clusters represented mainly the CRSsNP
phenotype, while with increasing IL-5 levels, the proportion of the
CRSwNP phenotype raised to 100 \%, with high IL-5 and positive SE-IgE.
The asthma prevalence increased with the elevation of IL-5 levels and
the presence of SE-IgE (12), but the presence and concentration of
chemokines were not analyzed so far.

Chemokines are small peptides (8-14 kDa) that induce chemotactic
activity in chemokine-receptor-positive cells. Chemokines influence the
activation status of the target cells and selectively regulate the
directed migration of specific inflammatory cells. They are also
involved in cell recruitment, inflammation, angiogenesis, type 1/ type 2
inflammation, wound healing, and lymphoid trafficking (13). The
classification of chemokines is based on the positioning of the first
two cysteine residues. Following chemokine families were defined: CXC,
CC, C, and CX3C (14). These families differ in function, e.g., CC
chemokines activate monocytes, basophils, eosinophils, T-lymphocytes,
and natural killer cells, whereas CXC chemokines stimulate mainly
neutrophils (15).

The general aim of the present study was analogous to previously
published GA2LEN study ''Chronic rhinosinusitis and nasal polyposis
cohort study'', aiming to match the immune patterns with the phenotypes
CRSwNP, CRSsNP, and comorbid asthma.

The primary aim of this substudy was to characterize chemokine patterns
in the tissue of CRS patients in relation to the levels of the Th2
related parameter IL-5 primarily, and subsequently assign them to the
phenotypes CRSwNP and CRSsNP and associated comorbidities asthma and
N-ERD. We analyzed the concentration of the CC-chemokines TARC/CCL17,
PARC/CCL18, eotaxin/CCL11, MCP-3/CCL7, MIP-1\selectlanguage{greek}α /\selectlanguage{english}CCL3, IP-10 /CXCL10, and
the CXC chemokine ENA-78/CCL5.

The secondary aim of the study was to examine the influences of the
additional parameters MPO, IL-17 (neutrophilic), IL-22, TNF-\selectlanguage{greek}α, \selectlanguage{english}and IFN-\selectlanguage{greek}γ
\selectlanguage{english}(Th1-associated) on chemokine patterns. The use of biologics for the
therapy of severe CRS with nasal polyps expands increasingly. Therefore,
improving the understanding of pathomechanisms behind CRS, and
performing systematic characterization of CRS endotypes could lead to
the discovery of new biomarkers, advancing the choice of specific
biologics for the treatment of uncontrolled severe CRSwNP.

\textbf{Patients and Methods}

\textbf{Patient population}

The Ethics Committee of the Charit\selectlanguage{ngerman}é University Hospital approved this
study (EA2/009/07). All investigations were conducted according to the
principles expressed in the Declaration of Helsinki. The patients
diagnosed with CRS according to the EPOS 2012 criteria (16) were
recruited to participate in the study. All subjects gave their written
informed consent. Control subjects were recruited from patients
undergoing inferior turbinate surgery but free of CRS symptoms, as per
EPOS 2012 (16). Steroids (oral/ nasal) were not taken four weeks before
surgery. Leukotriene inhibitors were not used two weeks before surgery.
Patients were considered allergic according to clinical allergy symptoms
(positive skin prick test results or elevated specific IgE-levels to
aeroallergens plus present allergy symptoms). Subjects were registered
as having asthma upon clinical diagnosis by a pulmonologist. N-ERD
diagnosis was made based on the history of reactions to NSAIDs. Of 151
patients from the Berlin cohort, 91 nasal biopsies were obtained from 66
CRS patients and 25 control subjects. The CRS group consisted of 66
patients (mean age 42.3 years {[}min 25, max 65{]} 26 females, 40
males). The control group consisted of 25 individuals (mean age 31.4
years {[}min 18; max 60{]}; 12 females; 13 males).

In detail, in the CRS group were included 26 CRSwNP patients (mean age
47.2 years {[}min 28, max 65{]} 11 females, 15 males ) and 40 patients
with CRSsNP (mean age 39.8 years {[}min 25, max 54{]}; 15 females, 25
males).

\textbf{Patients' material}

Samples from ethmoidal mucosa in the case of CRSsNP and nasal polyps
from CRSwNP or mucosal samples from the inferior turbinates in case of
controls were collected. The tissues were immediately snap-frozen in the
liquid nitrogen and stored at -80\textsuperscript{o}C. Control-tissues
were not used in cluster analyses but served as a source for standard
values.

\textbf{Measurement of inflammatory markers}

According to the study protocol, 100 mg of tissue was diluted in 1 ml of
0.9\% NaCl solution containing protease inhibitor cocktail (cOmplete\selectlanguage{english}
Protease Inhibitor Cocktail, Roche Diagnostics, Mannheim, Germany).
Next, the tissue was homogenized (1000 rpm, 5 minutes, 4degC) and
centrifuged at 1500 g for 10 minutes at 4\textsuperscript{o}C. The
protein concentration was measured in each sample using Coomassie Plus
(Bradford) assay kit (ThermoFischer Scientific, Darmstadt, Germany).

The chemokines Eotaxin/ CCL11, MIP-1\selectlanguage{greek}α/ \selectlanguage{english}CCL3, MCP-3/ CCL7, ENA-78/ CXCL5,
TARC/ CCL17, PARC/ CCL18 and IP-10/ CXCL10 (\textbf{Table 1} ) were
investigated. The chemokine concentrations were measured by using
commercially available LEGENDplex Multiple Chemokine Assay (Biolegend
San Diego USA).

The IL-5- and TNF-\selectlanguage{greek}α \selectlanguage{english}concentrations were measured with the Luminex
100-system (Luminex, Austin, Texas, USA). The concentration of the
additional cytokines (IL-22, IL-17, and IFN-\selectlanguage{greek}γ\selectlanguage{english}) was measured by using
commercially available ELISA kits from R\&D Systems (Minneapolis, USA).
The concentrations of tissue eosinophil cationic protein (ECP) and total
IgE and IgE directed against a mixture of enterotoxins from
Staphylococcus aureus (S. aureus enterotoxin A, C, and toxic shock
syndrome toxin-1) were examined using UniCAP-system (Phadia, Uppsala,
Sweden). Myeloperoxidase (MPO) was measured with ELISA (BioCheck, Foster
City, Calif., USA). The parameters measured and detection limits are
presented in \textbf{Table 2} . Values below the limit of detection were
considered to be negative.

\emph{\textbf{In agreement with the general study protocol (12), the
IL-5 levels were retrospectively defined as follows: negative IL-5
level} when} under the concentration of 12.98 pg/mL (assay detection
limit), low IL-5 level (IL-5 \textless{} 100 pg/mL), moderate IL-5 level
(IL-5 100 to 151 pg/mL) or high IL-5 level (IL-5 \textgreater{} 151
pg/mL).

\textbf{Statistical methods}

Component analysis and cluster analysis were performed following the
first study (12). The optimal number of clusters was based on the scree
plot, and additional for visualization a ``cluspot'' was drawn, plotting
subjects in two dimensions after multidimensional scaling. The variables
SAE, TNF\selectlanguage{greek}α, \selectlanguage{english}IL-17, and IFNg, \selectlanguage{english}were omitted from the cluster analysis
because they were not or barely detected in the samples. We calculated
differences between patients and controls and clusters and controls
using the Mann-Whitney U test. Between-cluster differences of all
parameters were tested by using the Kruskal-Wallis test with multiple
group-comparisons. Overall, the p-values of [?]0.05 were set to be
significant.

\textbf{Results}

\textbf{Comparison of CRS and control subjects}

The control subjects (n=25) were significantly younger (p=0.001; 31 yrs
vs. 40.0 yrs) than all CRS subjects (n=66). The proportion of gender,
smoking status, allergies, asthma, or N-ERD did not significantly differ
between CRS cases and controls. Eotaxin/ CCL11, TARC/ CCL17, total IgE,
IL-5, and ECP concentrations were significantly higher in CRS compared
to the control group (all p\textless{}0.005). The other parameters were
not significantly different in CRS cases vs. controls (\textbf{Table 3}
).

\textbf{Cluster analysis reveals 7 clusters of the chemokine expression
pattern.}

Cluster analysis of the data obtained from CRS patients, based on
chemokine measurements only (independent of clinical phenotype) resulted
in the identification of 7 distinct clusters (\textbf{Figure 1} ). The
clusters were well separated from each other. Means and ratios of
chemokines/ cytokines and phenotype data were calculated and tabulated
as a heat map (\textbf{Table 4 and Table 5} ) to characterize the
clusters. From the examined CC- and CXC chemokines, ENA-78/CCL5 revealed
no significant difference in the cluster analysis.

\textbf{Characterization of chemokines, other parameters, and clinical
phenotypes and association with the severity of IL-5 values as the main
parameter (see Tables 4 and 5):}

\textbf{Cluster group with negative/ low IL-5 levels consisted of
clusters 1-4:}

The first cluster group was categorized by undetectable IL-5 in
cluster-1 and low IL-5 levels in clusters 2-4. IL-5 revealed no
significant differences in cluster-1-3. Also, the type 2-associated
parameters ECP and total IgE did not differ significantly in clusters 1
and 2, ECP raised and was significantly elevated in clusters 3 and 4,
IL-5, and total IgE were significantly elevated in cluster-4.

\textbf{Characterization of clusters:}

\textbf{In cluster-1 (9 CRS cases)} , no significant differences were
detectable between the chemokine levels. Regarding the additional
parameters, MPO was significantly elevated compared to two or more
clusters, IL-22 was significantly higher than in three or more clusters,
and IFN-\selectlanguage{greek}γ \selectlanguage{english}(not included in the cluster analysis) was present in
cluster-1 (\emph{neutrophilic type 1-biased pattern).}

\begin{itemize}
\tightlist
\item
  \textbf{In cluster-2 (11 CRS cases)} , the MCP-3/CCL7 (expressed in
  type1/ type 2) level was significantly higher than in three or more
  other clusters. The neutrophilic additional parameter IL-17 (not
  included in the cluster analysis) was present \emph{(mixed type 1
  -\textgreater{} type 2-inflammatory pattern).}
\item
  \textbf{In cluster-3 (8 CRS cases)} , the IP-10/CXCL10 (expressed by
  type 1), MCP-3/CCL7, and MIP-1\selectlanguage{greek}α/\selectlanguage{english}CCL3 (type 1/ type 2) levels were
  significantly higher than 5 or more other clusters. The TARC/CCL17)
  and eotaxin/CCL11 (type 2) levels were significantly higher than in
  two or more other clusters \emph{(mixed type 1- \textgreater{} type
  2-inflammatory pattern).}
\item
  \textbf{In cluster-4 (13 CRS cases)} , the levels of type 2-related
  chemokines TARC/CCL17 and eotaxin/CCL11 were significantly higher than
  in two or more other clusters. Other chemokines revealed no
  significant differences. The neutrophilic type 1-biased pattern of the
  additional parameters was significantly elevated (MPO \textgreater{} 3
  other clusters; IL-22 \textgreater{} 2 other clusters), and IL-17,
  IFN-\selectlanguage{greek}γ , \selectlanguage{english}and TNF\selectlanguage{greek}α \selectlanguage{english}(not included in the cluster analysis) were present
  in cluster-4 (\emph{type 1- \textgreater{} type 2-inflammatory
  pattern} ).
\end{itemize}

\textbf{Association of endotypes to phenotypes in the negative/ low IL-5
levels cluster group:}

Nasal polyps were present in clusters 1-4 between 0\% in cluster-2 to
31\% in cluster-4. Asthma, but not N-ERD, was reported in 0 to 23\% in
clusters 1 to 4.

\textbf{Cluster group with high levels of IL-5 consisted of clusters
5-7} :

The concentration of IL-5 and also total IgE values were significantly
elevated in more than in three other clusters in cluster-5 and more than
in five other clusters in clusters 6 and 7, ECP was significantly
elevated in \textgreater{} 5 other clusters in cluster-5 and 7 and
significantly elevated in \textgreater{} than in three other clusters in
cluster-6.

\textbf{In cluster-5 (7 CRS cases)} , the levels of type-2 related
chemokines eotaxin/ CCL11, TARC/ CCL17, and PARC/ CCL18 were
significantly higher than in \textgreater{} 5 other clusters. Also, the
Th1/ Th2 chemokines MCP-3/ CCL7 and MIP-1\selectlanguage{greek}α/ \selectlanguage{english}CCL3 were significantly
higher than \textgreater{} 5 other clusters. IP-10/CXCL10 and
ENA-78/CXCL5 did not differ between the clusters. MPO and IL-22 were
significantly elevated (\textgreater{} 5 other clusters), and IFN\selectlanguage{greek}γ \selectlanguage{english}(not
included in the cluster analysis) was present in cluster-5 \emph{(strong
Th1/Th2 pattern).}

\begin{itemize}
\tightlist
\item
  \textbf{In cluster-6 (16 CRS cases)} , the eotaxin/ CCL11
  (\textgreater{}than five other clusters), PARC/CCL18, and TARC/CCL17
  levels (\textgreater{} 3 other clusters) were significantly elevated.
  SE-IgE was also significantly elevated (\textgreater{} 5 other
  clusters). MPO was significantly elevated (\textgreater{} 2 other
  clusters), and IFN-\selectlanguage{greek}γ \selectlanguage{english}(not included in the cluster analysis) was
  present in cluster-6 (\emph{type 2-inflammatory pattern} ).
\item
  \textbf{In cluster-7 (2 CRS cases)} , the chemokines TARC/CCL17,
  PARC/CCL18 were \textgreater{} 5 times and eotaxin/CCL11
  \textgreater{} 3 times significantly higher than other clusters. All
  other chemokines revealed no significant differences. SE-IgE was also
  significantly elevated in \textgreater{}5 other clusters. MPO
  (included in the cluster analysis) was significantly elevated, and
  TNF\selectlanguage{greek}α \selectlanguage{english}was present in cluster-7 (\emph{type 2-inflammatory pattern} ).
\end{itemize}

\textbf{Association of endotypes to phenotypes in the high IL-5 levels
cluster group:}

CRS with polyp formation ranged from 71 to 100\%, and asthma was present
in 43\% to 50\% of subjects. N-ERD was diagnosed in clusters 5 and 6
only, with a proportion of 19 to 29\%.

\textbf{Discussion}

Previous research (12) demonstrated that CRS could be subdivided into
endotypes based on inflammatory markers. The division of CRS into
endotypes is useful to understand the natural course of the disease and
to make a treatment choice. Here, we examined the chemokine expression
in the CRS tissues to cluster and match with the CRS phenotypes CRSwNP/
CRSsNP and the comorbidities asthma and N-ERD. To the best of our
knowledge, this is the first study analyzing this panel of chemokines in
the context of CRS. The results of the current study allow associating
chemokines signatures to the endotypes mentioned above, with type
1-related biomarkers such as IP-10/ CXCL10 and TARC/ CCL17 as well as
eotaxin/ CCL11 as early-type 2-related biomarkers. Additional
neutrophilic and type 1-related inflammatory parameters were detected in
IL-5 negative/ low and also in IL-5 high cluster groups, respectively.

Based on the chemokine expression pattern, we have identified seven CRS
clusters in the dataset of the multicenter cooperation GA2LEN ''Chronic
rhinosinusitis and nasal polyposis cohort study``(12). As analytical
targets, we selected type-2-associated CC-chemokines and a neutrophil
CXC-chemokine (\textbf{Table 1} ). Previous studies revealed that the
concentration of MIP-1\selectlanguage{greek}α/ \selectlanguage{english}CCL3 (17), eotaxin/ CCL11 (18), TARC/CCL12
(19), and PARC/CCL18 (20) were higher in tissue samples obtained from
CRSwNP patients, as opposed to the patients with CRSsNP. In addition,
MCP-3/CCL7 was found to be increased in CRS patients but not providing
distinction into CRSwNP and CRSsNP (21). Similarly, the concentration of
IP-10/CXCL10 was reported to increase in response to viral infection in
CRSwNP tissue (22). There was no difference reported in the levels of
ENA-78/CCL5 between CRS patients and healthy subjects (23). However,
none of these studies provided the classification of CRS endotypes,
which we performed here using the expression of the type 2-cytokine IL-5
as basis for differentiation.

In the clusters 1-4, IL-5 levels were negative or low in the
low\emph{inflammatory} or low mixed Th1/ Th2 \emph{inflammatory}
endotype groups. Corroborating the results of Tomassen et al. (12), we
detected a low inflammatory pattern in cluster 1 with negative IL-5
levels. In cluster 2 with low IL-5 levels, we found elevated
concentrations of a single chemokine (MCP-3/CCL7) in all patients who
suffered from CRSsNP. The MCP-3/CCL7 receptor CCR1 is known to be
expressed during Th1- and Th2 inflammatory responses, pointing to Th1
associated effects of MCP-3/CCL7 in cluster 2. In cluster 3 with mixed
Th1/Th2 inflammatory patterns, eotaxin/CCL11 - a typical Th2-chemokine -
and IP-10/ CXCL10 - a typical Th1- - were significantly elevated.
Interestingly\emph{,} in cluster-3, the levels of eotaxin/ CCL11 and
TARC/CCL17 were significantly higher than in other clusters. At the same
time, IL-5 and IgE concentrations showed no significant difference,
suggesting a possibility for using eotaxin/ CCL11 and TARC/ CCL17 as
early biomarkers of type-2 related inflammation. Eotaxin/ CCL11 serves
as eosinophilic chemokine (24) for CRSwNP patients compared to controls
(18, 25). TARC/ CCL17 is associated with a type-2-profile (19) and was
found to be elevated in fibroblasts isolated from CRSwNP patients,
following stimulation with IL-4 (26). In addition, its concentration was
higher in nasal secretions from the CRSwNP compared to CRSsNP patients
(27).

In clusters 5-7, we observed elevated concentrations of IL-5 in
association with the type-2 inflammatory profile. In addition to TARC/
CCL17 and eotaxin/ CCL11, also the concentration of PARC/ CCL18 was
significantly higher in these clusters. Type-2 cytokines upregulate
PARC/ CCL18 in the sputum of asthma patients, and PARC/ CCL18 markedly
correlates with the number of eosinophils in sputum (28). Elevated PARC
levels were found in the CRSwNP tissues (20). PARC/ CCL18 and TARC/
CCL17 are considered to be typical chemokines produced by dendritic
cells and are essential for the regulation of type-2 immune response as
well as for trafficking of memory T cells, as described in atopic
dermatitis (29). An increased number of dendritic cells were described
in nasal polyps (30). In a study of Jonstam et al. the levels of PARC/
CCL18 and the eotaxin-group eotaxin-2 and -3 (CCL24/ CCL26) decreased
significantly in nasal secretions following 16 weeks of therapy with
dupilumab (31) and also reduced in serum in recent phase 3 studies in
dupilumab \emph{versus} placebo-treated CRSwNP patients (8).

Further, the concentration of MCP-3/ CCL7 and MIP-1\selectlanguage{greek}α/ \selectlanguage{english}CCL3 (32) was
significantly greater in the ``high IL-5 group'', pointing at additional
type-2 inflammatory effects of these chemokines. Both CC chemokines are
known to chemoattract eosinophils in the type-2 inflammatory environment
(33, 34). MCP-3/ CCL7 and MIP-1\selectlanguage{greek}α/ \selectlanguage{english}CCL3 predominantly attract
type-1/type-2 cells with MCP-3/ CCL7 attracting monocytes, dendritic
cells, lymphocytes, NK cells, basophils, neutrophils, and eosinophils
via CCR1 and CCR3 (35). In contrast, MIP-1\selectlanguage{greek}α/ \selectlanguage{english}CCL3 attracts
IFN-\selectlanguage{greek}γ\selectlanguage{english}-\selectlanguage{english}activated neutrophils as well as a small subpopulation of
CCR1-expressing eosinophils (33, 34).

Of interest, a typical marker of neutrophilic inflammation -- MPO - was
significantly elevated in all clusters, stressing the impact of
neutrophiles in the not-Th2 CRSwNP with comorbid neutrophilic asthma,
and also in the Th2 CRSwNP with eosinophilic asthma (35, 36). Similarly,
the type-1 related cytokines IL-22and IFN-\selectlanguage{greek}γ \selectlanguage{english}were overexpressed in low-
and high-IL-5 clusters (clusters 1, 4, and 5; IFN-\selectlanguage{greek}γ \selectlanguage{english}also in cluster 6).
These results are comparable to the results of Tomassen et al. (12);
however, in contrast to Tomassen et al., IL-17 and TNF\selectlanguage{greek}α \selectlanguage{english}were only
present in the low IL-5 cluster group. Yet, the relatively small number
of patients per identified cluster in our study has to be considered a
shortcoming. More studies should follow to confirm our results and add
statistical weight to the findings.

Corroborating previous results (12), also in our study, the phenotype of
CRSwNP correlated positively with the IL-5 concentrations, increasing
from 11\% in cluster-1 to 100\% in cluster-7. This observation confirms
recent data about CRSwNP subjects with a negative IL-5 profile, which is
of importance to consider before making the therapeutic decision
regarding use of type 2-related biologicals. Additionally, also
non-type-2 asthma exists in CRSwNP subjects (9\% in cluster-2), in
contrast to the type-2-biased inflammatory patterns with an asthma
prevalence of 50\% in cluster-7. We diagnosed N-ERD only in type-2
endotypes, consistent with the results of Tomassen et al. The relevance
of IFN-\selectlanguage{greek}γ \selectlanguage{english}being present in N-ERD-patients was described before (37).

In conclusion, the endotype clustering of chronic rhinosinusitis based
on chemokine expression pattern has confirmed and extended previous
findings of CRS being a heterogeneous inflammatory disease (12). We
identified detailed CRS endotypes characterized by a wide diversity of
inflammatory markers. Increased levels of TARC/ CCL17, eotaxin/ CCL11,
and PARC/ CCL18 associated with type-2 biomarkers in CRSwNP, and TARC
and eotaxin may serve as early markers in patients without upregulation
of IL-5 and IgE. Additional neutrophilic biomarkers contribute to
endotype characterization in CRSsNP and CRSwNP. Our observations should
be further expanded to increase the general knowledge about CRS, to
augment the diagnostic and monitoring possibilities, and tune-up the
therapeutic approach. The recent expansion of biologics drugs used to
treat upper respiratory tract conditions fully justifies the individual,
patient-directed diagnostic approach to warrant a successful treatment
and monitoring of CRS.

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\textbf{Table 1} Origin and functions of analyzed chemokines.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Chemokines}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Origin\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Function\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
\textbf{TARC/CCL17} (\textbf{CC chemokine}) thymus and
activation-regulated chemokine CCR4, CCR8 receptor\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
secreted from monocytes, dendritic and epithelial cells (19)\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
attracts T lymphocytes (Th2)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{PARC/CCL18} (\textbf{CC chemokine}) pulmonary and activation-
regulated chemokine CCR8 receptor (38)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
secreted from antigen-presenting cells, mast cells, eosinophils and
neutrophils (39)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
attracts naive CD4\textsuperscript{+} and CD8\textsuperscript{+} T
cells, B cells, and immature dendritic cells (38, 40)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Eotaxin-1/CCL11} (\textbf{CC chemokine)} eosinophil chemotactic
protein CCR3 receptor\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
secreted from eosinophils (41)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
attracts eosinophils (42), selective on CCR3 receptor (24)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{MCP-3/CCL7} (\textbf{CC chemokine)} monocyte chemotactic
protein-3 CCR1, CCR3 receptor\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
secreted from mononuclear cells (43)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
attracts monocytes, dendritic cells, lymphocytes, natural killer cells,
eosinophils, basophils and neutrophils (44)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{MIP1α/CCL3}\selectlanguage{english} (\textbf{CC chemokine)} macrophage inflammatory
protein-1\selectlanguage{greek}α \selectlanguage{english}CCR1, CCR5 receptors\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
secreted from T- and B-lymphocytes, neutrophils, dendritic cells, mast
cells and NK cells (34)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
attracts IFN\selectlanguage{greek}γ-\selectlanguage{english}activated neutrophils and only small subpopulations of
CCR1 expressing eosinophils (34)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{ENA-78/CCL5} (\textbf{CXC chemokine)} the epithelial
cell-derived neutrophil-activating peptide CXCR2 receptor\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
secreted from monocytes, endothelial cells, mast cells, keratinocytes,
fibroblasts (45, 46)\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
attracts neutrophils (47)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{IP-10 /CXCL10} (\textbf{CC chemokine)} IFN\selectlanguage{greek}γ-\selectlanguage{english}inducible protein 10
CXCR3 receptor\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
secreted from monocytes, T-lymphocytes, fibroblasts, keratinocytes (48)
in response to the Th1 cytokine IFN\selectlanguage{greek}γ\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
attracts activated Th1 lymphocytes (49)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2} Markers used for the component and cluster analysis,
the cut-off value for markers that were analyzed as categorical.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Marker} & \textbf{Cutoff values*} & \textbf{Interpretation of
increased concentrations}\tabularnewline
Eotaxin/ CCL11 & NA & Type 2 activity\tabularnewline
MCP-3/ CCL7 & NA & Type 1 and type 2 activity\tabularnewline
MIP-1 \selectlanguage{greek}α/\selectlanguage{english}CCL3 & NA & Type 1 and type 2 activity\tabularnewline
ENA-78/CXCL5 & NA & Neutrophilic inflammation\tabularnewline
TARC/CCL17 & NA & Type 2 activity\tabularnewline
PARC/CCL18 & NA & Type 2 activity\tabularnewline
IP-10/ CXCL10 & NA & Type 2 activity\tabularnewline
ECP & NA & Type 2 activity\tabularnewline
IgE & NA & Adaptive immunity marker\tabularnewline
SE-IgE & 3.85 kU\textsubscript{A}/L* & Marker or superantigen effect on
local mucosa\tabularnewline
IL-5 & 12.98 pg/ml* & Type 2 activity\tabularnewline
MPO & NA & Neutrophilic activity\tabularnewline
IL-22 & & Type 1activity\tabularnewline
TNF-\selectlanguage{greek}α & 38.94 pg/ml* & Type 1activity\tabularnewline
IL-17 & 25.06 pg/ml* & TH17 activity\tabularnewline
IFN-g & 85.8 pg/ml* & Type 1 activity\tabularnewline
\bottomrule
\end{longtable}

*Cutoff values according to Tomassen et al. 2016 (12)

\textbf{Table 3} Descriptive statistics of CRS patients and control
subjects.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& & \textbf{Patients} & \textbf{Controls} &
\textbf{Significance}\tabularnewline
\textbf{Clinic} & Number & 66 & 25 &\tabularnewline
& Age MV & MV 40.04 & MV 30.96 & \textbf{p=0.001}\tabularnewline
& Gender male \% & 65.3\% & 56.0\% & p=0.474\tabularnewline
& Smoker former \% & 69.4\% & 64.0\% & p=0.626\tabularnewline
& Smoker current \% & 33.3\% & 48.0\% & p=0.232\tabularnewline
& Allergy \% & 26.4\% & 16.0\% & p=0.415\tabularnewline
& Asthma \% & 2\% & 26.4\% & p=0,.088\tabularnewline
& N-ERD \% & 8.3\% & 0\% & p=0.334\tabularnewline
\textbf{Laboratory Findings} & Eotaxin/ CCL11 pg/ml & MV 37.98 & MV
11.68 & \textbf{p=0.002}\tabularnewline
& MCP-3/ CCL7 pg/ml & MV 3.98 & MV 5.73 & p=0.226\tabularnewline
& MIP-1\selectlanguage{greek}α/ \selectlanguage{english}CCL3pg/ ml & MV 4.18 & MV 5.94 & p=0.334\tabularnewline
& ENA-78/ CXCL5pg/ml & MV 43.97 & MV 113.19 & p=0.063\tabularnewline
& TARC/ CCL17 pg/ml & MV 8.42 & MV 5.86 &
\textbf{p=0.000}\tabularnewline
& PARC/ CCL18pg/ml & MV 383.61 & MV 119.03 & p=0.038\tabularnewline
& IP-10/CXCL10 pg/ml & MV 424.12 & MV 274.25 & p=0.287\tabularnewline
& Total IgE kU/l & MV 337.34 & MV 30.00 &
\textbf{p=0.002}\tabularnewline
& ECP pg/ml & MV 5122.77 & MV 194.37 & \textbf{p=0.000}\tabularnewline
& IL-5 pg/ml & MV 412.83 & MV 2.91 & \textbf{p=0.000}\tabularnewline
& SAE kU/l & MV 8.34 & MV 0.00 & p=0.073\tabularnewline
& IL-22 pg/ml & MV 629.97 & MV 484.64 & p=0.283\tabularnewline
& TNF-\selectlanguage{greek}α \selectlanguage{english}pg/ml & MV 13.29 & MV 10.06 & p=0.715\tabularnewline
& IL-17 pg/ml & MV 14.56 & MV 14.49 & p=0.067\tabularnewline
& IFN-\selectlanguage{greek}γ \selectlanguage{english}pg/ml & MV 55.23 & MV 163.51 & p=0.227\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 4} Endotype profiles: Modified heat map of clustering of
individual cases. Rows define clusters of patients with CRS. Variables
used in cluster analysis are given in columns. Some parameters were used
in a categorical manner according to the high rate of measurements under
the detection limit (SAE, TNF-alpha, IL-17 and IFN-gamma). Significantly
elevated values between the CRS cluster differences are underlined.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllllllllll@{}}
\toprule
& & \textbf{Variables analyzed in cluster analysis} & \textbf{Variables
analyzed in cluster analysis} & \textbf{Variables analyzed in cluster
analysis} & \textbf{Variables analyzed in cluster analysis} &
\textbf{Variables analyzed in cluster analysis} & \textbf{Variables
analyzed in cluster analysis} & \textbf{Variables analyzed in cluster
analysis} & \textbf{Variables analyzed in cluster analysis} &
\textbf{Variables analyzed in cluster analysis} & \textbf{Variables
analyzed in cluster analysis} & \textbf{Variables analyzed in cluster
analysis} & \textbf{Variables analyzed in cluster analysis} &
\textbf{Variables not analyzed in cluster analysis} & \textbf{Variables
not analyzed in cluster analysis} & \textbf{Variables not analyzed in
cluster analysis} & \textbf{Variables not analyzed in cluster analysis}
& \textbf{Variables not analyzed in cluster analysis} &
\textbf{Variables not analyzed in cluster analysis}\tabularnewline
\midrule
\endhead
& & \textbf{ENA\_78 / CXCL5} & \textbf{MPO} & \textbf{IP-10 / CXCL10} &
\textbf{MIP1a / CCL3} & \textbf{MCP-3 / CCL7} & \textbf{TARC / CCL17} &
\textbf{PARC / CCL18} & \textbf{Eotaxi / CCL11} & \textbf{ECP} &
\textbf{IL-5} & \textbf{Total-IgE} & \textbf{IL-22} & \textbf{SAE} &
\textbf{SAE ratio} & \textbf{Ratio IL5 \%} &
\textbf{TNF-alpha-categorial} & \textbf{IL-17-categorial} &
\textbf{IFN-gamma-categorial}\tabularnewline
& & MV & MV & MV & MV & MV & MV & MV & MV & MV & MV & MV & MV & MV &
Ratio positive & Ratio positive & Ratio positive & Ratio positive &
Ratio positive\tabularnewline
\textbf{Cluster} & \textbf{1} & 32.91463 & 2866.261 & 332.7771 &
1.260987 & 1.363239 & 3.334867 & 80.5841 & 6.566953 & 686.4611111 & 6.49
& 42.74111 & 693.7425 & 0 & 0 & 0 & 0 & 0 & 0.285714\tabularnewline
& \textbf{2} & 53.37202 & 1982.33 & 346.3294 & 2.909686 & 3.019662 &
3.164666 & 127.0965 & 9.585244 & 373.86 & 30.69 & 17.185 & 372.8626 & 0
& 0 & 0.181818 & 0 & 0.090909 & 0\tabularnewline
& \textbf{3} & 82.2664 & 2426.655 & 1110.102 & 8.489828 & 6.601246 &
6.324046 & 168.232 & 18.46822 & 1294.98875 & 17.7375 & 66.88688 &
530.6479 & 0 & 0 & 0.25 & 0 & 0 & 0\tabularnewline
& \textbf{4} & 45.04413 & 4034.732 & 264.4073 & 2.613834 & 2.647437 &
6.773338 & 155.4964 & 21.36967 & 2673.592308 & 83.46462 & 142.835 &
640.023 & 0 & 0 & 0.538462 & 0.076923 & 0153846 &
0.076923\tabularnewline
& \textbf{5} & 74.70387 & 5507.627 & 173.8526 & 7.15083 & 5.415359 &
23.5649 & 881.4973 & 140.0067 & 11930.28571 & 178.0429 & 265.3933 &
931.37 & 1.241429 & 0.142857 & 0.857143 & 0 & 0 &
0.166667\tabularnewline
& \textbf{6} & 10.73267 & 2795.157 & 87.71971 & 3.10241 & 2.65226 &
9.569145 & 742.6069 & 54.08828 & 9846.44375 & 346.2869 & 524.4044 &
312.8451 & 1.459333 & 0.266667 & 1 & 0 & 0 & 0.125\tabularnewline
& \textbf{7} & 7.239 & 35603.54 & 33.57911 & 3.494613 & 2.263917 &
14.00733 & 1739.438 & 26.53524 & 15812.5 & 298.265 & 2046 & 1233.43 &
6.18 & 1 & 1 & 0.5 & 0 & 0\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 5} Association of the endotypes with the clinical
phenotypes: Summary graph.

Simplified schema of the clusters and their characteristic chemokines,
as well as the proportion of CRSwNP and CRSsNP, asthma and N-ERD. One
cluster with negative IL-5, 3 clusters with low IL-5 and 3 clusters with
high IL-5 values are given.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllllllllll@{}}
\toprule
\begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{Cluster}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{Number of cases}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{IL-5}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{IL-5 positive Ratio \%}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{IL-5}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{ECP}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{Total IgE}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{PARC}\\
\textbf{(CCL18)}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{TARC}\\
\textbf{(CCL17)}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{Eotaxin}\\
\textbf{(CCL11)}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{MCP3}\\
\textbf{(CCL7)}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\selectlanguage{greek}\textbf{MIP1α}\selectlanguage{english}\\
\textbf{(CCL3)}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{ENA-78}\\
\textbf{(CCL5)}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{MPO}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{IP-10}\\
\textbf{(CXCL10)}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{IL-22}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{NP \%}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{Asthma \%}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{N-ERD \%}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{1}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{IL-5} \textbf{Negative}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0.0\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}3\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
11\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
22\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{2}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
11\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{IL-5 low}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
18.2\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}3\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{3}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
25.0\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
13\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
0\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{4}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
13\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
53.8\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}3\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
31\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
23\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{5}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textbf{IL-5 high}\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
85.7\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}3\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}3\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
71\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
43\strut
\end{minipage} & \begin{minipage}[t]{0.03\columnwidth}\raggedright\strut
29\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{6}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
16\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
100\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}3\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}5 +SE IgE\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}3\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}3\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}2\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
81\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
19\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
19\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textbf{7}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
100\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}5 +SE IgE\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}3\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\textgreater{}5\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
100\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
50\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
0\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure Legends:}

\textbf{Figure 1} Scree plot:

The scree plot was used to determine the optimal number of clusters to
be retained in further analysis, as described elsewhere (12).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Conflict of interest:}

The authors declare no conflict of interest.

\textbf{Contributions:}

UFR data collection and laboratory measurements, analyzed and
interpreted data, manuscript draft

GP data collection, critical input to the manuscript

AJS data interpretation, manuscript draft and final version

JWF critical input to the manuscript

MA laboratory measurements, critical input to the manuscript

TZ critical input to the manuscript

CB study concept and design, critical input to the manuscript

HO study concept and design, study lead, data analysis and
interpretation, manuscript draft and final version

\selectlanguage{english}
\FloatBarrier
\end{document}

