10.14639/0392-100X-N1800
Objective. We analysed calprotectin in sinonasal secretions of different chronic rhinosinusitis with nasal polyps (CRSwNP) endotypes to assess its role as a biomarker of non-type 2 inflammation. Methods. We included primary diffuse CRSwNP patients (n = 41) and three different control groups [non-allergic rhinitis (NAR) (n = 13), non-allergic eosinophilic syndrome (NARES) (n = 10) and healthy subjects (n = 12)]. Calprotectin levels were detected in nasal secretions using a chemiluminescent immunoassay (CLIA). Results. Calprotectin levels in nasal secretions were significantly higher in all non-type 2 endotypes of CRSwNP compared to healthy controls (p &lt; 0.05). In contrast, in type-2 CRSwNP calprotectin was significantly lower compared to controls (p &lt; 0.05). A significant correlation between calprotectin levels and neutrophilic count/HPF was found in CRSwNP (p &lt; 0.01). Clinically, mean levels of calprotectin and neutrophilia were significantly higher in patients who previously underwent 3 or more endoscopic sinus surgeries (p &lt; 0.05). Conclusions. Calprotectin in nasal secretions may be a biomarker of non-type 2 inflammation. Low levels of calprotectin are indicative of a type-2 immune response in both CRSwNP and non-allergic rhinitis. We observed that calprotectin levels significantly increased based on the number of previous surgeries.
text/xml
1827-675X
1800
en
Jacopo Galli
http://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
precision medicine
Calprotectin in nasal secretion: a new biomarker of non-type 2 inflammation in CRSwNP
Text.Serial.Journal
https://www.actaitalica.it/
42
Rhinology
2022-06-30
2021-08-25
2022-10-12
2022-10-12
355-363
1
Jacopo Galli
2022/06/30
10.14639/0392-100X-N1800
355
A. Gemelli Hospital Foundation IRCCS, Unit of Otorhinolaryngology, Head and Neck Surgery, Rome, Italy; Catholic University of the Sacred Hearth, Faculty of Medicine and Surgery, Rome, Italy
1827-675X
precision medicine
en
363
Calprotectin in nasal secretion: a new biomarker of non-type 2 inflammation in CRSwNP
https://www.actaitalica.it/article/download/1800/810
42
ACTA OTORHINOLARYNGOLOGICA ITALICA 2022;42:355-363; doi: 10.14639/0392-100X-N1800




Rhinology


Calprotectin in nasal secretion: a new biomarker
of non-type 2 inflammation in CRSwNP
Calprotectina nel secreto nasale: nuovo biomarker di infiammazione non-tipo 2
nella poliposi nasale
Eugenio De Corso1, Silvia Baroni2,3, Maria Elisabetta Onori3, Laura Tricarico3, Stefano Settimi1, Giacomo Moretti3,
Eliana Troiani3, Rodolfo Francesco Mastrapasqua3, Daniela Furno3, Fabrizio Crudo3, Andrea Urbani2,3, Jacopo Galli1,3
1
 A. Gemelli Hospital Foundation IRCCS, Unit of Otorhinolaryngology, Head and Neck Surgery, Rome, Italy; 2 A. Gemelli Hospital
Foundation IRCCS, Unit of Chemistry, Biochemistry and Clinical Molecular Biology, Rome, Italy; 3 Catholic University of the Sacred
Hearth, Faculty of Medicine and Surgery, Rome, Italy


SUMMARY
Objective. We analysed calprotectin in sinonasal secretions of different chronic rhinosi-
nusitis with nasal polyps (CRSwNP) endotypes to assess its role as a biomarker of non-type
2 inflammation.
Methods. We included primary diffuse CRSwNP patients (n = 41) and three different
control groups [non-allergic rhinitis (NAR) (n = 13), non-allergic eosinophilic syndrome
(NARES) (n = 10) and healthy subjects (n = 12)]. Calprotectin levels were detected in nasal
secretions using a chemiluminescent immunoassay (CLIA).
Results. Calprotectin levels in nasal secretions were significantly higher in all non-type
2 endotypes of CRSwNP compared to healthy controls (p < 0.05). In contrast, in type-2
CRSwNP calprotectin was significantly lower compared to controls (p < 0.05). A significant
correlation between calprotectin levels and neutrophilic count/HPF was found in CRSwNP
(p < 0.01). Clinically, mean levels of calprotectin and neutrophilia were significantly higher
in patients who previously underwent 3 or more endoscopic sinus surgeries (p < 0.05).
Conclusions. Calprotectin in nasal secretions may be a biomarker of non-type 2 inflam-                Received: August 25, 2021
mation. Low levels of calprotectin are indicative of a type-2 immune response in both                 Accepted: January 15, 2022
CRSwNP and non-allergic rhinitis. We observed that calprotectin levels significantly in-              Published online: June 30, 2022
creased based on the number of previous surgeries.
                                                                                                      Correspondence
KEY WORDS: sinusitis, nasal polyps, rhinitis, immunophenotyping, precision medicine                   Stefano Settimi
                                                                                                      Head and Neck Department, Faculty of Medicine
RIASSUNTO                                                                                             and Surgery Catholic University of Sacred Heart,
Obiettivo. Abbiamo analizzato i livelli di calprotectina nelle secrezioni nasali di diversi           largo F. Vito 1, 00168 Rome, Italy
                                                                                                      Tel. +39 06 30154439. Fax +39 06 3051194
endotipi di CRSwNP, per valutarne il ruolo di marcatore di infiammazione non-tipo 2.
                                                                                                      E-mail: settimi.stefano90@gmail.com
Metodi. Abbiamo incluso pazienti con CRSwNP diffusa primaria (n = 41) e tre diversi
gruppi di controllo [rinite non allergica (NAR) (n = 13), rinite eosinofila non allergica (NA-
RES) (n = 10) e soggetti sani (n = 12)]. I livelli di calprotectina sono stati dosati mediante          How to cite this article: De Corso E, Bar-
test di chemoluminescenza.                                                                              oni S, Onori ME, et al. Calprotectin in na-
Risultati. I livelli di calprotectina sono risultati significativamente più alti in tutti gli endo-     sal secretion: a new biomarker of non-
tipi non-tipo 2 di CRSwNP, rispetto ai controlli sani (p < 0,05). Al contrario, nella CRSwNP            type 2 inflammation in CRSwNP. Acta
                                                                                                        Otorhinolaryngol Ital 2022;42:355-363. https://
tipo 2 essa è risultata inferiore rispetto ai controlli (p < 0,05). È stata riscontrata una cor-
                                                                                                        doi.org/10.14639/0392-100X-N1800
relazione significativa tra i livelli di calprotectina e la conta dei neutrofili/HPF (p < 0.01).
I livelli medi di calprotectina aumentano inoltre nei pazienti in precedenza sottoposti a 3 o
                                                                                                      © Società Italiana di Otorinolaringoiatria
più ESS (p < 0,05).                                                                                   e Chirurgia Cervico-Facciale
Conclusioni. La calprotectina nelle secrezioni nasali può essere considerata un biomar-
catore di infiammazione non-tipo 2 mentre bassi livelli di calprotectina sono indicativi di                          OPEN ACCESS
immunoflogosi di tipo 2. Abbiamo infine osservato che i livelli di calprotectina aumentano
significativamente in relazione al numero di interventi chirurgici pregressi.                         This is an open access article distributed in accordance with
                                                                                                      the CC-BY-NC-ND (Creative Commons Attribution-Non-
PAROLE CHIAVE: rinosinusite, polipi nasosinusali, rinite, immunofenotipizzazione,                     Commercial-NoDerivatives 4.0 International) license. The
                                                                                                      article can be used by giving appropriate credit and mentio-
medicina di precisione                                                                                ning the license, but only for non-commercial purposes and
                                                                                                      only in the original version. For further information: https://
                                                                                                      creativecommons.org/licenses/by-nc-nd/4.0/deed.en




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E. De Corso et al.




Introduction                                                      tients compared to healthy controls, reflecting lower epithe-
                                                                  lial production of these peptides. Furthermore, they observed
Calprotectin is a member of the S100 protein family, con-         that calprotectin levels correlated with levels of neutrophils,
sisting of two sub-units (S100A8/S100A9), produced by             as assessed by means of quantification of neutrophil elastase.
neutrophils, monocytes and epithelial cells, which partici-       Recently, Sumsion 9 reviewed several manuscripts highlight-
pate in the innate immune response by binding specific re-        ing the expression patterns of S100 proteins in CRS hypoth-
ceptors (TLR4 and RAGE) 1. Calprotectin may have several          esising that they may have an important role in mediating
functions in both the internal and the extracellular compart-     immunity in different subtypes of CRS. Unfortunately, stud-
ments by promoting anti-microbial and pro-inflammatory            ies investigating differential expression of S100A8/9 in en-
responses. Its exact function depends on the local tissue         dotypes of CRSwNP are lacking.
environment in which it is released. Firstly, it may be in-       The objective of this manuscript was to analyse calprotec-
volved in the recruitment and trans-endothelial migration         tin (S100A8/S100A9) levels in sino-nasal secretions of dif-
of neutrophils and monocytes by stimulating epithelial            ferent CRSwNP endotypes and investigate the relationship
cells to produce IL-8 and TNF-a, an important chemoat-            with neutrophilia. We tried to verify the hypothesis that cal-
tractant for these cells. Secondly, it has direct antimicrobial   protectin may be a biomarker of non-type 2 CRSwNP. Fur-
activity against bacteria and fungi, sequestering extracel-       thermore, we evaluated the expression of nasal calprotectin
lular divalent metal cations that are essential for bacterial     based on clinical characteristics of CRSwNP.
growth 2. Furthermore, cytoplasmic calprotectin is crucial
for cytoskeleton reorganisation and phagocyte migration
by conferring resistance to intracellular invading bacte-         Materials and methods
ria 3,4. Finally, the importance of this protein in maintaining
epithelium barrier integrity has been demonstrated, which         Study design and study population
is related to its tissue-protective functions by inhibiting the   This was a non-profit analytical case-control observation-
production of reactive oxygen species (ROS) and activity          al study performed at our institution “A. Gemelli Hospi-
of matrix metalloproteinases (MMPs) that cleave a vast            tal Foundation IRCCS”, Otorhinolaryngology, Rhinology
range of cytokines, chemokines, receptors, proteases, and         Unit, Catholic University of Sacred Heart-Rome, Italy. Pa-
adhesion molecules to alter their function 5.                     tients were screened and enrolled between October 2019
There are conflicting reports in the literature regarding         and January 2021.
whether calprotectin may play protective or pathologic roles      Inclusion criteria:
in patients with chronic rhinosinusitis (CRS). The expres-        • male or female 18-65 years old;
sion patterns of calprotectin in CRS patients may signifi-        • primary diffuse CRSwNP according to EPOS 2020 10;
cantly differ if measured in nasal secretions, polyp tissue       • significant nasal symptoms (SNOT-22 > 20);
and normal sinonasal mucosa at different subsites. Richer 6       • willingness and ability to provide written informed con-
showed that the gene expression of S100A7 and S100A8                 sent and to comply with all study related procedures.
in epithelial cells of sinonasal mucosa was significantly de-     Exclusion criteria:
creased in CRS compared with controls, resulting in a de-         • acute exacerbation of CRS (AECRS) as defined in the
creased proliferative or aberrant regenerative capacity of the       EPOS 2020 10;
respiratory epithelium, particularly in response to injury. On    • local or systemic steroid treatment less than 4 weeks be-
the other hand, Van Crombruggen 7 showed that tissue levels          fore the inclusion;
of S100A8/A9 were significantly higher in nasal polyp tis-        • active smokers;
sue from CRSwNP patients compared to inferior turbinates          • previous nasal surgery (septoplasty, cosmetic surgery
of controls. The authors observed a significant correlation          etc.);
between tissue levels of IL-8 and S100A8/A9, demonstrat-          • current malignancy;
ing that IL-8 produced by activated epithelium or resident        • previous radiotherapy for head neck cancer.
macrophages was a strong chemoattractant for neutrophils.         Based on inclusion and exclusion criteria we enrolled 41
Finally, Tieu 8 showed that the expression of S100A8/A9           primary diffuse CRSwNP (17/41 females; mean age 42.6;
was significantly increased in polyp tissue compared to tur-      range 18-65). We identified 3 different groups as controls:
binate and uncinate tissue from chronic rhinosinusitis with       13 non-allergic rhinitis (NAR) patients, 10 non-allergic
nasal polyps (CRSwNP), chronic rhinosinusitis without na-         eosinophilic syndrome (NARES) patients, and 12 healthy
sal polyps (CRSsNP) and controls; however, its levels were        subjects (no history of rhinitis/CRS, negative allergy tests,
significantly lower in the nasal lavage fluid of CRSwNP pa-       no nasal inflammation at nasal cytology, no evidence of na-



356
                                                                                                                         Calprotectin: biomarker of non-type 2 inflammation




Table I. Epidemiological data and clinical features of patients and controls.
                                                     Primary diffuse                              NAR                           NARES                     Healthy controls
                                                         CRSwNP                                 (n = 13)                       (n = 10)                      (n = 12)
                                                          (n = 41)
 Age, years, median (IQR)                                       42.6                            52 (17)                         39 (15)                        44 (14)
 Sex, female, n/total (%)                                   17/41 (41.5%)                    4/13 (30.7%)                     6/10 (60%)                     6/12 (50%)
 Asthma, n/total (%)                                        14/41 (34.1%)                        None                         3/10 (30%)                        None
 ASA                                                        9/41( 21.9%)                         None                         2/10 (20%)                        None
 intolerance, n/total (%)
 Atopy,                                                     17/41 (41.5%)                         None                           None                       4/12 (33.3%)
 n/total (%)
 Peripheral blood iper-eosinophilia, n/total (%)            10/41 (24.4%)                         None                        5/10 (50%)                         None

 Smoking, yes n/total (%)                                   16/41 (39%)                        None                         2/10 (20%)                     4/12 (33.3%)
 SNOT-22, mean                                                  33.2                            12.5                           21.9                            77.22
 LKES, mean                                                     13.3                             0                               0                               0
 Mean CT Lund Mackay score                                      13.3                             0                               0                             None
 Eosinophil count/HPF, mean±SD                               35.3 ± 5.9                          0                          52.4 ± 19.4                          0
 Neutrophil count/HPF, mean±SD                               27.9 ± 9.8                       3 ± 6.3                            0                           2.5 ± 4.5
 Mean number of surgery±SD                                    1.6 ±1.0                       0.1 ± 0.3                           0                               0
 Calprotectin ng/mL, mean±SD                             207.8 ± 97.6 ng/ml              118.6 ± 46.1 ng/ml              62.8 ± 31.2 ng/ml               77.3 ± 31.6 ng/ml
CRSwNP: Chronic Rhinosinusitis with Nasal Polyps; HPF: High Power Field; LKES: Lund Kennedy Endoscopic Score; NAR: non-allergic rhinitis NARES: Non-infectious non-allergic rhinitis
with eosinophilia syndrome; Non-EOS: Non-Eosinophilic; SNOT: sinonasal outcome test; SD standard deviation; ASA: acetylsalicylic acid; IQR: interquartile range.



sal polyps at nasal endoscopy). Clinical features of patients                                 (Farmark snc, Milan). Samples mounted on a slide and
and control groups are shown in Table I.                                                      stained by the May-Grunwald-Giemsa method. Cell
                                                                                              counts were expressed as mean value per high-power-
Study protocol                                                                                field (HPF) on a mean of at least 10 richest observed fields
All enrolled patients underwent:                                                              at high magnification (x 400). Percentage of eosinophils
• baseline interview including previous treatments, fam-                                      and neutrophils on total cellularity was also provided 14.
   ily history, asthma (based on previous pulmonologists                                      Eosinophilic inflammation was assumed if eosinophil
   evaluation), hypersensitivity to non-steroidal anti-in-                                    count was ≥ 10/HPF (x400) and eosinophils accounted
   flammatory drugs (NSAID) (based on a reported history                                      for more than 20% of total cellularity. To date, there is no
   of adverse reactions or on previous allergological evalu-                                  specific landmark to define type 2 inflammation by nasal
   ation). Eosinophilia was assumed for blood eosinophil                                      cytology; for this reason, in order to be more rigorous,
   count > 500/microliter. We divided patients into aller-                                    we adopted that used for histological findings (eosinophil
   gic/non-allergic and asthmatic/non-asthmatic according                                     count: > 10/HPF) combined with the one used for nasal
   to the ARIA project 11. Data on recently performed CT                                      cytology (eosinophils 20% of total cellularity) suggestive
   scans were collected;                                                                      of significant eosinophilia. Neutrophilic inflammation
• allergy testing: total immunoglobulinE (IgE), skin prick                                    was assumed if the neutrophil count was ≥ 20/HPF (x400)
   testing (SPT) for common inhalant allergens (at least 18),                                 with neutrophil accounting for over 50% of total cellular-
   specific IgE in serum by Radio-Allergo-Sorbent-Test                                        ity. Mixed inflammation if both eosinophilic count ≥ 10/
   (RAST) and, if indicated, nasal provocation test to ex-                                    HPF and neutrophilic count ≥ 20/HPF were observed;
   clude local allergy in patients who were negative by SPT;                                • nasal lavage fluid collection was obtained from subjects
• symptom score analysis by the validated Italian version                                     with the head bent down, as previously described 15-17. We
   SNOT-22 (Score range: 0-110 12);                                                           instilled 5 ml of saline solution (NaCl 0.9%) pre-warmed to
• nasal endoscopy bilaterally: findings were noted using                                      35°C into each nostril and used it to rinse the nostrils sever-
   the Lund-Kennedy Endoscopic Score (LKES) 13. A com-                                        al times. All fluid was then collected by asking the subjects
   bined score (right + left side) of 0-20 was possible;                                      to lean forward and blow the nasal contents gently into a
• nasal cytology: material was obtained by scraping of                                        funnel connected to a 30 ml universal container. The lavage
   lower/middle turbinate mucosa bilaterally by rhinoprobe                                    fluid was filtered to remove any nasal mucus and centri-



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E. De Corso et al.




   fuged immediately at 4000 revolutions per minute (RPM)         following incubation, a wash cycle is performed to remove
   for 5 minutes. It was then divided in aliquots and frozen at   any unbound material. An isoluminol conjugated monoclo-
   -80°C until the assays were performed.                         nal antibody that recognises calprotectin is then added to the
Eligible patients were grouped, according to nasal disorder,      reaction and incubated. The unbound conjugate is removed
as follows:                                                       with a second wash step. Starter reagents are then added,
• primary diffuse CRSwNP was defined according to                 and a flash chemiluminescent reaction is initiated. The light
   EPOS 2020 10. All patients underwent CT scan if not per-       signal is measured using a photomultiplier as relative light
   formed in the last 6 months and staged by Lund-Mackay          units (RLU), which is proportional to the concentration of
   score (LMS). Endotypes were established based on the           calprotectin present in the calibrators, controls, or samples.
   cellular pattern (defined by nasal cytology) and bio-          The kit measures the level of calprotectin in faeces in μg/g,
   chemical pathways measuring most relevant cytokines            then dividing it by 7500, with a value that corresponds to
   (IL-4, IL-5, IL-17, INF-g and TNF-a) in nasal secretion        mg/L calprotectin in nasal lavage fluid. In our study, we re-
   by ELISA 10. Type-2 was defined in case of eosinophilic        port the calprotectin value in ng/ml.
   inflammation at nasal cytology and if IL-4/IL-5 were
   measured in nasal secretion according to literature da-        Statistical analysis
   ta 15,18,19 (Tab. I). In case of neutrophilic inflammation,    Statistical analysis was performed using SPSS.25 for Win-
   Type-1 was defined when IFN-gamma/TNF-alpha were               dows (Chicago, Illinois). Continues values such as levels of
   detected, whereas Type-3 in case of IL-17 detection;           calprotectin, symptom scores, endoscopic scores, cell count,
   mixed endotypes were also described as shown in Ta-            are expressed as mean ± standard deviation (SD). Compari-
   ble II. CRSwNP patients were further divided according         sons between groups were performed using the Mann-Whit-
   to the dominant cellular pattern as follows:                   ney U-test for non-normally distributed data and t-test for
• eosinophilic CRSwNP (EOS-CRSwNP) (n = 15): pure                 paired samples in normally distributed values; when com-
   Type-2;                                                        paring more than two groups we used 1-way ANOVA and
• non-eosinophilic (Non-EOS-CRSwNP) (n = 12): pure                Bonferroni post-hoc analysis. The Chi-square test was used
   Type-1 or Type-3;                                              to compare categorical data such as prevalence and incidence.
• mixed CRSwNP (n = 14): mixed Type 2-3, Type 1-2.                The strength of the correlation between the two parameters
• nAR (n = 13): persistent rhinitis, negative allergy tests in    was obtained by Spearman’s rank correlation test. The results
   vivo/vitro, LKES = 0, LMS = 0; no evidence of eosino-          were considered significant for p-values < 0.05.
   philic inflammation at nasal cytology;
• NARES (n = 10): persistent rhinitis, no evidence of ato-
                                                                  Results
   py allergy in vivo/vitro, marked nasal hypereosinophilia
   at nasal cytology.                                             Calprotectin levels in nasal secretion of different endotypes
                                                                  of CRSwNP
Biochemical assays                                                Calprotectin levels based on dominant endotype of
Multiple cytokines were detected in nasal secretions by           CRSwNP are shown in Table II and Figure 1. Mean levels in
high-sensitivity ELISA (HS Quantikine Human Cytokine              nasal secretions of type-1 and type-3 CRSwNP were signif-
Immunoassay; R&D Systems, Minneapolis, US). Assays                icantly higher compared to healthy controls: 170.37 ± 70.2
were performed in duplicate during a single session and the       vs 77.3 ± 31.6 ng/ml (p < 0.05) and 434.73 ± 100.2 vs
mean values were calculated. The coefficient of variation         77.3 ± 31.6 ng/ml (p < 0.05), respectively. Contrarily, in
(CV) of duplicates was always less than 3%. The sensitivity       type-2 CRSwNP calprotectin was significantly lower com-
of cytokines assays were specifically: 1.2 pg/ml for IL-4,        pared to controls (31.6 ± 15.8 ng/ml vs 77.3 ± 31.6 ng/ml;
1.08 pg/mL for IL-5, 0.51 pg/mL for IL-17, 5.1 pg/ml for          p < 0.05). The highest levels were observed in type-3 endo-
IFN-g and 6.3 pg/ml for TNF-a.                                    type vs type-1 and 2 (p < 0.05). Significantly higher levels
Calprotectin was determined by DiaSorin-LIAISON as-               of calprotectin were also detected in mixed type 2-3 and
say, an in vitro diagnostic chemiluminescent immunoassay          type 1-2 CRSwNP compared to healthy controls (p < 0.05).
(CLIA) intended for the quantitative measurement of faecal        The inter-group comparisons revealed that mean calprotec-
calprotectin. This is a sandwich assay using two monoclonal       tin levels in type-1 and type-3 endotype dominant CRSwNP
antibodies for capture and detection of the molecule. The         patients were significantly higher compared to type-2:
samples are incubated with assay buffer and paramagnetic          170.37 ± 70.2 vs 31.6 ± 15.8 (p < 0.05) and 434.73 ± 100.2
particles coated with a monoclonal antibody that specifical-      vs 31.6 ± 15.8 (p < 0.05), respectively. Furthermore, mean
ly recognises the heterometric calprotectin (S100A8/A9);          calprotectin levels were significantly higher in mixed



358
                                                                                                                 Calprotectin: biomarker of non-type 2 inflammation




Table II. Endotype dominance of patients included with primary diffuse chronic rhinosinusitis with nasal polyps. Values of cytokines are indicated as mean ± SD.
               No. of     Eosinophil        Neutrophil      IL-5 (ng/L)           IL-4            IL-17         TNF-a            INF-g          Calprotectin
              patients    count/HPF         count/HPF                            (ng/L)           (ng/L)         (ng/L)         (ng/L)              ng/ml

 Type 1             5           3 ± 6.7          37.4 ± 8.0        0.6 ± 0.2          0.9 ± 0.7         0          1.4 ± 0.6      1.7 ± 0.6       170.37 ± 70.2
 Type 2            15         36.2 ± 8.2         0.6 ± 1.2         3.0 ± 2.0          2.9 ± 1.3     0.1 ± 0.2      0.1 ± 0.2     0.05 ± 0.2        31.6 ± 15.8
 Type 3             6              0              47 ± 6.7         0.9 ± 0.4          1.2 ± 0.9     1.2 ± 0.3      0.4 ± 0.3     0.4 ± 0.3       434.73 ± 100.2
 Type 2-3           5         16.7 ± 5.1        41.3 ± 10.6        3.3 ± 2.8          1.6 ± 0.3      2 ± 0.8       0.5 ± 0.9     0.5 ± 0.9       270.86 ± 89.2
 Type 1-2          10          27 ± 3.5           39 ± 6.9         3.7 ± 1.8          1.9 ± 0.9         0          0.4 ± 0.7     0.4 ± 0.7       268.42 ± 87.2



                                                                                        By Spearman’s rank test, we observed a significant corre-
                                                                                        lation between calprotectin levels and neutrophilic count/
                                                                                        HPF in all patients (rs: 0.692; p < 0.01) (Fig. 2) and in all
                                                                                        CRSwNP patients (rs: 0.882; p < 0.01) (Fig. 3). A signifi-
                                                                                        cant correlation was found between calprotectin levels and
                                                                                        eosinophil count/HPF (rs: 0.127; p > 0.05).

                                                                                        Calprotectin levels in nasal secretion based on different
                                                                                        clinical characteristics of CRSwNP
                                                                                        The mean calprotectin in nasal lavage of CRSwNP patients
                                                                                        was significantly higher compared to healthy subjects
                                                                                        (200.2 ± 71.3 ng/ml vs 77.3 ± 31.6 ng/ml; p < 0.05). Ana-
                                                                                        lysing concomitant factors, calprotectin levels were high-
                                                                                        er in non-atopic versus atopic (237.1 ± 109.3 ng/ml vs
                                                                                        153.9 ± 98.2 ng/ml; p > 0.05) and in non-asthmatic ver-
                                                                                        sus asthmatic (213.4 ± 98.9 ng/ml vs 186.2 ± 101.1 ng/ml;
                                                                                        p > 0.05) even though the difference was not statistically
Figure 1. Box plots showing mean levels of calprotectin (ng/ml) based on
CRS endotyping. The box plot shows the median and interquartile range and               significant. No significant correlation was found between
the error bars show the 5th and 95th percentiles. Statistical significance is given     calprotectin levels and SNOT-22 (p > 0.05), LMS (p > 0.05)
as p < 0.05.                                                                            and LKES (p > 0.05) by Spearman’s rank test.


type 2-3 and type 1-2 compared to type-2: 270.86 ± 89.2
vs 31.6 ± 15.8 (p < 0.05) and 268.42 ± 87 vs 31.6 ± 15.8
(p < 0.05), respectively.
Mean concentrations of calprotectin in nasal lavage of dif-
ferent subtypes of CRSwNP based on the cellular dominant
pattern is reported in Table III. The mean concentration of
calprotectin in non-EOS-CRSwNP and Mixed-CRSwNP
was significantly higher compared to healthy subjects:
324.5 ± 159.7 ng/ml vs 77.3 ± 31.6 ng/ml (p < 0.05) and
269.6 ± 94.5 ng/ml vs 77.3 ± 31.6 ng/ml (p < 0.05), re-
spectively. Mean levels of calprotectin in EOS-CRSwNP
were significantly lower compared to healthy controls
(31.6 ± 15.8 ng/ml vs 77.3 ± 31.6 ng/ml; p < 0.05).
At the inter-group comparison, we observed higher mean
levels of calprotectin in non-EOS-CRSwNP and in mixed-
CRSwNP compared to EOS-CRSwNP (p < 0.05). A dif-                                        Figure 2. Scatter plot showing intra-individual correlations between calpro-
                                                                                        tectin and neutrophil count/HPF in all patients (NAR; NARES and CRSwNP) ex-
ference was observed between non-EOS-CRSwNP and                                         cluding healthy subjects (rs = correlation coefficient assessed by Spearman’s
mixed-CRSwNP (p > 0.05).                                                                rank correlation test. Statistical significance is given as p < 0.05).



                                                                                                                                                               359
E. De Corso et al.




                                                                                          performed a subgroup analysis in Type-2 patients observ-
                                                                                          ing that levels of calprotectin were significantly higher in
                                                                                          patients who underwent 3 or more surgeries compared with
                                                                                          those who underwent fewer than 3 surgeries (280.4 ± 109.3
                                                                                          ng/ml vs 92.5 ± 41.2 ng/ml; p < 0.05). In conclusion, the
                                                                                          levels of calprotectin significantly increased in both Type-2
                                                                                          patients with multiple surgeries and in non-Type 2, but the
                                                                                          difference was not statistically significant (280.4 ± 109.3 ng/
                                                                                          ml ng/ml vs 222.9 ± 92.1 ng/ml; p > 0.05).

                                                                                          Nasal lavage calprotectin in CRSwNP vs
                                                                                          non-allergic-rhinitis
                                                                                          In order to compare CRSwNP patients to upper respiratory
                                                                                          diseases with overlapping pathophysiology, but without na-
Figure 3. Scatter plot showing intraindividual correlations between calpro-               sal polyp development, we evaluated calprotectin levels in
tectin and neutrophil count/HPF in CRSwNP patients (rs = correlation coeffi-
cient assessed by Spearman’s rank correlation test). Statistical significance is          non-allergic-rhinitis patients with eosinophilic inflamma-
given as p < 0.05).                                                                       tion (NARES) or without it (NAR). The average concen-
                                                                                          tration of calprotectin in non-EOS CRSwNP was signifi-
                                                                                          cantly higher compared to NAR (324.5 ± 159.7 ng/ml vs
Finally, we observed that levels of calprotectin were signifi-                            118.6 ± 46.1 ng/ml; p < 0.05) and NARES (324.5 ± 159.7 ng/
cantly different based on the number of previously performed                              ml vs 62.8 ± 31.2 ng/ml; p < 0.05). The difference between
endoscopic sinus surgeries (ESS) (Tab. IV). Calprotectin pro-                             EOS-CRSwNP and NAR was significant (31.6 ± 15.8 ng/
gressively increased with the number of surgeries, as shown                               ml vs 118.6 ± 46.1 ng/ml; p < 0.05), whereas the difference
in Figure 4. The inter-group comparison revealed that mean                                between EOS-CRSwNP and NARES was not (31.6 ± 15.8
levels of calprotectin and neutrophilia were significantly                                ng/ml vs 62.8 ± 31.2 ng/ml; p > 0.05). Box plots summaris-
higher in patients who previously underwent 3 or more ESS                                 ing the levels of calprotectin in all groups are reported in
compared to the other groups (p < 0.05). Furthermore, we                                  Figure 5.

Table III. Clinical features of patients based on cellular dominant patter of inflammation associated to CRSwNP.
                                                               Non-EOS                  Eosinophilic                Mixed                                   Healthy
                                                               CRSwNP                     CRSwNP                  CRSwNP                                    controls
                                                            (Type 1; Type 3)              (Type 2)           (Type 1-2; Type 2-3)                           (n = 12)
                                                                (n = 12)                  (n = 15)                 (n = 14)
 Age, years, median (IQR)                                          44 (17)                       38 (12)                      46 (14)                      44 (14)
 Sex, female, n/total (%)                                       2/12 (16.6%)                 11/15 (73.3%)                4/14 (28.5%)                   6/12 (50%)
 Asthma, n/total (%)                                            2/12 (16.6%)                  5/15 (33.3%)                 7/14 (50%)                       None
 ASA intolerance, n/total (%)                                    1/12 (8.3%)                   3/15 (20%)                 5/14 (35.7%)                      None
 Atopy, n/total (%)                                              3/12 (25%)                   5/15 (33.3%)                9/14 (64.3%)                  4/12 (33.3%)
 Peripheral blood hyper-eosinophilia, n/total (%)                   None                       6/15 (40%)                 4/14 (28.5%)                      None
 Smoking, yes n/total (%)                                       5/12 (41.6%)                  5/15 (33.3%)                6/14 (42.8%)                  4/12 (33.3%)
 SNOT-22, mean                                                      29.12                         39.77                       40.05                         77.22
 LKES, mean                                                         11.1                          13.3                         14.9                           0
 Mean CT Lund Mackay score                                           11.1                          13.3                        14.9                         None
 Eosinophil count/HPF, mean ± SD                                      0                         36 ± 5.0                     34 ± 7.3                         0
 Neutrophil count/HPF, mean ± SD                                  43 ± 12.8                     0.6 ± 2.5                   40 ± 14.9                     2.5 ± 4.5
 Mean number of surgery ± SD                                       1 ± 1.0                      1.4 ± 0.7                   2.2 ± 1.0                         0
 Calprotectin ng/mL, mean ± SD                               324.5 ± 159.7 ng/ml            31.6 ± 15.8 ng/ml           269.6 ± 94.5 ng/ml            77.3 ± 31.6 ng/ml
CRSwNP: Chronic Rhinosinusitis with Nasal Polyps; HPF: High Power Field; LKES: Lund Kennedy Endoscopic Score; NAR: non-allergic rhinitis NARES: Non-infectious non-allergic rhi-
nitis with eosinophilia syndrome; Non-EOS: Non-Eosinophilic; SNOT: sinonasal outcome test; SD standard deviation; ASA: acetylsalicylic acid; IQR: interquartile range.




360
                                                                                                       Calprotectin: biomarker of non-type 2 inflammation




Table IV. Mean levels of calprotectin (ng/mL) based on number of previous
surgeries.
 Number of previous      Number of         Neutrophils       Mean
 surgeries                patients          (N)/HPF       calprotectin
                                                             ng/ml
 0                            36              29.4             110.5
 1                            22              22.5             141.5
 2                            10              23.5             184.4
 3                             5              43.3             341.7
 4                             4              42.5             363.2




                                                                              Figure 5. Box plots showing mean levels of calprotectin (ng/ml) in nasal
                                                                              secretions (NAR: non-allergic rhinitis; NARES: non-allergic eosinophilic syn-
                                                                              drome; CRSwNP chronic rhinosinusitis with nasal polyps). The box plot shows
                                                                              the median and interquartile range and the error bars show the 5th and 95th
                                                                              percentiles. Statistical significance is given as p < 0.05.


                                                                              ers of Type-1 or Type-3 inflammation are especially lacking
                                                                              because they have received less attention, and much informa-
                                                                              tion has not been confirmed at the protein level 22,23.
                                                                              To our knowledge, this is the first study analysing calpro-
                                                                              tectin in nasal secretions of different CRSwNP endotypes.
                                                                              According to our previous experience 15-17, we believe that
Figure 4. Box plot showing mean levels of calprotectin (ng/ml) of CRSwNP      nasal secretion may be representative of the different in-
patients based on the number of previous endoscopic sinus surgery per-
formed. The box plot shows the median and interquartile range and the error
                                                                              flammatory cytokine patterns associated with CRSwNP.
bars show the 5th and 95th percentiles.                                       We firstly demonstrated that levels of calprotectin were
                                                                              significantly higher compared to controls in all endotypes
                                                                              associated with significant neutrophilia (Type-1, Type-3,
Discussion                                                                    mixed Type 1-2 and Type 2-3). The highest levels were ob-
                                                                              served in type-3 consistent with literature data showing a
CRSwNP is a complex and heterogeneous disorder with
                                                                              more pronounced neutrophilia in this endotype 10. Accord-
distinct pathophysiologic mechanisms. With the advent of                      ingly, we observed a significant correlation between calpro-
more individualised treatments, the efforts of researchers                    tectin levels and neutrophil count in all patients (rs:0.692;
have been focused on identifying the molecular pathway(s)/                    p < 0.01) and a stronger one considering only CRSwNP
inflammatory endotypes that have been activated in an in-                     (rs:0.882; p < 0.01). Interestingly, calprotectin levels ob-
dividual patient rather than analysis of the unknown and                      served in Type-2 CRSwNP were significantly lower than in
aetiologic factors that may cause CRSwNP 10.                                  healthy subjects.
Over the last years, significant progress has been made in char-              The analyses based on cellular dominant pattern of inflam-
acterising the inflammatory endotypes in CRSwNP. Nowa-                        mation associated with CRSwNP further support our hy-
days, it is clear that chronic inflammatory response in CRS                   pothesis that calprotectin may be considered as a biomark-
may utilise Type-1, 2 or 3 pathways of the immune response.                   er of non-Type 2 inflammation. In fact, we observed that
The selective or combined expression of Type-1, 2, or 3 im-                   the levels of calprotectin in nasal secretions of non-EOS
mune responses which gives rise to significant disease het-                   CRSwNP (Type 1-3) were significantly higher compared to
erogeneity 20. Type-2 inflammation associated with CRSwNP                     EOS-CRSwNP (Type-2). In addition, we demonstrated that
has been widely investigated and seems to respond better to                   mixed CRSwNP (type 1-2 and 2-3) had significantly higher
biologic therapy compared to non-Type 2 driven CRSwNP                         levels of calprotectin compared to the control group and
(Type-1 and Type-3) 21. Nevertheless, it is still unclear which               to EOS-CRSwNP; these patients showed the same magni-
biomarkers are most accurate at predicting specific endotypes                 tude of calprotectin levels compared to non-EOS CRSwNP,
and their impact on clinical disease. In particular, biomark-                 probably due to a comparable mean neutrophil count/HPF



                                                                                                                                                     361
E. De Corso et al.




in the two groups. Accordingly, in the past, some authors 8        recent evidence 10 suggests that they may be associated with
demonstrated an increased level of calprotectin in nasal           Type-2 or non-Type 2 inflammation as well as CRSwNP.
polyp tissue which reflects neutrophil recruitment as a            In conclusion, our data suggest that the role of nasal secreted
compensatory mechanism. Our data support the hypoth-               calprotectin in CRS seems to be promising as biomarker of
esis that levels of calprotectin in nasal secretions reflect       non-Type 2 inflammation; our data suggest that increased
the magnitude of neutrophil recruitment. As demonstration          levels of calprotectin in nasal secretions may be the expres-
of this, mean calprotectin levels in NARES patients were           sion of a pure non-type 2 mechanism or of the neutrophilic
comparable to those in EOS-CRSwNP and significantly                component in a mixed pattern of inflammation. To our knowl-
lower compared to non-EOS CRSwNP. NARES patients                   edge, this is the first study analysing calprotectin levels in
have overlapping pathophysiology with EOS-CRSwNP,                  nasal secretions based on different CRSwNP endotypes. The
and neither are typically associated with neutrophilia.            conflicting data in the literature regarding calprotectin in na-
The analyses of calprotectin based on the clinical character-      sal secretions may be explained by the heterogeneity of the
istics of CRSwNP revealed higher mean calprotectin levels          disease and the different proportion of non-type 2 endotypes
in atopic and asthmatic CRSwNP compared to non-atopic              included in different series. For this reason, we suggest that
and non-asthmatic, even though the differences were not            the levels of calprotectin should always be defined based on
statistically significant. The relationship between atopy and      endotypes of the disease and on the proportion of neutro-
calprotectin is still matter of discussion. Interestingly, Kato    philic infiltration in mixed patterns.
et al. 24 observed in vitro on cultured scraped epithelial cells   The role of biomarkers in the identification of different endo-
that the intracellular calprotectin concentration in unstimu-      types of CRSwNP seems to be crucial to improve the results
lated cells from EOS-CRS patients was significantly lower          of targeted therapy and the efforts of researchers should focus
than those from non EOS-CRS patients. Nevertheless, they           on this goal 23. The most prevalent endotype of CRSwNP in
observed that allergen stimulation significantly induced           Italy shows a type-2 inflammatory response 15,18,19 and excit-
calprotectin production in epithelial cells from EOS-CRS           ing and promising new treatments with biologics, primarily
patients. Our data seem to be in contrast with those results,      developed as treatments for asthma, have shown good results
although a direct comparison cannot be performed because           for type-2 CRSwNP 19. Our data support the hypothesis that
we measured calprotectin levels in nasal secretions in vivo.       calprotectin may be a diagnostic biomarker of non-type 2 in-
Furthermore, literature data 6-9 suggest that expression pat-      flammation (type-1 and 3 inflammation); in fact, low levels
terns of calprotectin in CRS patients may significantly dif-       of calprotectin in nasal lavage seem to be more indicative of
fer if measured in nasal secretions, polyp tissue, or normal       a type-2 immune response. Bearing in mind that biologics
sinonasal mucosa. For this reason, we believe that future          may offer greater efficacy in type-2 inflammatory disease,
studies are required to further define a correlation between       future studies should be performed to determine if high lev-
atopy and calprotectin secretion.                                  els of calprotectin in nasal secretions may be predictive of
We finally observed that a significant increase of calprotectin    poor response to type-2 biologics in CRSwNP, whereas low
level in nasal secretions was correlated with a high number        levels might be predictive of good response.
of previous ESS. Patients who underwent multiple ESS (> 3)
showed more pronounced neutrophilia and a corresponding            Conflict of interest statement
significantly higher level of calprotectin in both Type 2 and      The authors declare no conflict of interest.
non-Type 2. Inter-group comparison revealed that the differ-
ence between multiple operated (> 3) type 2 and non-type 2         Funding
patients was not statistically different. We previously hypoth-    This research did not receive any specific grant from fund-
esised that calprotectin levels in these cases are related to      ing agencies in the public, commercial, or not-for-profit
the increased neutrophilia as a consequence of progressive         sectors.
epithelial barrier breakdown that is generally observed in pa-
tients who underwent multiple surgeries 25,26.                     Author contributions
Among the limitations of this study there is the absence           EDC and JG: conception and design of the study, acqui-
of confirmation of the endotype by histological analysis.          sition of the data, analysis and interpretation of the data;
However, this limitation has been exceeded by combining            drafted the article and revised it for important intellectual
nasal cytological features and cytokine dosage in nasal se-        content; gave final approval of the version to be submitted;
cretion. Another limitation of the study is the exclusion of       agree to be accountable for all aspects of the work. SB,
CRSsNP patients. Future investigations should confirm if           MEO, LT, SS, GM, ET, RFM, DF, FC and AU: acquisition
our conclusions may also be applied to CRSsNP; in fact,            of the data, analysis and interpretation of the data; drafted



362
                                                                                                          Calprotectin: biomarker of non-type 2 inflammation




the article and revised it for important intellectual content;                   11
                                                                                      Bousquet JJ, Schünemann HJ, Togias A, et al. Next-generation ARIA
                                                                                      care pathways for rhinitis and asthma: a model for multimorbid chron-
gave final approval of the version to be submitted; agree to                          ic diseases. Clin Transl Allergy 2019;9:44. https://doi.org/10.1186/
be accountable for all aspects of the work.                                           s13601-019-0279-2
                                                                                 12
                                                                                      Mozzanica F, Preti A, Gera R, et al. Cross-cultural adaptation and
Ethical consideration                                                                 validation of the SNOT-22 into Italian. Eur Arch Otorhinolaryngol
                                                                                      2017;274:887-895. https://doi.org/10.1007/s00405-016-4313-x
This study was approved by the Institutional Ethics Com-                         13
                                                                                      Psaltis AJ, Li G, Vaezeafshar R, et al. Modification of the Lund-
mittee (“A. Gemelli Hospital Foundation IRCCS”, Otorhi-                               Kennedy endoscopic scoring system improves its reliability and
nolaryngology, Rhinology Unit, Catholic University of Sa-                             correlation with patient-reported outcome measures. Laryngoscope
cred Heart-Rome, Italy) (protocol n. 11177/20; ID number:                             2014;124:2216-23. https://doi.org/10.1002/lary.24654
3036).
                                                                                 14
                                                                                      De Corso E, Lucidi D, Battista M, et al. Prognostic value of nasal
                                                                                      cytology and clinical factors in nasal polyps development in patients
The research was conducted ethically, with all study pro-                             at risk: can the beginning predict the end? Int Forum Allergy Rhinol
cedures being performed in accordance with the require-                               2017;7:861-7. https://doi.org/10.1002/alr.21979
ments of the World Medical Association’s Declaration of                          15
                                                                                      De Corso E, Baroni S, Battista M, et al. Nasal fluid release of eotax-
Helsinki.                                                                             in-3 and eotaxin-2 in persistent sinonasal eosinophilic inflammation.
                                                                                      Int Forum Allergy Rhinol 2014;4:617-624. https://doi.org/10.1002/
All subjects signed a written informed consent form.                                  alr.21348
                                                                                 16
                                                                                      De Corso E, Baroni S, Lucidi D, et al. Nasal lavage levels of granulo-
                                                                                      cyte-macrophage colony-stimulating factor and chronic nasal hypere-
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