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 (Table 1 ). Previous studies revealed that the
concentration of MIP-1α/ 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 lowinflammatory or low mixed Th1/ Th2 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, 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 versus placebo-treated CRSwNP patients (8).
Further, the concentration of MCP-3/ CCL7 and MIP-1α/ 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-1α/ 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-1α/ CCL3 attracts
IFN−γ−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−γ were overexpressed in low- and high-IL-5 clusters
(clusters 1, 4, and 5; IFN−γ 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 TNFα 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-γ 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.