Discussion
This is the first study to show that both CD40-CD40L and ICOS-ICOSL are
upregulated in the nasal polyps of CRS patients. Our results demonstrate
that increased expression of CD40-CD40L and ICOS-ICOSL in CRS nasal
tissues is linked to high eosinophils infiltration and disease severity.
Then, we found CD40-CD40L and ICOS-ICOSL pathways do take effect on the
activation of eosinophils from ECRS patients. Additionally, we
illustrated that TNF-ɑ induces CD40 expression on eosinophils via the
activation of the p38 MAPK signaling pathway, and IL-5 further augments
TNF-ɑ stimulated CD40 expression on eosinophils. Our findings indicate
that CD40-CD40L and ICOS-ICOSL are potential clinical biomarkers of
disease activity in patients with CRS, particularly in the population
with high-level eosinophils.
For the first time, our findings show that levels of CD40-CD40L and
ICOS-ICOSL are markedly increased in the nasal tissue of ECRS patients
compared with that in Non-eCRS patients. Our subsequent correlation
analyses showed that high nasal tissue CD40-CD40L and ICOS-ICOSL levels
were strongly correlated in CRS. Besides, based on the classification of
histopathologic phenotypes, we observed similar upregulation of CD40 and
ICOS-ICOSL in nasal polyps of edematous CRS. Consistently, a strong
correlation with CD40-CD40L and ICOS-ICSOL levels was observed in
edematous CRS nasal tissues. It has been reported that edematous CRS was
commonly observed in eosinophilic inflammation1,45.
However, our findings that groupings based on ECRS/Non-eCRS and
groupings based on histopathologic phenotypes do not completely overlap.
We think that this is mainly related to the uneven distribution and the
small number of patient cases in different pathological subtypes, so
statistical analysis cannot be conducted. Given that the ICOS-ICOSL
signal can strengthen CD40-CD40L interaction thus providing a
co-stimulatory signal for B cell activation30,31, as
well as the allergic characteristics of CRS. Importantly and novel, our
findings indicate that high CD40-CD40L and ICOS-ICOSL expression in
nasal tissues are potential immunoregulatory factors for the development
of CRS, especially in patients with high eosinophil levels.
Then, our subsequent correlation analyses showed that high CD40-CD40L
and ICOS-ICOSL expression was linked to high eosinophils infiltration in
the nasal tissue of CRS patients. We further observed that both
augmented CD40 and ICOSL expression was primarily on eosinophils in the
local tissue of ECRS.
So far, several studies have shown that not only CD40 but also CD40L is
expressed on the surface of human eosinophils20,46,47.
And close to what we found, Ohkawara Y et al. also found that CD40 was
mainly expressed on the surface of eosinophils in the nasal polyp
tissues of allergic subjects. They only compared the expression of CD40
in nasal polyp tissues20. In this study, we further
found the different CD40-CD40L expression in Non-ECRS and ECRS nasal
polyps and also their correlation with clinical feature of CRS. We
assume that the high nasal tissue eosinophils proportion of CRS mainly
contributed to the high CD40 expression levels. As CD40L is
predominantly expressed on activated CD4+ T cells, it
has been shown that there is a large number of T cells infiltrating in
nasal tissue of CRS32,48-50. Thus, we suspect that
there is a “T-eosinophils-centered function” of CD40-CD40L in the
nasal tissue of CRS with high-level eosinophils, which is worthy of
further study.
As for the expression of ICOS-ICOSL in nasal tissues, we reported it for
the first time. Andreas Hutloff et al. reported that there is no ICOS
expression on granulocytes using F44 (specific monoclonal antibody to
ICOS)51. And no research has studied the expression of
ICOSL on eosinophils so far. Our co-location staining showed first that
ICOSL but no ICOS expression on eosinophils. Considering that ICOS is
mainly expressed on activated CD4+T cells, especially
activated TH2 cells52,53. ECRS found
worldwide is characterized by a type 2 immune response involving
TH2 cells, type 2 innate lymphoid cells, eosinophils,
mast cells, and M2 macrophages50,54-58. Thus, we
speculate that activated CD4+ cells, especially
TH2 cells, can exert influence on eosinophils
mono-directionally, through ICOS-ICOSL ligation signal pathway in ECRS.
Further studies are still needed.
Then, our clinical correlation analysis shown that blood eosinophils
count was significantly higher in ECRS subset compared with that in
Non-eCRS subset (Supplementary Fig. 3A), which is consistent with
previous studies59-61. As shown in Supplementary Fig.
3B-C, we further observed that blood eosinophil count was positively
correlated with disease activity assessed by Lund-Mackay score as well
as nasal tissue eosinophils count in our CRS patients. Developing from
progenitors in bone marrow, eosinophils can be recruited to diseased
nasal tissue from peripheral circulation by chemokines and cytokines,
which resulting a specific correlation between them. Then, positive
correlations between blood eosinophil count and tissue CD40-positive
cell numbers as well as CD40L-positive cell numbers were found, and the
same findings were with ICOS-ICOSL-positive cell numbers. Importantly,
we noticed that high ICOS-ICOSL expression levels was positively
correlated with Lund-Mackay score of patients with CRS patients. Recent
studies have reported the pathological effect of ICOS-ICOSL signals
widely participate in inflammatory responses, particularly
ICOS+ T cells, including TH1,
TH2, TH17 as well as T follicular helper
(Tfh), T follicular regulatory cells (Tfr) and regulatory T cells
(Treg), with the increased generation, proliferation, and survival
abilities62-66. Thus, the ICOS-ICOSL pathway may
associate with the local immune microenvironment and then contribute to
the development of CRS, especially ECRS. Interestingly, ICOSL positive
cells also had positive correlation with blood basophils (Supplementary
Fig. 3D). Therefore, our above data indicate that CD40-CD40L and
ICOS-ICOSL signals may involve in the pathogenies of CRS by modulating
the function of eosinophils.
Next, we confirmed whether CD40-CD40L and ICOS-ICOSL axis function on
eosinophils by using CD40 and ICOSL protein in ECRS. We found CD40
protein stimulation upregulated the expression of CD69, which is an
important marker of activation for eosinophils. In addition, CD69 levels
were also increased in response to ICOSL protein stimulation. These
results show that both CD40-CD40L and ICOS-ICOSL signals activate
eosinophils, and then contributes to the development of ECRS. Cause
recent evidence suggests that activated eosinophils have an axial role
in symptomology of CRS, especially ECRS. Studies have shown the
association between activated eosinophil count and the development of
ECRS. Moreover, some reports demonstrated a significant drop of blood
eosinophils from before to after FESS61,67-69. In the
advantage of great local cytokines and chemokines production,
eosinophils are characterized by increased production, enhanced
activation and prolonged survival. These factors promote the eosinophils
accumulation, which ultimately contributing to the increased destroy of
epithelial barrier and hyper-activity in nasal
mucosa70-72.
TNF-α and IL-5 are closely related to CRS. Previously many researchers
have reported the high levels of TNF-ɑ and IL-5 in patients with CRS and
positive correlation with disease activity33,34. In
addition, TNF-ɑ and IL-5 are critical for the function of eosinophils
including antigen presentation, cytokine or chemokine production, and
secretion of granule mediators42,73,74. Furthermore,
clinical studies of anti-IL-5 antibody (Ab), anti-IL-5 receptor (IL-5R)
Ab have been performed for severe CRSwNP. Several placebo-controlled
double-blind study of anti-IL-5 (mepolizumab) and anti-IL-5RA
(benralizumab) demonstrated to decrease nasal polyps and to improve CT
findings in patients with large nasal polyps, especially in
ECRS75-77. Then, we observed that TNF-ɑ stimulation
significantly upregulated CD40 expression on eosinophils, which was
further markedly enhanced by combined incubation with IL-5. However,
TNF-ɑ, IL-5, or TNF-ɑ plus IL-5 stimulation feebly affected ICOSL
expression on eosinophils, no significant difference was observed
compared with that in control groups. These results indicated that TNF-ɑ
and IL-5 mainly affected the expression of CD40 on eosinophil. As for
the expression of eosinophils derived ICOSL, the specific mechanism
needs to be further explored in the future. For example, is there a
synergistic effect of cytokines? Or other potential, unknown mediators?
Since previously, it has been described that p38 MAPK is activated in
eosinophils by TNF-α44,73. Thus, in discerning the
individual contributions of specific signaling pathways, we observed
that inhibitor that target the pathway mediated by the p38 MAPK. The
present study shows that the specific p38 MAPK inhibitor SB203580 could
largely inhibit TNF-α and IL-5 induced CD40 expression on eosinophils.
These data indicated the important role of the activation of p38 MAPK in
the mechanism of TNF-α and IL-5 induced CD40 expression on eosinophils.
Therefore, modulation of TNF-ɑ/IL-5/CD40/p38 MAPK pathways might be
useful for the treatment of CRS. Besides, we found that SB203580 did not
fully inhibit the CD40 expression on eosinophils. These findings
indicate that pathways other than p38 MAPK are also involved in TNF-ɑ
and IL-5 induced inhibition of CD40 expression on eosinophils. Since p38
MAPK is required for NF-kB-dependent gene expression and CD40 gene
expression could partly mediated by NF-kB, it is reasonable that the
inhibition of p38 MAPK can down-regulate the expression of
CD40.78-81. Therefore, it may be possible that the
inhibition of p38 MAPK by SB203580 can block TNF-ɑ and IL-5 induced
eosinophil-derived CD40 by indirect inhibiting NF-kB activity and
subsequently suppress the eosinophil activation. Further investigation
is required to explore other signaling pathways involved in TNF-ɑ and
IL-5 mediated modulation of CD40 expression on eosinophil.
The limitations of our study are its retrospective, cross-sectional
design, the univariate and descriptive nature of the analyses performed,
the lack of a large cohort of CRS patients, and the not yet identified
relevant mechanisms underlying.
In summary, we observed that the high levels of CD40-CD40L, ICOS-ICOSL
in local nasal tissues are closely associated with high eosinophils
infiltration and high disease activity in CRS. We demonstrated a
previously unrecognized role for CD40-CD40L and ICOS-ICOSL pathways,
most remarkably in eosinophil activation of ECRS. Our data has shown
that TNF-ɑ and IL-5 mediate CD40 upregulation in human eosinophils in
part via activation of p38 MAPK. In view of the above findings, we
conclude that blocking of the activation of eosinophils by targeting
CD40-CD40L and ICOS-ICOSL pathways, especially manipulation of TNF-ɑ/p38
MAPK pathways targeting eosinophils activation might be useful for the
treatment of CRS with high-level eosinophils.