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.