Alteration of blood monocyte subsets in chronic rhinosinusitis with regard to anti-inflammatory 1,8-Cineol treatment*

Background: Chronic rhinosinusitis (CRS) affects about 10% of the european population causing considerable disease burden. The inflammatory microenvironment is mainly Th2 driven, but the impact of monocytes is still poorly understood. Aim of this study was to comprehensively investigate the composition of circulating monocytes and T cells in CRSwNP and CRSsNP patients, particularily with regard to the therapeutic herbal monoterpene 1,8-Cineol. Methodology: We analyzed the distribution of CD14 and CD16 classified monocyte subsets and the T-cell subset composition with respect to their PD-1 and PD-L1 expression in the peripheral blood of CRS patients using flow cytometry. Additionally, the M1/M2 like macrophage infiltration in nasal tissue and polyps was examined by immunofluorescence staining. Results: Data revealed a decrease of classical monocytes accompanied by a significant increase of intermediate CD16+ monocytes in CRSwNP and CRSsNP patients compared to healthy donors. PD-L1 expression on overall monocytes was also significantly increased in CRSwNP and CRSsNP patients. CRS patients with a severe drop of the proportion of classical monocytes showed a significant restoration of this subset in response to two-week 1,8-Cineol treatment. Conclusions: Our data indicate a CRS-induced shift of peripheral monocyte subsets to more inflammatory phenotypes that might be reversed by the herbal drug 1,8-Cineol.


Introduction
Chronic Rhinosinusitis (CRS) is a common disease worldwide and affecting about 10% of the European population (1,2) . On epidemiologic grounds, different factors have been associated with CRS prevalence, such as air pollution, active cigarette smoking, perennial allergic rhinitis, individual immune barrier dysfunctions or alterations in the eicosanoid pathway (3)(4)(5) . In addition, various fungi and microbial pathogens such as Staphylococcus aureus are supposed to trigger CRS emergence (6,7) . Chronic rhinosinusitis can occur without (CRSsNP) or with a manifestation of nasal polyps (CRSwNP).
Although our knowledge of the pathogenesis, inflammatory processes as well as the cellular progression of this disease grows permanently, the exact multifactorial mechanisms still remain elusive (8) . In fact, the inflammatory milieu of the sinonasal mucosa in CRSsNP patients is characterized by a mixed T helper (Th) Th1/Th2 or a Th1 phenotype, whereas in CRSwNP a Th2 polarization is dominant (9,10) . Th2 cytokines promote rhinorrhoea, mucus production and hyperplasia. Monocyte-derived macrophages in the sinunasal tissue can directly and indirectly induce a Th2 cell response, but mechanisms in CRS are still poorly understood (11) . Moreover, nasal polyps are also known to be infiltrated by eosinophilic granulocytes (12) .
Most common therapies of chronic rhinosinusitis comprise administration of corticosteroids or antibiotics and surgery (13) .
Furthermore, novel biologicals targeting IgE, IL-5 or IL-4 and IL-13 have been introduced recently (14,15) . The anti-inflammatory monoterpene 1,8-Cineol is a natural plant-based therapeutic agent that is commonly applied to treat various chronic and acute airway diseases as well as patients with CRS. It has been shown that 1,8-Cineol is able to significantly reduce the production of proinflammatory mediators such as TNF-α, IL-1β, and IL-6 from monocytes (16,17) as well as the IL-4 and IL-5 production from lymphocytes (18) . There are only few studies on systemic changes in monocyte or T-cell composition in peripheral blood from CRS patients.
CD14 ++ CD16are referred to as "classical" monocytes and comprise the biggest compartment with about 85% of monocytes.
Classical monocytes are the most migrative and phagocytotic cytokine producers among monocyte subsets. CD14 ++ CD16 + "intermediate" monocytes are with 5-10% considered as the superior antigen presenting cells. The remaining 5-10% constitute CD14 dim+ CD16 + "non-classical" monocytes which exert a marked patrolling behaviour. Some studies hypothesised that both CD16 + subsets emerge from the classical monocyte population and thus are more closely related to each other (19) , whereas others assume that the non-classical subset is solely different regarding the gene expression profile (22) . Although classical monocytes constitute always the mayor compartment of monocytes in the periphery, an increase of CD16 + monocytes has been described in many chronic inflammatory conditions as persistent viral infections (23) , rheumatoid arthritis (24) , asthma (25) , and obstructive sleep apnea syndrome (26) . All monocyte subsets are able to acquire macrophage morphology and characteristics, but the exact differentiation potential of the different subsets to macrophages remains incomplete (27) . The polarization of monocytes/macrophages to the classic pro-inflammatory type (M1-like) or the alternative protective type (M2-like) resembles an important factor in inflammatory and neoplastic diseases (28) .
It remains to be determined comprehensively whether and how the function and phenotypes (M1 vs. M2) of macrophages participate in the CRSwNP pathogenesis. It has been shown that CRS is associated with M2 polarization which propagate a Th2 dominant response (11) .
T cells infiltrate and differentiate in inflammatory tissue to competent effector cell subsets depending on the inflammatory milieu and stimuli provided by innate immune cells such as monocytes. Nevertheless, effector, memory and specific cytokine producing helper cell subtypes can be found in blood as well.
Following encounter to foreign antigens a single naïve T cells is able to generate multiple subsets of effector and memory cells with distinct differentiation states and functionality. Analysis of peripheral T-cell subsets can provide comprehensive information on an individual's health and disease status, respectively or any immune disturbances (29)(30)(31) .
Only a few studies have investigated the peripheral changes of monocyte subsets or T-cell composition in chronic rhinosinusitis, not to speak of the therapeutic impact of 1,8-cineol on these cells. Therefore, the aim of this study was to understand the differentiation patterns of circulating monocyte subsets in the peripheral blood of CRS patients, also with regard to the the-

Staining of T-cell subsets in isolated PBMC
PBMC were isolated from the remaining blood by density  T-cell subsets was performed as described before (26) . In brief, CD45 was used as a pan leukocyte marker to facilitate whole blood measurement and monocytes were first roughly gated by their FSC/SSC characteristics and the positivity for CD14 and CD16. Remaining neutrophil granulocytes, NK cells and B cells were excluded by help of HLA-DR expression, which is specific for monocytes. Remaining real monocytes were then subgated into CD14 ++ CD16 -(classical), CD14 ++ CD16 + (intermediate) and CD14 dim+ CD16 + (non-classical) monocytes. Lymphocytes were also gated by their FSC/SSC characteristics and T cells were   Figure   1C).

Alteration of T-cell subsets in chronic rhinosinusitis patients
The T-cell subset distribution as well as their PD-1 and PD-L1 expression were also analysed in CRSwNP as well as CRSsNP patients compared to healthy donors. The T-cell differentiation  Figure 4B).
In addition, nasal polyp tissue probes before and after 1,8-Cineol treatment were analysed for the distribution of tissue infiltrating M1 and M2 macrophages. Therefore, tissue samples were cryosectioned and analysed by immunofluorescence microscopy using specific primary antibodies for human CD68 and CD163 to recognize M1 and M2 macrophages as described before. Data revealed strong individual deviations, but no significant changes in the abundance of M1/M2 macrophages in response to twoweek treatment with 1,8-Cineol ( Figure 4C).

Discussion
The present study was undertaken to investigate the impact of chronic rhinosinusitis with (CRSwNP) and without (CRSsNP) nasal polyps on die distribution of circulating monocyte subsets and T-cell immune alterations, distinguishing between CD4 + and CD8 + T cells. Additionally, the expression levels of PD-1 and PD-L1 on the peripheral T cells of CRSwNP and CRSsNP patients and healthy donors were analysed for CD3 + CD4 + and the CD3 + CD8 + subsets and monocytes using flow cytometry. Furthermore, the M1/M2 macrophage distribution in nasal tissue and polyps was investigated by immunofluorescence staining.
Zagolski and colleagues found that in CRSwNP, peripheral blood leukocytes, neutrophils, monocytes and eosinophils were higher than in CRS without nasal polyps, thus further subtyping of different immune cells was not performed (35) . Our study revealed a significant decrease of the percentage of classical monocytes accompanied by increased CD16 + monocyte subsets in the peripheral blood of both CRSwNP as well as CRSsNP patients compared to healthy donors. The decrease of classical monocytes was even more pronounced in CRSsNP patients by tendency.
An increase of CD16 + subsets was repeatedly linked to various chronic inflammatory conditions, including asthma (25) . About 28% of our patients (21 out of 76) suffer from asthma and 25% (19 out of 76) have also allergies. Unexpectedly, there was no correlation or differences of the monocyte subset distribution between patients with these comorbidities and those without.
Our findings indicate that the monocyte subset composition in peripheral blood is primarily affected by the chronic rhinosinusitis itself.
The life cycle and cellular interplay of circulating monocyte subsets has been the subject of several recent studies. Basically, these studies suggest a linear differentiation relationship between the three monocyte subsets. In two studies a sequential enrichment of classical monocytes, then intermediate monocytes and finally non-classical monocytes in the peripheral blood has been shown using in vivo cell labelling with 6,6-2H2-glucose in healthy volunteers (21,36) . Furthermore, classical monocytes reappeared in the blood 7 days after an autologous stem cell transplantation, followed by intermediate monocytes and then non-classical monocytes after 10 days (37) . These observations indicate a differentiation process from classical to CD16 + monocyte subsets. Our study revealed a reconstitution of classical monocytes after 1,8-Cineol intake in patients with less than 75% classical monocytes pre 1,8-Cineol treatment. Questions remain whether 1,8-Cineol is able to block the differentiation to CD16 + monocyte subsets or if only those increasingly disappear from circulation and how long the effect remains. It has been shown that circulating classical monocytes have a short lifespan of about 1-2 days, before differentiating into CD14 + CD16 + monocytes or migrating into tissue and differentiate into macrophages (21,36,38) . These data suggest a short-term restorative influence of anti-inflammatory therapeutic approaches regarding the healthy balance of circulating monocyte subsets.
It has been repeatedly reported that the inflamed sinunasal and polyp tissue is predominantly infiltrated by M2 macrophages that induce and maintain a Th2 type inflammation which mediates the humoral immune cell response. But there was also no significant difference within the M1/M2 macrophage distribution in our examined polyp tissues after oral 1,8-Cineol therapy.
Possible explanations might be the lacking effect of 1,8-Cineol on the macrophage differentiation within the nose or a rather slow-working long-term influence. We investigated the macrophage distribution after two-week 1,8-Cineol treatment. Tissue resident macrophages are known to stay present for several months, so the impact might not be visible after two weeks. This has to be investigated in further studies.
With respect to monocyte immune functions and their immunological interplay our study revealed some new insights.
We found a positive correlation between the percentage of CD14 + CD16classical monocytes and serum IgE levels in CRSwNP patients (p = 0.048). One study likewise reported elevated serum IgE levels correlating with monocytic CD14 expression in patients with allergic asthma which was also validated in-vitro (39) . They showed an increased monocytic secretion of the pro-inflammatory cytokines TNF-α and IL-6 after IgE cross-linking and impaired phagocytotic functions of these monocytes.
Furthermore, our data revealed a significant increase of PD-L1 expression on monocytes in CRS patients, suggesting a crucial mechanism for the regulation of T-cell responses preventing overreaction. Recently, significantly higher levels of CD80, CD274, and CD273 have been identified in nasal polyp tissues compared to peripheral blood from patients with CRSwNP, indicating an important role of the PD-1/PD-L1 pathway as a potential therapeutic target in CRS (40) . In addition, we found an increase of PD-L1 by tendency and low significance in naïve and effector CD4 + and CD8 + T cells, respectively which implies an activated but regulatory state of these T cells (41,42) . An imbalanced PD-L1 signalling has been repeatedly associated with chronic viral or bacterial infections that also exist during chronic rhinosinusitis (43,44) . Astonishingly, our data revealed no significant alterations of peripheral CD4 + and CD8 + T-cell subset percentages, neither in CRSwNP nor in CRSsNP patients, since recent studies corroborated alterations of T-cell differentiation within the microenvironment of nasal polyps (32) . All analysed patients and healthy donors showed a very comparable and stable distribution of circulating T-cell subsets, unaffected by CRS and observed monocyte subset alterations. These data suggest a spatial separation of monocyte and T-cell subset redistribution in the peripheral blood of CRS patients compared to the inflamed sinunasal tissue and nasal polyps, respectively.

Conclusion
The present study revealed new insights into the peripheral monocyte as well as tissue macrophage composition in CRS patients with respect to oral 1,8-Cineol treatment. Our data hint to a beneficial, anti-inflammatory effect of 1,8-Cineol since monocyte subsets were restored to healthy levels in some patients. Thus, further studies need to be performed in order to understand the immunological interplay and to select profiting patient cohorts.

Authorship contribution
CP and KL performed the molecular studies, data analysis and statistical evaluation. CI, DW and MH conducted the medical examination and sample collection. KLB and RP participated in the design, coordination and evaluation of the study and helped to draft the manuscript. All authors read and approved the final manuscript.