Asthma and COPD are the most common obstructive lung diseases characterized by inflammation in the lower airways which contribute to airflow limitation. Different inflammatory mediators are thought to play a key role in these diseases. This study was conducted in 13 patients with asthma, 12 patients with COPD, and 13 control subjects. The expression of mRNA of IL-6, IL-13, CXCL8, TSLP, IL-33, IL-25, IL-17, ECP, mast cell tryptase, CCL24, and CCL26 was assessed in induced sputum cells by real time PCR. We found that CXCL8 was strongly related to the neutrophil percentage but differed significantly in COPD and asthma patients. The expression of IL-17 was lower in patients with atopic asthma compared to non-atopic asthma. The percentage of macrophages correlated negatively with the expression of mast cell tryptase and ECP in COPD, and with CXCL8 in asthma. The expression of ECP correlated negatively with the severity of COPD symptoms measured by CAT. We conclude that asthma and COPD demonstrate a significant overlap in the airway cytokine profile. Thus, differentiation between the two diseases is difficult as based on a single cytokine, which suggests the coexistence of phenotypes sharing a common cytokine network in these obstructive lung diseases.

The underlying inflammation present in chronic airway diseases is orchestrated by increased secretion of CC and CXC chemokines that selectively recruit the leukocyte populations into the pulmonary system. Human chemokines, eotaxins (CCL11 and CCL26), RANTES, and interleukin (IL)-8, are dramatically upregulated through G-protein receptors in cell inflammation, including human asthma. In previous studies, a series of new glucocorticoid antedrugs (GCAs) were synthesized as derivatives of isoxazoline and oxime, and their pharmacological properties based on the antedrug concepts were evaluated. Utilizing both human airway epithelium (HAE) and eosinophil (EOS) cell culture models, we carried out studies to test the hypothesis that new GCA cell treatment would ameliorate Th-1/Th-2-driven secretion of these asthmatic biomarkers, eotaxins (CCL11 and CCL26), RANTES, and IL-8 chemokines, that would in turn decrease recruitment, proliferation, and activation of EOS cells. Results demonstrate that isoxazoline and oxime derivatives exhibit concentration-dependent inhibition, and specifically the compound No. 7 decreases significantly the secretion of eotaxins, RANTES, and IL-8 in cytokine-stimulated HAE cells. It was shown that EOS proliferation and activation were reduced considerably, and cell apoptosis occurred when exposed to nonfluorinated isoxazoline derivatives. These results provide evidence that concentration and structural manipulation of GCAs could increase the anti-inflammatory potency in treatment of chronic diseases, including asthma.

The development of siRNA-based asthma therapeutics is currently hampered by a paucity of relevant biomarkers and the need to ascertain tissue-specific gene targeting in the context of active disease. Epithelial STAT6 expression is fundamental to asthma pathogenesis in which inflammatory changes are found throughout the respiratory tract. Therefore, to improve preclinical evaluation, we tested the efficacy of STAT6-targeting siRNA within nasal epithelial cells (NEC's) obtained from asthmatic and non-asthmatic donors. STAT6 expression was invariant in both donor groups and amenable to suppression by siRNA treatment. In addition, STAT6 mRNA was also suppressible by apically delivered siRNA treatment in comparative differentiated nasal epithelial cell-line monolayer cultures. Analysis of donor NEC's showed consistent elevation in CCL26 (eotaxin-3) mRNA within the asthmatic group suggesting potential as a relevant biomarker. Furthermore, targeting of STAT6 with siRNA attenuated IL-13-driven CCL26 expression in these cells, pointing to the utility of this approach in preclinical testing. Finally, siRNA-mediated suppression of STAT6 was independent of donor disease phenotype or epithelial cell differentiation status, signifying therapeutic potential.

BACKGROUND

Chemokines play a pivotal role in immune regulation and response, and previous studies suggest an association between immune deficiency and non-Hodgkin lymphoma (NHL).

METHODS

We evaluated the association between NHL and polymorphisms in 18 genes (CCL1, CCL2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL18, CCL20, CCL24, CCL26, CCR1, CCR3, CCR4, CCR6, CCR7, CCR8, and CCR9) encoding for the CC chemokines using data from a population-based case-control study of NHL conducted in Connecticut women.

RESULTS

CCR8 was associated with diffuse large B-cell lymphoma (DLBCL; P = 0.012), and CCL13 was associated with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL; P = 0.003) at gene level. After adjustment for multiple comparisons, none of the genes or single-nucleotide polymorphisms (SNP) were associated with risk of overall NHL or NHL subtypes.

CONCLUSIONS

Our results suggest that the genes encoding for CC chemokines are not significantly associated with the risk of NHL, and further studies are needed to verify these findings.

CONCLUSIONS

Our data indicate that CC chemokine genes were not associated with NHL risk.

Atopic dermatitis (AD) is a common inflammatory skin disease predominately related to Type 2 helper T (Th2) immune responses. In this study, we investigated whether piperine is able to improve AD symptoms using a trimellitic anhydride (TMA)-induced AD-like mouse model. Topical treatment with piperine reduced ear swelling (ear thickness and epidermal thickness) induced by TMA exposure. Furthermore, piperine inhibited pro-inflammatory cytokines such as TNF-α and IL-1β in mouse ears, compared with the TMA-induced AD group. In measuring allergic immune responses in draining lymph nodes (dLNs), we found that IL-4 secretion, GATA3 mRNA level, and STAT6 phosphorylation were suppressed by piperine treatment. In an ex vivo study, piperine also inhibited the phosphorylation of STAT6 on the CD4⁺ T cells isolated from splenocytes of BALB/c mice, and piperine suppressed IL-4-induced CCL26 mRNA expression and STAT6 phosphorylation in human keratinocytes resulting in the inhibition of infiltration of CCR3⁺ cells into inflammatory lesions. These results demonstrate that piperine could ameliorate AD symptoms through suppression of Th2-mediated immune responses, including the STAT6/GATA3/IL-4 signaling pathway. Therefore, we suggest that piperine is an excellent candidate as an inhibitor of STAT6 and may help to improve AD symptoms.

BACKGROUND

Mycobacterium tuberculosis is a pulmonary pathogen responsible for tuberculosis. Tuberculosis (TB) is characterized histologically by granulomas at the site of disease activity. Primary pathologic feature of TB is formation of a granuloma, and chemokines are known to play an important role in the formation of granulomas during infection. Therefore, the aim of this study was to evaluate the serum levels of CCL11, CCL24, and CCL26 in the TB patients in comparison to healthy controls.

METHODS

The population of this cross-sectional study included 300 patients suffering from TB and 100 healthy controls. Concentrations of CCL11, CCL24, and CCL26 were measured by enzyme linked immunosorbent assay (ELISA) technique. The results were analyzed using SPSS software package version 18. Differences were considered significant where p was less than 0.05.

RESULTS

The results showed significant elevated serum levels of CCL11, CCL24, and CCL26 in TB patients compared to controls.

CONCLUSIONS

According to the present results it can be concluded that CCL11, CCL24, and CCL26 (which are produced by Th2 cells and other cells which induce Th2 development) are increased in TB patients; hence, it seems that TB suppresses Th1 and the classic function of macrophages subsequently by inducing the chemokines' expression.

The expression pattern of chemokines and chemokine receptors is specific to certain organs and cells. Therefore, chemokines are important to elucidate the mechanism of organ-specific human diseases such as cutaneous lymphoma, characterized by proliferation of clonally expanded lymphocytes in skin without detectable systemic involvement. The most popular type of cutaneous lymphoma is T cell lymphoma, including mycosis fungoides and Sezary syndrome. We have reported that CCL17, CCL27, CCL11, and CCL26 are involved in progression of these diseases. The above chemokines are highly expressed in the lesional skin and serum levels of the chemokines are elevated as the disease progressed. Moreover, CXCL9 and CXCL10 are associated with epidermotropism of tumor cells, CCL21 is important for tumor invasion to lymph nodes, and CXCL12 may explain downregulation of CD26 on the cell surface. CXCL13 expression in lymphoid follicular formation in skin and CCR3 expression on tumor cells in CD30(+) lymphoproliferative disorders are also discussed. Biologics targeting chemokines and their receptors are promising strategies for cutaneous lymphoma. Indeed, humanized anti-CCR4 monoclonal antibody showed potent antitumor activity against CCR4(+) lymphoma cells both in vitro and ex vivo. This antibody may also be useful for allergic diseases such as hay fever. Further study on chemokines and chemokine receptors will be helpful for new classification of cutaneous lymphoma, elucidation of pathogenesis, and development of new therapeutic strategies.

Minocycline/tetracycline is clinically used for the treatment of bullous pemphigoid (BP), and its clinical benefits are superior to those of prednisolone when considering adverse events. Although the clinical benefits of minocycline/tetracycline are well known, its immunosuppressive mechanisms are still unclear. In this study, we investigated the immunomodulatory effects of traditional anti-BP drugs (minocycline, nicotinic acid amide, dexamethasone and cyclosporine) on CD163+ M2 macrophages in vitro, with special focus on the production of CCL18 and CCL22, both of which are produced by CD163+ M2 macrophages in the lesional skin of BP and are increased in the serum of BP patients. Minocycline decreased the production of CCL22, CCL24 and CCL26 as well as CCL2 from M2 macrophages. CCL18 from M2 macrophages was decreased by dexamethasone and cyclosporine, but not decreased by minocycline. These data suggest that the clinical benefit of minocycline is partially explained by its suppressive effects against the production of specific Th2 chemokines from M2 macrophages, which should contribute to the recruitment of Th2 cells and eosinophils in the lesional skin of BP patients.

OBJECTIVE

This study aimed to characterize DNA methylation (DNA-me) in promoter region of IL33, IL1RL1 and CCL26 in asthma and their impacts on transcriptional activity in bronchial epithelial cells (BECs).

METHODS

We performed bis-pyrosequencing, quantitative real-time PCR and sequencing in BECs from ten asthmatic and ten control individuals.

RESULTS

We detected lower DNA-me levels of IL33 and CCL26 in asthmatic than control BECs. No correlation was found between methylation and expression levels. Interestingly, carriers of a mutative allele in a haplotype within the promoter of IL33 had a lower IL33 DNA-me level and CCL26 gene expression correlated with eosinophil count.

CONCLUSIONS

These findings highlight the importance of investigating both epigenetic and genetic mechanisms in understanding the epithelial immune response in asthma.

Eosinophilic chronic rhinosinusitis (ECRS), a subgroup of chronic rhinosinusitis with nasal polyps, is recognized as a refractory eosinophilic disorder characterized by both upper and lower airway inflammation. In some severe cases, disease control is poor, likely due to local steroid insensitivity. In this study, we focused on protein phosphatase 2A (PP2A), a key factor regulating glucocorticoid receptor (GR) nuclear translocation, and examined its association with local responses to corticosteroids in eosinophilic airway inflammation. Our results indicated reduced responses to corticosteroids in nasal epithelial cells from ECRS patients with asthma, which were also associated with decreased PP2A mRNA expression. Eosinophil peroxidase stimulates elevated PP2A phosphorylation levels, reducing PP2A protein expression and activity. In addition, mRNA levels of inflammatory mediators (TSLP, IL-25, IL-33, CCL4, CCL5, CCL11, and CCL26) associated with eosinophilic airway inflammation in epithelial cells were increased in nasal polyps (eosinophil-rich areas) compared with those in uncinate process tissues (eosinophil-poor areas) from the same patients. PP2A reduction by siRNA reduced GR nuclear translocation, whereas PP2A overexpression by plasmid transfection, or PP2A activation by formoterol, enhanced GR nuclear translocation. Collectively, our findings indicate that PP2A may represent a promising therapeutic target in refractory eosinophilic airway inflammation characterized by local steroid insensitivity.

Parkinson's disease (PD) is one of the most common neuroinflammatory disorders and inflammatory processes seem to play an important role in the pathogenesis of PD. Chemokines as inflammatory mediators, which are involved in the recruitment of leukocytes, can play a role in the pathogenesis of PD. The aim of this study was to examine the serum level of eotaxins (CCL11, CCL24, and CCL26) and the expression of C-C chemokine receptor type 3 (CCR3) in patients with PD compared with healthy subjects. In this study, we measured the serum levels of CCL11, CCL24, and CCL26 with ELISA. In addition, gene and protein expression of CCR3 were measured by RT-PCR and flow cytometry techniques in PD patients (n = 30) and age- and sex-matched healthy subjects (n = 30). All patients suffering from PD were assessed clinically through Unified Parkinson's Disease Rating Scale, Motor Examination (UPDRS ME). The results of this study showed that there was no significant alteration in the serum level of these chemokines and also their receptor among patients with PD and healthy subjects. No significant correlation was observed between the eotaxins serum levels and the clinical measures of PD severity. Based on the results, it can be concluded that eotaxins cannot be considered as appropriate targets for the diagnosis or treatment of PD.

Eotaxins are proteins which belong to the group of cytokines. These small molecules are secreted by cells that are mainly involved in immune-mediated reactions in the course of allergic diseases. Eotaxins were discovered in 1994 and their main role was considered to be the selective recruitment of eosinophils. As those blood cells are involved in the course of all inflammatory diseases, including cancer, we decided to perform an extensive search of the literature pertaining to our investigation via the MEDLINE/PubMed database. On the basis of available literature, we can assume that eotaxins can be used as markers for the detection and determination of origin or type of allergic disease. Many publications also confirm that eotaxins can be used in the determination of allergic disease treatment. Moreover, there are also studies indicating a connection between eotaxins and cancer. Some researchers revealed that CCL11 (C-C motif chemokine ligand 11, eotaxin-1) concentrations differed between the control and tested groups indicating their possible usefulness in cancer detection. Furthermore, some papers showed usefulness of eotaxins in determining the treatment efficacy as markers of decreasing inflammation. Therefore, in this paper we present the current knowledge on eotaxins in the course of allergic and cancerous diseases.

Keywords: CCL11; CCL24; CCL26.

Tumor resection represents the only curative treatment option for patients with biliary tract cancers (BTCs), including intrahepatic cholangiocarcinoma (CCA), perihilar and extrahepatic CCA and gallbladder cancer. However, many patients develop early tumor recurrence and are unlikely to benefit from surgery. Therefore, markers to identify ideal surgical candidates are urgently needed. Circulating programmed cell death 1 ligand 1 (PD-L1) has recently been associated with different malignancies, including pancreatic cancer which closely resembles BTC in terms of patients' prognosis and tumor biology. Here, we aim at evaluating a potential role of circulating PD-L1 as a novel biomarker for resectable BTC.

Methods: Serum levels of PD-L1 were analyzed by ELISA in 73 BTC patients and 42 healthy controls.

Results: Circulating levels of preoperative PD-L1 were significantly lower in patients with BTC compared to controls. Patients with low PD-L1 levels displayed a strong trend towards an impaired prognosis, and circulating PD-L1 was negatively correlated with experimental markers of promalignant tumor characteristics such as CCL1, CCL21, CCL25 and CCL26. For 37 out of 73 patients, postoperative PD-L1 levels were available. Interestingly, after tumor resection, circulating PD-L1 raised to almost normal levels. Notably, patients with further decreasing PD-L1 concentrations after surgery showed a trend towards an impaired postoperative outcome.

Conclusion: Circulating PD-L1 levels were decreased in patients with resectable BTC. Lack of normalization of PD-L1 levels after surgery might identify patients at high risk for tumor recurrence or adverse outcome.

Keywords: PD-1; PD-L1; biliary tract cancer; biomarker; cholangiocarcinoma.

Background. Despite considerable interest in the search for a spinal cord injury (SCI) therapy, there is a critical need to develop a panel of diagnostic biomarkers to determine injury severity. In this regard, there is a requirement for continuing research into the fundamental processes of neuroinflammatory and autoimmune reactions in SCI, identifying changes in the expression of cytokines. Methods. In this pilot study, an extended multiplex analysis of the cytokine profiles in the serum of patients at 2 weeks post-SCI (n = 28) was carried out, together with an additional assessment of neuron-specific enolase (NSE) and vascular endothelial growth factor (VEGF) levels by enzyme-linked immunosorbent assay. A total of 16 uninjured subjects were enrolled as controls. Results. The data obtained showed a large elevation of IFNγ (>52 fold), CCL27 (>13 fold), and CCL26 (>8 fold) 2 weeks after SCI. The levels of cytokines CXCL5, CCL11, CXCL11, IL10, TNFα, and MIF were different between patients with baseline American Spinal Injury Association Impairment Scale (AIS) grades of A or B, whilst IL2 (>2 fold) and MIP-3a (>6 fold) were significantly expressed in the cervical and thoracic regions. There was a trend towards increasing levels of NSE. However, the difference in NSE was lost when the patient set was segregated based on AIS group. Conclusions. Our pilot research demonstrates that serum concentrations of cytokines can be used as an affordable and rapid detection tool to accurately stratify SCI severity in patients.

Keywords: blood serum; clinical trial; cytokine profile; inflammation; traumatic spinal cord injury.

Background: The anti-interleukin 13 (IL-13) monoclonal antibody lebrikizumab improves lung function in patients with moderate to severe uncontrolled asthma, but its effects on airway inflammation and remodelling are unknown. CLAVIER was designed to assess lebrikizumab's effect on eosinophilic inflammation and remodelling.

Objective: To report safety and efficacy results from enrolled participants with available data from CLAVIER.

Methods: We performed bronchoscopy on patients with uncontrolled asthma before and after 12 weeks of randomised double-blinded treatment with lebrikizumab (n=31) or placebo (n=33). The pre-specified primary endpoint was relative change in airway subepithelial eosinophils per mm² of basement membrane (cells/mm² ). Pre-specified secondary and exploratory outcomes included change in IL-13-associated biomarkers and measures of airway remodelling.

Results: There was a baseline imbalance in tissue eosinophils and high variability between treatment groups. There was no discernible change in adjusted mean subepithelial eosinophils/mm² in response to lebrikizumab (95% CI, -82.5%, 97.5%). As previously observed, FEV₁ increased after lebrikizumab treatment. Moreover, subepithelial collagen thickness decreased 21.5% after lebrikizumab treatment (95% CI, -32.9%, -10.2%), and fractional exhaled nitric oxide, CCL26, and SERPINB2 mRNA expression in bronchial tissues also reduced. Lebrikizumab was well tolerated, with a safety profile consistent with other lebrikizumab asthma studies.

Conclusions & clinical relevance: We did not observe reduced tissue eosinophil numbers in association with lebrikizumab treatment. However, in pre-specified exploratory analyses, lebrikizumab treatment was associated with reduced degree of subepithelial fibrosis, a feature of airway remodelling, as well as improved lung function and reduced key pharmacodynamic biomarkers in bronchial tissues. These results reinforce the importance of IL-13 in airway pathobiology and suggest that neutralisation of IL-13 may reduce asthmatic airway remodelling.

Locally advanced rectal cancer is treated with neoadjuvant-chemoradiotherapy; however, only ~22% of patients achieve a complete response, and resistance mechanisms are poorly understood. The role of inflammation and immune cell biology in this setting is under-investigated. In this study, we profiled the inflammatory protein secretome of normal (non-cancer) (n = 8) and malignant rectal tissue (n = 12) pre- and post-radiation in human ex vivo explant models and examined the influence of these untreated and treated secretomes on dendritic cell biology (n = 8 for cancer and normal). These resultant profiles were correlated with patient clinical characteristics. Nineteen factors were secreted at significantly higher levels from the rectal cancer secretome when compared to the normal rectal secretome; Flt-1, P1GF, IFN-γ, IL-6, IL-10, CCL20, CCL26, CCL22, CCL3, CCL4, CCL17, GM-CSF, IL-12/IL-23p40, IL-17A, IL-1α, IL-17A/F, IL-1RA, TSLP and CXCL10 (p < 0.05). Radiation was found to have differential effects on normal rectal tissue and rectal cancer tissue with increased IL-15 and CCL22 secretion following radiation from normal rectal tissue explants (p < 0.05), while no significant alterations were observed in the irradiated rectal cancer tissue. Interestingly, however, the irradiated rectal cancer secretome induced the most potent effect on dendritic cell maturation via upregulation of CD80 and PD-L1. Patient's visceral fat area correlated with secreted factors including CCL20, suggesting that obesity status may alter the tumour microenvironment (TME). These results suggest that radiation does not have a negative effect on the ability of the rectal cancer TME to induce an immune response. Understanding these responses may unveil potential therapeutic targets to enhance radiation response and mitigate normal tissue injury. Tumour irradiation in this cohort enhances innate immune responses, which may be harnessed to improve patient treatment outcome.

Keywords: dendritic cells; inflammation; radiotherapy; rectal cancer; tumour immunology.

Background: Atopic dermatitis (AD) is the most common inflammatory skin disease. It is highly heterogeneous in clinical presentation, treatment response, disease trajectory and associated atopic comorbidities. Immune biomarkers are dysregulated in skin and peripheral blood.

Aims: We used non-invasive skin and peripheral biomarkers to observe the effects of real-world topical corticosteroid treatment (TCS) in infants with AD, by measuring skin and blood biomarkers before and after therapy.

Methods: 74 treatment-naïve infants with AD underwent six-weeks TCS treatment. Stratum corneum (SC) and plasma blood biomarkers as well as SC natural moisturising factor (NMF) were measured before and after TCS therapy. Immune markers included innate, Type 1 and 2 immunity, angiogenesis, and vascular factors. AD severity was assessed by SCORAD and skin barrier function by TEWL. 20 healthy infants were recruited as controls.

Results: TCS therapy predictably led to improvement in disease severity. Levels of immune markers in the skin and in the peripheral blood showed significant change from baseline, though most did not reach healthy control levels. The most prominent change from baseline in SC was in markers of innate activation: IL-18, CXCL8, IL-1α and Th2 chemokines CCL17 and CCL22. In blood, the largest changes were in Type 2 skewed biomarkers: CCL17, IL-13, CCL22, IL-5, and CCL26. TEWL decreased after therapy; no significant changes from baseline were found for NMF.

Conclusions: Profound impact of topical therapy on systemic biomarkers suggests that the skin compartment generates a major component of dysregulated systemic cytokines in infant AD. There may be long term beneficial effects of correcting systemic immune dysregulation through topical therapy.

<AbstractText>15-Lipoxygenase 1 (15LO1) is expressed in airway epithelial cells in patients with type 2-high asthma in association with eosinophilia. Chronic rhinosinusitis with nasal polyps (CRSwNP) is also associated with type 2 inflammation and eosinophilia. <em>CCL26</em>/eotaxin 3 has been reported to be regulated by 15LO1 in lower airway epithelial cells. However, its relation to 15LO1 in patients with CRSwNP or mechanisms for its activation are unclear.</AbstractText><AbstractText>We sought to evaluate 15LO1 and <em>CCL26</em> expression in nasal epithelial cells (NECs) from patients with CRSwNP and healthy control subjects (HCs) and determine whether 15LO1 regulates <em>CCL26</em> in NECs through extracellular signal-regulated kinase (ERK) activation.</AbstractText><AbstractText>15LO1, <em>CCL26</em>, and phosphorylated ERK were evaluated in NECs from patients with CRSwNP and HCs. 15LO1/<em>CCL26</em> and <em>CCL26</em>/cytokeratin 5 were colocalized by means of immunofluorescence. IL-13-stimulated NECs were cultured at an air-liquid interface with or without 15-lipoxygenase 1 gene (ALOX15) Dicer-substrate short interfering RNAs (DsiRNA) transfection, a specific 15LO1 enzymatic inhibitor, and 2 ERK inhibitors. Expression of 15LO1 and <em>CCL26</em> mRNA and protein was analyzed by using quantitative RT-PCR, Western blotting, and ELISA.</AbstractText><AbstractText>15LO1 expression was increased in nasal polyp (NP) epithelial cells compared with middle turbinate epithelial cells from patients with CRSwNP and HCs. 15LO1 expression correlated with <em>CCL26</em> expression and colocalized with <em>CCL26</em> expression in basal cells of the middle turbinate and NPs from patients with CRSwNP. In primary NECs in vitro, IL-13 induced 15LO1 and <em>CCL26</em> expression. 15LO1 knockdown and inhibition decreased IL-13-induced ERK phosphorylation and <em>CCL26</em> expression. ERK inhibition (alone) similarly decreased IL-13-induced <em>CCL26</em>. Phosphorylated ERK expression was increased in NECs from CRSwNP subjects and positively correlated with both 15LO1 and <em>CCL26</em> expression.</AbstractText><AbstractText>15LO1 expression is increased in NP epithelial cells and contributes to <em>CCL26</em> expression through ERK activation. 15LO1 could be considered a novel therapeutic target for CRSwNP.</AbstractText>

Background: Stimulator of interferon genes (STING) activation favors effective innate immune responses against viral infections. Its role in chronic rhinosinusitis with nasal polyps (CRSwNP) remains unknown.

Objective: To explore the expression, regulation, and function of STING in CRSwNP.

Methods: STING expression in sinonasal mucosal samples was analyzed by means of quantitative RT-PCR, immunohistochemistry, flow cytometry and western blotting. STING expression regulation and function were explored by using cultured primary human nasal epithelial cells (HNECs) and BEAS-2B cell lines in vitro.

Results: STING expression was reduced in eosinophilic nasal polyps compared with that in noneosinophilic nasal polyps and control tissues. STING was predominantly expressed by epithelial cells in nasal tissue and was downregulated by IL-4 and IL-13 in a STAT6-dependent manner. HNECs derived from eosinophilic polyps displayed compromised STING-dependent type I interferon production, whereas heightened IL-13-induced STAT6 activation and CCL26 production as compared to those from noneosinophilic polyps and control tissues, which were rescued by exogenous STING overexpression. Knocking down or overexpressing STING decreased or enhanced SOCS1 expression in BEAS-2B cells, respectively, independent of canonical STING pathway elements, TBK1 and IRF3. Knocking down SOCS1 abolished the inhibitory effect of STING on IL-13 signaling in BEAS-2B cells. STING expression positively correlated with SOCS1 expression, but negatively correlated with CCL26 expression in nasal epithelial cells in CRSwNP patients.

Conclusions: Reduced STING expression caused by the type 2 milieu not only impairs STING-dependent type I interferon production, but also amplifies IL-13 signaling by decreasing SOCS1 expression in nasal epithelial cells in eosinophilic CRSwNP.

Keywords: Chemokine (C-C motif) ligand 26; interleukin 13; nasal polyp; signal transducer and activator of transcription 6; stimulator of interferon genes; suppressor of cytokine signaling 1.

In vitro models of differing macrophage functions are useful since human monocyte-derived macrophages are short-lived, finite and vary from donor to donor. Published protocols using the promonocytic cell line THP-1 have tended to result in cells that closely resemble classically-activated macrophages, differentiated in IFNγ and LPS. However, no protocol, to date, has fully recapitulated polarization of THP-1 to the M(IL-4) or M(IL-10) macrophage phenotypes seen when human monocyte-derived macrophages are exposed to each cytokine. Here we present protocols that can be used to prepare M(IL-4) polarized THP-1 that transcribe CCL17, CCL26, CD200R and MRC1 and M(IL-10) cells which transcribe CD163, C1QA and SEPP1. We show that the inhibitory Fcγ Receptor IIb is preferentially expressed on the surface of M(IL-4) cells, altering the balance of activating to inhibitory Fcγ Receptors. Adoption of standardized experimental conditions for macrophage polarization will make it easier to compare downstream effector functions of different macrophage polarization states, where the impact of PMA exposure is minimized and rest periods and cytokine exposure have been optimized.

Background: While mucus plugging is a well reported feature of asthma, it is unknown whether asthma and type-2 inflammation affect mucociliary clearance (MCC).

Research question: Does type 2 inflammation influence mucus clearance rates in mild asthmatics who are not receiving corticosteroids?

Study design and methods: The clearance rates of inhaled radiolabeled particles were compared between mild asthmatics with low (n=17) and high (n=18) levels of T2 inflammation. Fraction exhaled nitric oxide (FeNO) was used to prospectively segregate subjects into T2 Lo (FeNO < 25 ppb) and T2 Hi (FeNO >35 ppb) cohorts. Bronchial brush samples were collected with fiberoptic bronchoscopy and quantitative PCR was performed to measure expression of genes associated with T2 asthma. MCC rate comparisons were also made with a historical group of healthy controls (HCs, n=12).

Results: The T2 Lo cohort demonstrated increased MCC when compared to both T2 Hi and historic HCs. MCC within the T2 Hi group varied significantly with some subjects having low or zero clearance. MCC decreased with increasing expression of several markers of T2 airway inflammation (CCL26, NOS2, and POSTN) and with FeNO. MUC5AC and FOXJ1 expression was similar between the T2Lo and T2Hi cohorts.

Interpretation: Increasing T2 inflammation was associated with decreasing MCC. High rates of MCC in T2 Lo subjects may indicate a compensatory mechanism present in mild disease but lost with high levels of inflammation. Future studies are required to better understand mechanisms and whether impairments in MCC in more severe asthma drive worse clinical outcomes.

Keywords: asthma; cilia; lung imaging; mucociliary clearance; mucus; scintigraphy; type 2 inflammation.