German cockroach extract prevents IL-13-induced CCL26 expression in airway epithelial cells through IL-13 degradation.

Inhaled aeroallergens can directly activate airway epithelial cells (AECs). Exposure to cockroach allergens is a strong risk factor for asthma. Cockroach allergens mediate some of their effects through their serine protease activity; protease activity is also a major contributor to allergenicity. The Th2 cytokine interleukin-13 (IL-13) induces upregulation of the eosinophil chemotactic factor CCL26. CCL26 induces eosinophil migration in allergic inflammation. In this work, we studied the effect of cockroach proteases on IL-13-induced effects. Immersed cultures of the human bronchial epithelial cell line BEAS-2B and air-liquid interface (ALI) cultures of primary normal human bronchial epithelial (NHBE) cells were stimulated with IL-13, Blattella Germanica cockroach extract (CE), or both. IL-13-induced genes were analyzed with qRT-PCR. IL-13 induced upregulation of CCL26, periostin, and IL-13Rα2 in bronchial epithelial cells which were decreased by CE. CE was heat-inactivated (HICE) or pre-incubated with protease inhibitors. HICE and CE preincubated with serine protease inhibitors did not prevent IL-13-induced CCL26 upregulation. CE-degraded IL-13 and specific cleavage sites were identified. CE also decreased IL-4-induced CCL26 upregulation and degraded IL-4. Other serine proteases such as bovine trypsin and house dust mite (HDM) serine proteases did not have the same effects on IL-13-induced CCL26. We conclude that CE serine proteases antagonize IL-13-induced effects in AECs, and this CE effect is mediated primarily through proteolytic cleavage of IL-13. IL-13 cleavage by cockroach serine proteases may modulate CCL26-mediated effects in allergic airway inflammation by interfering directly with the pro-inflammatory effects of IL-13 in vivo.

Amniotic fluid modifies esophageal epithelium differentiation and inflammatory responses.

The interplay between genetic and environmental factors during pregnancy can predispose to inflammatory diseases postnatally, including eosinophilic esophagitis (EoE), a chronic allergic disease triggered by food. Herein, we examined the effects of amniotic fluid (AF) on esophageal epithelial differentiation and responsiveness to proallergic stimuli. Multiplex analysis of AF revealed the expression of 66 cytokines, whereas five cytokines including IL-4 and thymic stromal lymphopoietin (TSLP) were not detected. Several proinflammatory cytokines including TNFα and IL-12 were highly expressed in the AF from women who underwent preterm birth, whereas EGF was the highest in term birth samples. Exposure of esophageal epithelial cells to AF resulted in transient phosphorylation of ERK1/2 and the transcription of early response genes, highlighting the direct impact of AF on esophageal epithelial cells. In a three-dimensional spheroid model, AF modified the esophageal epithelial differentiation program and enhanced the transcription of IL-13-target genes, including CCL26 and CAPN14, which encodes for a major genetic susceptibility locus for eosinophilic esophagitis. Notably, CAPN14 exhibited upregulation in spheroids exposed to preterm but not term AF following differentiation. Collectively, our findings call attention to the role of AF as a potential mediator of the intrauterine environment that influences subsequent esophageal disorders.NEW & NOTEWORTHY The interaction between amniotic fluid and the esophageal epithelium during pregnancy modifies esophageal epithelial differentiation and subsequent responsiveness to inflammatory stimuli, including interleukin 13 (IL-13). This interaction may predispose individuals to inflammatory conditions of the esophagus, such as eosinophilic esophagitis (EoE), in later stages of life.

Th2 cytokine signaling through IL-4Rα increases eotaxin-3 secretion and tension in human esophageal smooth muscle.

In esophageal epithelial cells in eosinophilic esophagitis (EoE), Th2 cytokines (IL-4, IL-13) signal through IL-4Rα, activating JAK to increase eotaxin-3 secretion, which draws eosinophils into the mucosa. We explored whether Th2 cytokines also might stimulate eotaxin-3 secretion and increase tension in esophageal smooth muscle (ESM), which might impair esophageal distensibility, and whether those events could be blocked by proton pump inhibitors (PPIs) or agents that disrupt IL-4Rα signaling. We established human ESM cell cultures from organ donors, characterizing Th2 cytokine receptor and P-type ATPase expression by qPCR. We measured Th2 cytokine-stimulated eotaxin-3 secretion by enzyme-linked immunosorbent assay (ELISA) and ESM cell tension by gel contraction assay, before and after treatment with omeprazole, ruxolitinib (JAK inhibitor), or IL-4Rα blocking antibody. CPI-17 (inhibitor of a muscle-relaxing enzyme) effects were studied with CPI-17 knockdown by siRNA or CPI-17 phospho(T38A)-mutant overexpression. ESM cells expressed IL-4Rα and IL-13Rα1 but only minimal H+-K+-ATPase mRNA. Th2 cytokines increased ESM eotaxin-3 secretion and tension, effects blocked by ruxolitinib and IL-4Rα blocking antibody but not consistently blocked by omeprazole. IL-13 increased ESM tension by increasing CPI-17 expression and phosphorylation, effects blocked by CPI-17 knockdown. Blocking IL-4Rα decreased IL-13-stimulated eotaxin-3 secretion, CPI-17 expression, and tension in ESM. Th2 cytokines increase ESM eotaxin-3 secretion and tension via IL-4Rα signaling that activates CPI-17. Omeprazole does not reliably inhibit this process, but IL-4Rα blocking antibody does. This suggests that ESM eosinophilia and impaired esophageal distensibility might persist despite elimination of mucosal eosinophils by PPIs, and IL-4Rα blocking agents might be especially useful in this circumstance.NEW & NOTEWORTHY We have found that Th2 cytokines increase eotaxin-3 secretion and tension in esophageal smooth muscle (ESM) cells via IL-4Rα signaling. Unlike esophageal epithelial cells, ESM cells do not express H+-K+-ATPase, and omeprazole does not inhibit their cytokine-stimulated eotaxin-3 secretion or tension. An IL-4Rα blocking antibody reduces both eotaxin-3 secretion and tension induced by Th2 cytokines in ESM cells, suggesting that an agent such as dupilumab might be preferred for patients with EoE with esophageal muscle involvement.

Increased SERPINB2 potentiates 15LO1 expression via STAT6 signalling in epithelial cells in eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: SERPINB2, a biomarker of Type-2 (T2) inflammatory processes, has been described in the context of asthma. Chronic rhinosinusitis with nasal polyps (CRSwNP) is also correlated with T2 inflammation and elevated 15LO1 induced by IL-4/13 in nasal epithelial cells. The aim of this study was to evaluate the expression and location of SERPINB2 in nasal epithelial cells (NECs) and determine whether SERPINB2 regulates 15LO1 and downstream T2 markers in NECs via STAT6 signalling. METHODS: SERPINB2 gene expression in bulk and single-cell RNAseq database was analysed by bioinformatics analysis. SERPINB2, 15LO1 and other T2 markers were evaluated from CRSwNP and HCs NECs. The colocalization of SERPINB2 and 15LO1 was evaluated by immunofluorescence. Fresh NECs were cultured at an air-liquid interface with or without IL-13, SERPINB2 Dicer-substrate short interfering RNAs (DsiRNAs) transfection, exogenous SERPINB2, 15-HETE recombinant protein and pSTAT6 inhibitors. 15LO1, 15-HETE and downstream T2 markers were analysed by qRT-PCR, western blot and ELISA. RESULTS: SERPINB2 expression was increased in eosinophilic nasal polyps compared with that in noneosinophilic nasal polyps and control tissues and positively correlated with 15LO1 and other downstream T2 markers. SERPINB2 was predominantly expressed by epithelial cells in NP tissue and was colocalized with 15LO1. In primary NECs in vitro, SERPINB2 expression was induced by IL-13. Knockdown or overexpression SERPINB2 decreased or enhanced expression of 15LO1 and 15-HETE in NECs, respectively, in a STAT6-dependent manner. SERPINB2 siRNA also inhibited the expression of the 15LO1 downstream genes, such as CCL26, POSTN and NOS2. STAT6 inhibition similarly decreased SERPINB2-induced 15LO1. CONCLUSIONS: SERPINB2 is increased in NP epithelial cells of eosinophilic CRSwNP (eCRSwNP) and contributes to T2 inflammation via STAT6 signalling. SERPINB2 could be considered a novel therapeutic target for eCRSwNP.

CX3CR1+ Macrophage Facilitates the Resolution of Allergic Lung Inflammation via Interacting CCL26.

Rationale: The resolution of inflammation is an active process coordinated by mediators and immune cells to restore tissue homeostasis. However, the mechanisms for resolving eosinophilic allergic lung inflammation triggered by inhaled allergens have not been fully elucidated. Objectives: Our objectives were to investigate the cellular mechanism of tissue-resident macrophages involved in the resolution process of eosinophilic lung inflammation. Methods: For the study, we used the institutional review board-approved protocol for human subsegmental bronchoprovocation with allergen, mouse models for allergic lung inflammation, and novel transgenic mice, including a conditional CCL26 knockout. The samples were analyzed using mass cytometry, single-cell RNA sequencing, and biophysical and immunological analyses. Measurements and Main Results: We compared alveolar macrophage (AM) subsets in the BAL before and after allergen provocation. In response to provocation with inhaled allergens, the subsets of AMs are dynamically changed in humans and mice. In the steady state, the AM subset expressing CX3CR1 is a relatively small fraction in bronchoalveolar space and lung tissue but drastically increases after allergen challenges. This subset presents unique patterns of gene expression compared with classical AMs, expressing high C1q family genes. CX3CR1+ macrophages are activated by airway epithelial cell-derived CCL26 via a receptor-ligand interaction. The binding of CCL26 to the CX3CR1+ receptor induces CX3CR1+ macrophages to secrete C1q, subsequently facilitating the clearance of eosinophils. Furthermore, the depletion of CX3CR1 macrophages or CCL26 in airway epithelial cells delays the resolution of allergic lung inflammation displaying prolonged tissue eosinophilia. Conclusions: These findings indicate that the CCL26-CX3CR1 pathway is pivotal in resolving eosinophilic allergic lung inflammation.

Expression of inducible nitric oxide synthase, nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3 in patients with gastroesophageal reflux disease, eosinophilic esophagitis, and in healthy controls: a semiquantitative image analysis of 3,3'-diaminobenzidine-stained esophageal biopsies.

Eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) share many histopathological features; therefore, markers for differentiation are of diagnostic interest and may add to the understanding of the underlying mechanisms. The nitrergic system is upregulated in GERD and probably also in EoE. Esophageal biopsies of patients with EoE (n = 20), GERD (n = 20), and healthy volunteers (HVs) (n = 15) were exposed to antibodies against inducible nitric oxide synthase (iNOS), nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3. The stained object glasses were randomized, digitized, and blindly analyzed regarding the expression of DAB (3,3'-diaminobenzidine) by a protocol developed in QuPath software. A statistically significant overexpression of iNOS was observed in patients with any of the two inflammatory diseases compared with that in HVs. Eotaxin-3 could differentiate HVs versus inflammatory states. Gastroesophageal reflux patients displayed the highest levels of nitrotyrosine. Neither iNOS nor nitrotyrosine alone were able to differentiate between the two diseases. For that purpose, eosinophil peroxidase was a better candidate, as the mean levels increased stepwise from HVs via GERD to EoE. iNOS and nitrotyrosine are significantly overexpressed in patients with EoE and GERD compared with healthy controls, but only eosinophil peroxidase could differentiate the two types of esophagitis. The implications of the finding of the highest levels of nitrotyrosine among gastroesophageal reflux patients are discussed.

Efficacy of dupilumab in patients with uncontrolled, moderate-to-severe asthma with fungal sensitization.

BACKGROUND: Fungal sensitization (FS) exacerbates asthma in patients who have elevated type 2 inflammatory response. Dupilumab, a fully human monoclonal antibody, blocks the shared receptor component for interleukin (IL)-4 and IL-13, key and central drivers of type 2 inflammation in multiple diseases. OBJECTIVE: This post hoc analysis, funded by the manufacturers of dupilumab, was conducted to assess dupilumab efficacy in patients from the phase 3 LIBERTY ASTHMA QUEST trial (NCT02414854) and TRAVERSE open-label extension (NCT02134028) study who had uncontrolled, moderate-to-severe asthma with type 2 inflammatory phenotype (defined as blood eosinophil count ≥150 cells/µL or FeNO ≥25 ppb) and with FS (defined as IgE specific to Alternaria alternata, Aspergillus fumigatus or Cladosporium herbarum >0.35 IU/mL). METHODS: We evaluated annualized rate of severe exacerbations (AER), change from baseline in pre-bronchodilator (BD) forced expiratory volume in 1 s (FEV1 ), asthma control (per 5-item Asthma Control Questionnaire [ACQ-5]) and biomarker levels (blood eosinophil count, fractional exhaled nitric oxide [FeNO], total IgE, fungal-specific IgEs, thymus and activation-regulated chemokine [TARC] and eotaxin-3). RESULTS: Dupilumab vs. placebo reduced AER, improved pre-BD FEV1 and asthma control (ACQ-5), and reduced serum IgE levels, blood eosinophil count, TARC, eotaxin-3 and FeNO in patients both with and without FS after 52 weeks of treatment in QUEST. Reductions in asthma exacerbation rates and improvements in all other variables were sustained over the TRAVERSE open-label extension study. CONCLUSION: Dupilumab demonstrated efficacy during prolonged treatment in patients with uncontrolled, moderate-to-severe asthma with FS.

High proportion of inflammatory CD62Llow eosinophils in blood and nasal polyps of severe asthma patients.

BACKGROUND: In mice models, eosinophils have been divided into different subpopulations with distinct phenotypes and functions, based on CD62L and CD101 patterns of membrane expression. Limited data are available in humans. OBJECTIVE: To investigate eosinophils subpopulations in peripheral blood (PB) and nasal polyp tissue (NP) from severe eosinophilic asthma (SEA) patients plus concomitant chronic rhinosinusitis with nasal polyps (CRSwNP). METHODS: We recruited 23 SEA patients (14 with CRSwNP); as controls, we enrolled 15 non-severe asthma patients, 15 allergic rhinitis patients without asthma and 15 healthy donors. Eosinophils were isolated from PB and NP and analysed by FACS. Eotaxin-3 and eotaxin-1 mRNA expression in NP tissue was also evaluated. RESULTS: A significantly higher percentage of circulating CD62Llow cells was observed in SEA, as compared with controls, expressing higher levels of CCR3, CD69 and lower levels of CD125 (IL-5R), CRTH2, CD86 and CD28 in comparison with CD62Lbright cells. In NP, eosinophils showed a high proportion of CD62Llow phenotype, significantly greater than that observed in PB. Surface expression of IL-3R, IL-5R, CD69 and CD86 was significantly higher in CD62Llow eosinophils from NP than in those from blood. Moreover, eotaxin-3 mRNA expression positively correlated with the percentage of CD62Llow cells in NP. CONCLUSION: Two different eosinophil subphenotypes can be identified in blood and NP of SEA patients, with a preferential accumulation of CD62Llow inflammatory cells in NP.

Assessing serum cytokine profiles in inflammatory breast cancer patients using Luminex® technology.

BACKGROUND: Inflammatory breast cancer (IBC), accounts for the majority of deaths associated with breast tumors. Because this form is aggressive from its appearance and has a strong metastatic potential. The majority of patients are not diagnosed until late stages, highlighting the need for the development of novel diagnostic biomarkers. Immune mediators may affect IBC progression and metastasis installation. AIM OF THE STUDY: Analysis of serum proteins to identify a panel of prognostic biomarkers for IBC. PATIENTS AND METHODS: Serum levels of 65 analytes were determined in IBC and Non-IBC patients with the ProcartaPlex Human Immune Monitoring 65-Plex Panel. RESULTS: Fifteen analytes: 5 cytokines (IL-8, IL-16, IL-21, IL-22 and MIF), 7 chemokines (Eotaxin, eotaxin-3, Fractalkine, IP-10, MIP-1α, MIP-1ß and SDF-1α), One growth factors (FGF-2) and 2 soluble receptors (TNFRII and Tweak); were significantly differentially expressed between the two groups. ROC curves showed that twelve of them (IL-8, IL-16, IL-21, IL-22, MIF, MIP-1α, MIP-1ß, SDF-1α, TNFRII, FGF-2, Eotaxin-3, and Fractalkine) had AUC values greater than 0.70 and thus had potential clinical utility. Moreover, seven cytokines: IL-8, IL-16, MIF, Eotaxin-3, MIP-1α, MIP-1ß, and CD-30 are positively associated with patients who developed distant metastasis. Ten analytes: Eotaxin-3, Fractalkine, IL-16, IL-1α, IL-22, IL-8, MIF, MIP-1α, MIP-1ß, and TNFRII are positively associated with patients who had Lymph-Nodes invasion. CONCLUSION: This study has uncovered a set of 8 analytes (Eotaxin-3, Fractalkine, IL-16, IL-8, IL-22, MIF, MIP-1α, MIP-1ß) that can be used as biomarkers of IBC, and can be utilized for early detection of IBC, preventing metastasis and lymph-Nodes invasion.

C-C Motif Chemokine Receptor 3-Mediated Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase Signaling: Promising Targets for Human Airway Epithelial Mucin 5AC Induction by Eotaxin-2 and Eotaxin-3.

INTRODUCTION: Eotaxin-2 and -3 of the C-C chemokine subfamily function as potent chemoattractant factors for eosinophil recruitment and various immune responses in allergic and inflammatory airway diseases. Mucin 5AC (MUC5AC), a major gel-forming secretory mucin, is overexpressed in airway inflammation. However, the association between mucin secretion and eotaxin-2/3 expression in the upper and lower airway epithelial cells has not been fully elucidated. Therefore, in this study, we investigated the effects of eotaxin-2/3 on MUC5AC expression and its potential signaling mediators. METHODS: We analyzed the effects of eotaxin-2 and -3 on NCI-H292 human airway epithelial cells and primary human nasal epithelial cells (HNEpCs) via reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. Along with immunoblot analyses with specific inhibitors and small interfering RNA (siRNA), we explored the signaling pathway involved in MUC5AC expression following eotaxin-2/3 treatment. RESULTS: In HCI-H292 cells, eotaxin-2/3 activated the mRNA expression and protein production of MUC5AC. A specific inhibitor of C-C motif chemokine receptor 3 (CCR3), SB328437, suppressed eotaxin-2/3-induced MUC5AC expression at both the mRNA and protein levels. Eotaxin-2/3 induced the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and p38, whereas pretreatment with a CCR3 inhibitor significantly attenuated this effect. Induction of MUC5AC expression with eotaxin-2/3 was decreased by U0126 and SB203580, specific inhibitors of ERK1/2 and p38 mitogen-activated protein kinase (MAPK), respectively. In addition, cell transfection with ERK1/2 and p38 siRNAs inhibited eotaxin-2/3-induced MUC5AC expression. Moreover, specific inhibitors (SB328437, U0126, and SB203580) attenuated eotaxin-2/3-induced MUC5AC expression in HNEpCs. CONCLUSION: Our results imply that CCR3-mediated ERK1/2 and p38 MAPK are involved in the signal transduction of eotaxin-2/3-induced MUC5AC overexpression.

In Esophageal Squamous Cells From Eosinophilic Esophagitis Patients, Th2 Cytokines Increase Eotaxin-3 Secretion Through Effects on Intracellular Calcium and a Non-Gastric Proton Pump.

BACKGROUND & AIMS: In upper airway cells, T helper 2 cytokines that signal through interleukin-4 (IL-4) receptor-α have been shown to stimulate eotaxin-3 secretion via a nongastric proton pump (ngH+,K+ATPase). To seek novel targets for eosinophilic esophagitis (EoE) treatments, we evaluated ngH+,K+ATPase expression in EoE squamous cells, and explored molecular pathways involved in eotaxin-3 secretion by IL-4 receptor-α signaling. METHODS: ngH+,K+ATPase expression in EoE cells was evaluated by quantitative real-time polymerase chain reaction and Western blotting. IL-4-stimulated eotaxin-3 secretion was measured by enzyme-linked immunosorbent assay after treatment with omeprazole, SCH 28080 (potassium-competitive acid blocker), ethylene glycol-bis(ß-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester (calcium chelator), 2-aminoethoxydiphenyl borate (inhibitor of endoplasmic reticulum calcium release), verapamil, and diltiazem (L-type calcium channel inhibitors). Intracellular calcium transients were measured by Fluo-4 fluorescence. Key experiments were confirmed in EoE primary cells and in RNA sequencing datasets from mucosal biopsies of patients with EoE and controls. RESULTS: EoE cells expressed ngH+,K+ATPase messenger RNA and protein. Omeprazole and SCH 28080 decreased IL-4-stimulated eotaxin-3 secretion. IL-4 increased intracellular calcium transients, and IL-4-stimulated eotaxin-3 secretion was blocked by ethylene glycol-bis(ß-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester, 2-aminoethoxydiphenyl borate, verapamil, and diltiazem. The combination of omeprazole and verapamil suppressed IL-4-stimulated eotaxin-3 secretion more than either agent alone. EoE biopsies expressed higher ngH+,K+ATPase and exhibited more calcium signaling than controls. CONCLUSIONS: EoE cells express a nongastric proton pump that mediates T helper 2 cytokine-stimulated eotaxin-3 secretion. IL-4 induces calcium release from the endoplasmic reticulum and calcium entry via L-type calcium channels, increasing intracellular calcium that contributes to eotaxin-3 secretion by EoE cells. L-type calcium channel inhibitors block T helper 2 cytokine-stimulated eotaxin-3 secretion, suggesting a potential role for these agents in EoE treatment.

Eotaxin-2 and eotaxin-3 in malaria exposure and pregnancy.

BACKGROUND: Eotaxin-1 concentrations in plasma have been inversely associated with malaria exposure, malaria infection and pregnancy, but the effect of these conditions on the levels of the related chemokines eotaxin-2 and eotaxin-3 remains unknown. METHODS: Eotaxin-2 and -3 concentrations were measured in 310 peripheral or placental plasma samples from pregnant and non-pregnant individuals from Papua New Guinea (malaria-endemic country) and Spain (malaria-naïve individuals) with previous data on eotaxin-1 concentrations. Correlations between eotaxin concentrations were examined with the Spearman's test. Differences in eotaxin concentrations among groups were evaluated with the Kruskal-Wallis or Mann Whitney tests. The pairwise Wilcoxon test was performed to compare eotaxin-2 concentration between peripheral and placental matched plasmas. Univariable and multivariable linear regression models were estimated to assess the association between eotaxins and Plasmodium infection or gestational age. RESULTS: Eotaxin-2 concentrations in plasma showed a weak positive correlation with eotaxin-3 (rho = 0.35, p < 0.05) concentrations. Eotaxin-2 concentrations in the malaria-exposed non-pregnant group were significantly lower than the in the malaria-naive non-pregnant and the malaria-exposed pregnant groups. Eotaxin-3 plasma concentrations were lower in malaria-exposed than in non-exposed groups (p < 0.05), but no differences were found associated to pregnancy. Eotaxin-2 and eotaxin-3 plasma concentrations were negatively correlated with anti-Plasmodium IgG levels: PfDBL5ε-IgG (rhoEo2 = - 0.35, p = 0.005; rhoEo3 =- 0.37, p = 0.011), and eotaxin-3 was negatively correlated with PfDBL3x-IgG levels (rhoEo3 =- 0.36; p = 0.011). Negative correlations of eotaxin-2 and 3 in plasma were also observed with atypical memory B cells (rhoEo2 = - 0.37, p < 0.001; rhoEo3= - 0.28, p = 0.006), a B cell subset expanded in malaria-exposed individuals. In addition, a borderline negative association was observed between eotaxin-3 concentrations and Plasmodium infection (adjusted effect estimate, ß = - 0.279, 95% CI - 0.605; 0.047, p = 0.091). Moreover, eotaxin-2 placental concentrations were significantly increased compared to peripheral concentrations in the malaria-exposed pregnant group whereas the contrary was observed in the non-exposed pregnant group (p < 0.005). CONCLUSION: Although a clear epidemiological negative association is observed between eotaxins concentrations and malaria exposure and/or infection, pregnancy may alter this association for eotaxin-2. Further research is required to understand the role of these chemokines in this disease and in combination with pregnancy.

Tissue eosinophils and mucous inflammatory cytokines for the evaluation of olfactory recovery after endoscopic sinus surgery in patients with nasal polyposis.

The etiologies of chronic rhinosinusitis with nasal polyps (CRSwNP)-associated olfactory dysfunction have several potentially overlapping hypotheses. Understanding the association of tissue eosinophils and mucous inflammatory cytokines with olfactory function and identifying predictors of olfactory outcomes in patients with nasal polyposis after surgery is fundamental for future clinical care and research. METHODS: Eighty-five patients who underwent endoscopic surgery for nasal polyposis were enrolled in this study. Olfactory measurements were performed before surgery and 3-6 months after surgery using a T&T olfactometer. Baseline characteristics of CRSwNP patients were collected, and Spearman's rho correlation was performed to assess the association of olfactory function with tissue eosinophils and mucous inflammatory cytokines. A multivariate logistic regression model was used to assess the independent predictors of olfactory outcomes after surgery. RESULTS: Here, 85 CRSwNP patients, including 25 patients without olfactory disorder and 60 patients with hypo-anosmia, were evaluated. Of the 60 patients with preoperative hypo-anosmia, 22 did not have improved olfactory function, and 38 demonstrated normal olfactory function after surgery based on the T&T olfactometer results. The levels of tissue eosinophil, interleukin-5 (IL-5), IL-13, eotaxin-3, and periostin in the preoperative hypo-anosmia group were higher than those in the preoperative normosmia group. Tissue eosinophil count, IL-5, and periostin levels in patients without olfactory improvement were higher than those in patients with olfactory improvement. The tissue eosinophil count, blood eosinophil count, and nasal mucus levels of IL-5, eotaxin-3, and periostin were significantly correlated with olfactory function in all patients with CRSwNP. The IL-5 level remained a strong predictor of poor olfactory outcomes after surgery. CONCLUSIONS: Both tissue eosinophils and mucous inflammatory cytokines, including IL-5, IL-13, eotaxin-3, and periostin, may contribute to the pathogenesis of CRSwNP-associated olfactory dysfunction. Higher IL-5 levels are associated with a lower chance of olfactory function recovery after each surgical revision.

The Role of Heparan Sulfate in CCL26-Induced Eosinophil Chemotaxis.

Proinflammatory chemokine ligand 26 (CCL26, eotaxin-3) mediates transendothelial cell migration of eosinophils by binding and activating the G-protein-coupled (GPC) chemokine receptor 3 on the surface of eosinophilic cells. Here we have investigated the role of glycosaminoglycans (GAGs) as potential co-receptors in the process of CCL26-induced eosinophil chemotaxis. For this purpose, we have first identified the GAG-binding site of CCL26 by a site-directed mutagenesis approach in the form of an alanine screening. A panel of GAG-binding-deficient mutants has been designed, generated, and analyzed with respect to their binding affinities to heparan sulphate (HS) by isothermal fluorescence titration studies. This showed that basic amino acids in the α-helical part of CCL26 are strongly involved in GAG-binding. In chemotaxis experiments, we found that decreased GAG-binding affinity correlated with decreased chemotactic activity, which indicates an involvement of GAGs in eosinophil migration. This was further proven by the negative impact of heparinase III treatment and, independently, by the incubation of eosinophils with an anti heparan sulfate antibody. We finally investigated eosinophils' proteoglycan (PG) expression patterns by real-time PCR, which revealed the highest expression level for serglycin. Including an anti-serglycin antibody in CCL26-induced eosinophil migration experiments reduced the chemotaxis of these immune cells, thereby proving the dependence of eosinophil mobilization on the proteoglycan serglycin.

Extensive serum biomarker analysis in the prethrombotic state of recurrent spontaneous abortion.

The prethrombotic state (PTS) is a possible cause of recurrent spontaneous abortion (RSA). The aim of this study was to identify serum biomarkers for the detection of RSA with PTS (PSRSA). A Quantibody array 440 was used to screen novel serum-based biomarkers for PSRSA/NRSA (RSA without PTS). Proteins differentially expressed in PSRSA were analysed using bioinformatics methods and subjected to a customized array and enzyme-linked immunosorbent assay (ELISA) validation. We used receiver operating characteristic to calculate diagnostic accuracy, and machine learning methods to establish a biomarker model for evaluation of the identified targets. 20 targets were selected for validation using a customized array, and seven targets via ELISA. The decision tree model showed that IL-24 was the first node and eotaxin-3 was the second node distinguishing the PSRSA and NRSA groups (an accuracy rate of 100% and an AUC of 1). Epidermal growth factor (EGF) as the node distinguished the PSRSA and NC groups (an accuracy rate of 100% and an AUC of 1). EGF as the node distinguished the NRSA and NC groups (an accuracy rate of 96.5% and an AUC of 0.998). Serum DNAM-1, BAFF, CNTF, LAG-3, IL-24, Eotaxin-3 and EGF represent a panel of promising diagnostic biomarkers to detect the PSRSA.

Dupilumab suppresses type 2 inflammatory biomarkers across multiple atopic, allergic diseases.

BACKGROUND: Type 2 inflammation is common in numerous atopic/allergic diseases and can be identified by elevated biomarker levels. Dupilumab, a fully human monoclonal antibody, blocks the shared receptor component for interleukin-4 and interleukin-13, key and central drivers of type 2 inflammation. OBJECTIVE: Assessment of dupilumab effect on type 2 inflammatory biomarkers in atopic dermatitis (AD), asthma, chronic rhinosinusitis with nasal polyps (CRSwNP) and eosinophilic esophagitis (EoE). METHODS: Data were extracted from three randomized placebo-controlled trials of dupilumab in AD (NCT02277743, N = 671; NCT02277769, N = 708; NCT02260986, N = 740); and one each in asthma (NCT02414854, N = 1902); CRSwNP (NCT02898454, N = 448); and EoE (NCT02379052, N = 47). Biomarkers assessed were serum thymus and activation-regulated chemokine (TARC), plasma eotaxin-3, serum total immunoglobulin E (IgE), serum periostin and blood eosinophil count. RESULTS: Dupilumab versus placebo significantly suppressed most type 2 inflammatory biomarker levels across all studies/indications where data were assessed. Reductions in serum TARC, plasma eotaxin-3 and serum periostin occurred rapidly, whereas reductions in serum total IgE were more gradual. Across diseases, at the end of treatment, median percentage change from baseline in TARC levels ranged from -24.8% to -88.6% (placebo +2.6% to -53.6%); -38.2% to -51.5% (placebo +8.3% to -0.16%) in eotaxin-3; -24.8% to -76.7% (placebo +8.3% to -4.4%) in total IgE; and -13.6% to -41.1% (placebo +10.1% to -6.94%) in periostin levels. Blood eosinophil responses to dupilumab varied by disease, with minimal changes in AD in the SOLO studies (median percentage change from baseline to end of treatment: 0% [95% CI: -15.8, 0]); transient increases followed by decreases to below-baseline levels in asthma (-14.6% [-20.0, -7.7]) and CRSwNP (-29.4% [-40.0, -16.3]); and significant decreases in EoE (-50.0% [-50.0, -33.3]). CONCLUSION AND CLINICAL RELEVANCE: Dupilumab reduced levels of type 2 biomarkers across clinical studies in patients with AD, asthma, CRSwNP and EoE.

Lymphadenopathy in IgG4-related disease: a phenotype of severe activity and poor prognosis, with eotaxin-3 as a new biomarker.

OBJECTIVE: To clarify relevant proteins and clinical characteristics of a phenotype of IgG4-related disease (IgG4-RD) with lymphadenopathy. METHODS: We enrolled patients newly diagnosed with IgG4-RD in our department between January 2000 and June 2018 and performed proteomic analysis to measure serum concentrations of 1305 proteins. We extracted proteins overexpressed in patients with IgG4-RD with lymphadenopathy by comparing between those with lymphadenopathy, those without lymphadenopathy and healthy controls. We further reviewed all the patients with IgG4-RD in our institution and investigated the characteristics and prognosis of the patients with IgG4-RD with lymphadenopathy. RESULTS: Eighty-five patients with IgG4-RD were enrolled, of which, 55% had lymphadenopathy. Proteomic analysis in 31 patients with IgG4-RD and 6 healthy controls revealed that eotaxin-3 was a potential serum biomarker in the patients with lymphadenopathy versus those without lymphadenopathy and healthy controls. A cohort of 85 patients with IgG4-RD demonstrated that patients with lymphadenopathy showed a significantly higher serum IgG4, IgG4:IgG ratio, IgG4-RD responder index and eosinophilia (P < 0.001 for all), irrelevant of the extent to which organ involvement developed. Patients with lymphadenopathy treated with glucocorticoid alone relapsed with significantly higher rates than those without lymphadenopathy (P = 0.03). CONCLUSION: Lymphadenopathy in IgG4-RD represents a phenotype associated with high disease activities, eosinophilia and relapsing disease. Eotaxin-3 is a novel biomarker related to IgG4-RD with lymphadenopathy.

Vaginal host immune-microbiome interactions in a cohort of primarily African-American women who ultimately underwent spontaneous preterm birth or delivered at term.

BACKGROUND: Recent studies suggest that alterations in the vaginal microbiome allow for the assessment of the risk for spontaneous preterm birth (PTB), the leading cause of neonatal morbidity and mortality worldwide. However, the associations between the local immune response and the vaginal microbiome are still poorly understood. Herein, we characterize the vaginal host immune-microbiome interactions in women who ultimately underwent PTB and in those who delivered at term. METHODS: Vaginal fluid samples from 52 pregnant women (of whom 18 underwent PTB and 34 delivered at term) were collected between 10 and 32 weeks of gestation in a case-control study. Concentrations of 33 immune mediators were determined using sensitive and specific immunoassays. The previously published 16S rRNA gene sequence and bacterial phylotype data of these subjects were utilized in this study. Linear mixed effects models were utilized to test associations between vaginal immune mediator concentrations and bacterial phylotype relative abundances. RESULTS: 1) In the overall study population, vaginal concentrations of CXCL10, CCL2, CCL3, SLP1 and VEGF negatively correlated with non-Lactobacillus, Community State Type IV (CST IV) members of the vaginal microbiome; 2) CXCL10, in particular, negatively correlated with 15 bacterial phylotypes, most of which are typical members of CST IV, such as Gardnerella vaginalis, Megasphaera spp., and Atopobium vaginae; 3) Gemella spp., also members of CST IV, negatively correlated with vaginal concentrations of VEGF, CCL2, CCL3, SLPI, and CXCL10; 4) when comparing PTB cases to term controls, five soluble immune mediators (CCL26, CCL22, CCL2, CXCL10, and IL-16), especially CCL26, were negatively correlated with five typical members of CST IV: Sneathia sanguinegens, Parvimonas micra, Veillonellaceae, BVAB2, and Gemella spp.; and 5) Sneathia sanguinegens had stronger negative associations with all five soluble immune mediators (CCL26, CCL22, CCL2, CXCL10, and IL-16) in PTB cases than in term controls. CONCLUSIONS: The assessment of vaginal host immune-microbiome interactions revealed that specific soluble immune mediators, mainly CXCL10, negatively correlated with typical members of CST IV of the vaginal microbiome. Sneathia sanguinegens, in particular, had stronger negative associations with different immune mediators, including CXCL10 and CCL26, in women who ultimately underwent PTB compared to those who delivered at term. These findings provide insight into the vaginal host immune-microbiome interactions in normal and complicated pregnancies.

Immunoinflammatory Biomarkers in Serum Are Associated with Disease Severity in Atopic Dermatitis.

BACKGROUND: A growing body of evidence links various biomarkers to atopic dermatitis (AD). Still, little is known about the association of specific biomarkers to disease characteristics and severity in AD. OBJECTIVE: To explore the relationship between various immunological markers in the serum and disease severity in a hospital cohort of AD patients. METHODS: Outpatients with AD referred to the Department of Dermatology, Bispebjerg Hospital, Copenhagen, Denmark, were divided into groups based on disease severity (SCORAD). Serum levels of a preselected panel of immunoinflammatory biomarkers were tested for association with disease characteristics. Two machine learning models were developed to predict SCORAD from the measured biomarkers. RESULTS: A total of 160 patients with AD were included; 53 (33.1%) with mild, 73 (45.6%) with moderate, and 34 (21.3%) with severe disease. Mean age was 29.2 years (range 6-70 years) and 84 (52.5%) were females. Numerous biomarkers showed a statistically significant correlation with SCORAD, with the strongest correlations seen for CCL17/thymus and activation-regulated chemokine (chemokine ligand-17/TARC) and CCL27/cutaneous T cell-attracting-chemokine (CTACK; Spearman R of 0.50 and 0.43, respectively, p < 0.001). Extrinsic AD patients were more likely to have higher mean SCORAD (p < 0.001), CCL17 (p < 0.001), CCL26/eotaxin-3 (p < 0.001), and eosinophil count (p < 0.001) than intrinsic AD patients. Predictive models for SCORAD identified CCL17, CCL27, serum total IgE, IL-33, and IL-5 as the most important predictors for SCORAD, but with weaker associations than single cytokines. CONCLUSIONS: Specific immunoinflammatory biomarkers in the serum, mainly of the Th2 pathway, are correlated with disease severity in patients with AD. Predictive models identified biomarkers associated with disease severity but this finding warrants further investigation.

CCL26 is upregulated by nab-paclitaxel in pancreatic cancer-associated fibroblasts and promotes PDAC invasiveness through activation of the PI3K/AKT/mTOR pathway.

Recently, the combined use of FOLFIRINOX (leucovorin and fluorouracil plus irinotecan and oxaliplatin) and gemcitabine plus nab-paclitaxel has significantly improved the prognosis of patients with pancreatic cancer. However, there is still a high proportion of patients who develop metastatic pancreatic cancer in the course of chemotherapy or within a short period after chemotherapy. Previous reports have shown that chemotherapy-driven cytokine storms or the direct effects of certain chemotherapeutics on stromal and/or immune cells collectively change the microenvironment of the primary tumor, thus indirectly promoting metastasis. However, the mechanism underlying chemotherapy-induced metastasis in the course of chemotherapy, and afterwards, remains elusive in pancreatic cancer. In the present study, we aimed to determine the expression of CCL26 in the pancreatic cancer-associated fibroblasts (CAFs) after nab-paclitaxel treatment and to explore the role of CCL26 in the pancreatic adenocarcinoma (PDAC) invasion. Our results showed that nab-paclitaxel increased CCL26 mRNA and protein expression levels in a dose- and time-dependent manner. Subsequently, PDAC cell lines were treated with recombinant CCL26 for 48 h. The transwell migration assay showed that recombinant CCL26 enhanced the invasion of PDAC cells. Western blot analysis showed that the protein expression levels of phospho-(p-)PI3K, p-AKT, and p-mTOR were increased by CCL26 in PDAC cells. CCL26 expressions in 95 PDAC tissues and adjacent normal tissues were evaluated using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. CCL26 was found to be overexpressed in PDAC samples, and upregulated CCL26 expression was significantly associated with advanced perineural invasion, lymph node metastasis, and poor differentiation. In summary, our results showed that nab-paclitaxel increased the expression of CCL26 in CAFs, and CCL26 enhanced the invasive potential of pancreatic cancer cells by activating the PI3K/AKT/mTOR axis. Thus, CCL26 may be a potential prognostic biomarker for pancreatic cancer.