(1) Background: The intra-tumoural heterogeneity (ITH) of hepatocellular carcinoma (HCC) and its microenvironment (TME) across primary and secondary disease is poorly characterised. (2) Methods: Intra-tumoural (IT) and peri-tumoural (PT) staining of matched primary and secondary samples was conducted to evaluate the distribution of CD4+/FOXP3+ and CD8+/PD1+ T-cells. Samples underwent PD-L1/2 immunostaining, tumour mutational burden (TMB) evaluation, and high-resolution T-cell receptor (TCR) sequencing to derive T-cell clonality and targeted transcriptomics. (3) Results: We analysed 24 samples from matched primary (n = 11) and secondary (n = 13; 5 synchronous, 6 metachronous) deposits, 11 being extrahepatic (84.6%). IT CD8+ density was lower than PT in both primary (p = 0.005) and secondary deposits (p = 0.01), consistent with immune exclusion. PD-L1+ tumours displayed higher IT and PT CD8+/PD1+ cell density compared to PD-L1- (p < 0.05), and primary IT infiltrate was enriched in CD4+/FOXP3+ cells, compared to PT regions (p = 0.004). TCR-sequencing demonstrated enrichment of the top T-cell clonotype in secondary versus primary HCC (p = 0.02), without differences in overall productive clonality (p = 0.35). TMB was similar across primary versus secondary HCC (p = 0.95). While directed gene set analysis demonstrated the uniformity of transcriptional signatures of individual immune cell types, secondary deposits demonstrated higher COLEC12 (p = 0.004), CCL26 (p = 0.02), CD1E (p = 0.02) and CD36 (p = 0.03) expression with downregulation of CXCL1 (p = 0.03), suggesting differential regulation of innate immunity. (4) Conclusion: Immune exclusion is a defining feature of the HCC TME. Despite evidence of homogeneity in somatic TMB, secondary HCC is characterised by the expansion of a distinct T-cell clonotype and differential regulation of innate immune pathways.

Keywords: hepatocellular carcinoma; intra-tumoural heterogeneity; tumour microenvironment.

We previously demonstrated that IL4, IL13, CLCA1, and CCL26 mRNA were significantly upregulated in the lungs of pigs given a low dose of all trans-retinoic acid (ATRA) and infected with Ascaris suum. We also demonstrated that in vitro ATRA induced a state of partial alternative activation in porcine macrophages (Mφs) and amplified certain aspects of M2a activation induced by IL-4. Given these results, we tested the effect of ATRA on IL-4 responses in two porcine intestinal epithelial cell lines, IPEC1 and IPEC-J2 and observed that ATRA increased mRNA for the IL-4 receptor alpha chain. ATRA also increased IL-4 induced phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mRNA expression of the chloride channel, calcium activated, family member 1 (CLCA1), important for mucus formation, and chemokine (C-C motif) ligand 26 (CCL26), a potent eosinophil chemoattractant. We extended these findings to human Mφ THP-1 cells and showed that ATRA synergistically increased IL-4-induced CCL2, CCL13, and CCL26 mRNA and protein levels. Transglutaminase 2 mRNA, protein, and enzyme activity were synergistically induced in THP-1 cells pretreated with ATRA and then treated with IL-4, thus, ATRA increased signaling in response to IL-4 in porcine epithelial cells and porcine and human Mφs. Given the prevalence of allergic and parasitic diseases worldwide and the close similarities in the porcine and human immune responses, these findings have important implications for the nutritional regulation of allergic inflammation at mucosal surfaces.

Keywords: all-trans-retinoic acid; human; interleukin 4; macrophage; porcine.

Gene expression patterns have not been extensively examined in the context of clinical features of eosinophilic esophagitis (EoE).

To assess whether gene expression is associated with clinically defined phenotypes in adults with EoE.

This was an analysis of prospectively collected esophageal biopsies in newly diagnosed EoE patients. We determined differential gene expression with a 94 gene panel in relation to clinical features and phenotypes. These included: endoscopic findings of esophageal rings, stricture, narrowing, linear furrows, exudates, edema, and dilation; an allergic phenotype; an inflammatory phenotype, and a fibrostenotic phenotype.

In 89 EoE cases analyzed, patients with exudates on endoscopy had multiple differences in gene expression compared to patients without exudates, though patients with exudates also had higher eosinophil counts (172 vs 106eos/hpf; p=.01). Genes associated with esophageal narrowing included CCL26 (q-value=0.028), ALOX15 (q=0.011), GRK5 (q=0.029), CPA3 (q=0.012), and TRIM2 (q=0.0027). TRIM2 was also associated with the fibrostenotic phenotype (q=0.0051). No genes were associated with the inflammatory or atopic phenotypes, or with dilation.

Multiple genes are associated with exudates, possibly related to higher eosinophil counts. However, a number of genes, including those related to both inflammation and remodelling, are associated with esophageal narrowing. In particular, TRIM2 is associated with clinical fibrotic phenotypes.

BACKGROUND Rhinitis is the most common clinical manifestation of allergy, affecting more than 400 million people around the world. Rhinitis increases the risk of developing bronchial hyper-responsiveness and asthma. Previous studies have shown that rhinitis is closely related with the physiology, pathology, and pathogenesis of asthma. We analyzed co-expressed genes to explore the relationships between rhinitis and asthma and to find biomarkers of comorbid rhinitis and asthma. MATERIAL AND METHODS Asthma- and rhinitis-related differentially-expressed genes (DEGs) were identified by bioinformatic analysis of GSE104468 and GSE46171 datasets from the Gene Expression Omnibus (GEO) database. After assessment of Gene Ontology (GO) terms and pathway enrichment for DEGs, a protein-protein interaction (PPI) network was conducted via comprehensive target prediction and network analyses. We also evaluated co-expressed DEGs and corresponding predicted miRNAs involved in the developing process of rhinitis and asthma. RESULTS We identified 687 and 1001 DEGs in bronchial and nasal epithelia samples of asthma patients, respectively. For patients with rhinitis, we found 245 DEGs. The hub-genes of PAX6, NMU, NTS, NMUR1, PMCH, and KRT6A may be associated with rhinitis, while CPA3, CTSG, POSTN, CLCA1, HDC, and MUC5B may be involved in asthma. The co-expressed DEGs of BPIFA1, CCL26, CPA3, and CST1, together with corresponding predicted miRNAs (e.g., miR-195-5p and miR-125a-3p) were found to be significantly correlated with rhinitis and asthma. CONCLUSIONS Rhinitis and asthma are related, and there are significant correlations of BPIFA1, CCL26, CPA3, and CST1 genes with novel biomarkers involved in the comorbidity of rhinitis and asthma.

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.

Keywords: 16S rRNA gene; CCL26; CXCL10; Chemokines; Cytokines; Pregnancy; Sneathia; Vaginal microbiota; β-defensins.

Nivolumab plus ipilimumab combined therapy is among the most effective therapies for advanced melanoma. However, this therapy is also associated with a high frequency of immune-related adverse events (irAEs). To avoid such severe irAEs caused by additional administration of anti-CTLA4 antibodies, biomarkers to distinguish responders from non-responders among patients treated with anti-PD1 antibodies are important. The purpose of this study is to evaluate the increased serum levels of CCL11, CCL24, and CCL26 as a predictive biomarker for the efficacy of anti-PD1 antibodies in advanced cutaneous melanoma patients. This study analyzed increased serum levels of CCL11, CCL24, and CCL26 in 46 cases of advanced cutaneous melanoma treated with anti-PD1 antibodies. Serum levels on day 42 were compared to baseline (day 0) and analyzed statistically. Receiver operating characteristic curves were established to evaluate the correlation between serum levels of CCL11, CCL24, and CCL26 and efficacy of anti-PD1 antibodies. Increased serum levels of CCL26 correlated significantly with the efficacy of anti-PD1 antibodies. In contrast, no significant correlations were seen between increased serum levels of CCL11 and CCL24 and efficacy of anti-PD1 antibodies. Increased serum levels of CCL26 may be a useful biomarker for identifying those patients with advanced cutaneous melanoma most likely to benefit from anti-melanoma immunotherapy.

Interleukin (IL)-13 is a signature cytokine of type 2 inflammation important for the pathogenesis of various diseases, including allergic diseases. Signal transducer and activator of transcription (STAT) 6 is a critical transcriptional factor for the IL-13 signals; however, it remains unknown how expression of the IL-13-induced genes is differentiated by the transcriptional machineries. In this study, we identified IL-13-induced transcriptional factors in lung fibroblasts using DNA microarrays in which SOX11 was included. Knockdown of SOX11 down-regulated expression of periostin and CCL26, both of which are known to be downstream molecules of IL-13, whereas enforced expression of SOX11 together with IL-13 stimulation enhanced expression of periostin. Moreover, we found that in DNA microarrays combining IL-13 induction and SOX11 knockdown there exist both SOX11-dependent and -independent molecules in IL-13-inducible molecules. In the former, many inflammation-related and fibrosis-related molecules, including periostin and CCL26, are involved. These results suggest that SOX11 acts as a trans-acting transcriptional factor downstream of STAT6 and that in lung fibroblasts the IL-13 signals are hierarchically controlled by STAT6 and SOX11.

Keywords: CCL26/eotaxin-3; basophils; eosinophils; eruptive pruritic papular porokeratosis; thymic stromal lymphopoietin.

Background: Eosinophilic esophagitis (EoE) is a food-allergen driven disease that is accompanied by interleukin (IL) 13 overexpression and esophageal barrier dysfunction allowing transepithelial food allergen permeation. Nutraceuticals, such as short-chain fatty acids (SCFAs) that restore barrier function and increase immune fitness may be a promising tool in the management of EoE. Here, we investigated the effects of the SCFAs acetate, propionate and butyrate on an IL-13-compromised human esophageal epithelial barrier, including the mechanisms involved.

Methods: An air-liquid interface culture model of differentiated human EPC2-hTERT (EPC2) was used to study whether SCFAs could restore barrier function after IL-13-induced impairment. Esophageal epithelial barrier function was monitored by transepithelial electrical resistance (TEER) and FITC-dextran paracellular flux, and was further examined by qPCR and immunohistochemical analysis. G protein-coupled receptor (GPR) 41, GPR43, GPR109a or histone deacetylase (HDAC) (ant)agonists were used to assess mechanisms of action of SCFAs.

Results: IL-13 stimulation decreased TEER and increased FITC flux, which was counteracted by butyrate and propionate, but not acetate treatment. Barrier proteins FLG and DSG1 mRNA expression was upregulated following butyrate and propionate treatment, whereas expression of eosinophil chemoattractant CCL26 and protease CAPN14 was downregulated. Similarly, butyrate and propionate restored FLG and DSG1 protein expression. Similar effects were observed with an HDAC antagonist but not with GPR agonists.

Conclusion: Nutraceuticals butyrate and propionate restore the barrier function of esophageal epithelial cells after an inflammatory insult and may be of therapeutic benefit in the management of EoE.

Keywords: barrier function; dietary intervention; eosinophilic esophagitis; interleukin 13; short-chain fatty acids.

The bronchial epithelium is a very important factor during the inflammatory response, it produces many key regulators involved in the pathophysiology of asthma and COPD. Local influx of eosinophils, basophils, Th2 lymphocytes and macrophages is the source of many cytotoxic proteins, cytokines and other mediators of inflammation. These cells are attracted by eotaxins (eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26). Inhibitors of phosphodiesterase 4 (PDE4) are new anti-inflammatory drugs which cause cAMP accumulation in the cell and inhibit numerous stages of allergic inflammation. The aim of our study was to evaluate the influence of PDE4 inhibitors: rolipram and RO-20-1724 on the expression of eotaxins in human primary bronchial epithelial cells. Cells were preincubated with PDE4 inhibitors for 1 hour and then stimulated with IL-4 or IL-13 alone or in combination with TNF-α. After 48 hours, eotaxin protein level was measured by ELISA and mRNA level by real time PCR. These cells produce CCL24 and CCL26. PDE4 inhibitors increased CCL24 and CCL26 mRNA level irrespectively of the used stimulators. Rolipram and RO-20-1724 had no effect on eotaxin protein production in our experimental conditions. Thus PDE4 inhibitors have no effect on eotaxin protein expression in human primary bronchial epithelial cells. In vitro experiments should be performed using a primary cell model rather than immortalized lines.

BACKGROUND

Chemokine ligands (CCLs) play a pivotal role in tissue injury before and after kidney transplantation. Meanwhile, transplantation improves patient's survival and diminishes morbidity. It is hypothesized, then, that kidney transplantation diminishes pre-transplant (pre-TX) levels of circulating inflammatory CCLs. This retrospective study compared circulating levels and profiles of CCLs before transplantation (pre-TX) and after transplantation (post-TX).

METHODS

Nineteen CCLs (1, 2, 3, 4, 5, 8, 11, 13, 15, 17, 21, 24, 26, 27, CXCL 5, 8, 10, 12 and 13) were measured in 47 stable post-TX recipients, and their stored pre-TX plasma was analyzed by multiplexed fluorescent bead-based immunoassay. Twenty normal controls were included for comparisons. Normalized data was presented as mean ± SD and non-normalized data as median (5-95 % CI). Significance was measured at p < 0.01. Arbitrary upper and lower margins for each CCL at the 95 % CI or 2SD levels in each group were chosen to calculate the percentile of patients in the other group who exceeded these limits. Significant CCL levels present in more than 75 % of patients in a group that exceeded the arbitrary upper or lower set margins in the other two groups were labeled as preferentially characteristic for the respective group.

RESULTS

More than 75 % of pre- and post-TX patients had levels that exceeded the upper control for CCL1, 11, 15 and CCL15, CCL26 and CXCL13 levels, respectively. More than 75 % of pre- and post-TX patients exceeded the lower control for CCL3, 21, and CCL5 limits, respectively. More than 75 % of post-TX patients demonstrated elevated levels of CCL2, 3, 21, 26 and CXCL13 above the upper pre-TX cut offs. Meanwhile, more than 75 % of post-TX patients exceeded the lower pre-TX levels for CCL1, 4, 5, 8, 13, 15, 17, 24 and CXCL8 and10. Pre-TX was preferentially characterized by elevated CCL1 and 15 and diminished CCL3 and 21. Post-TX was preferentially characterized by elevated CCL26 and CXCL13 and diminished CCL4 and 5.

CONCLUSIONS

End stage kidney disease is associated with enhanced circulating inflammatory chemokine levels. Stable kidney transplantation is associated with 1) lowered burden of circulating inflammatory chemokine levels and, 2) elevation in the pro T-helper2 chemokine, CCL26 and the homeostatic CXCL13.

Chronic neuroinflammation is strongly associated with AD and altered peripheral and central levels of chemokines and cytokines have been frequently described in those with AD. Given the increasing evidence of ethnicity-related differences in AD, it was of interest to determine if those altered chemokine and cytokine levels are ethnicity-related. Because African Americans exhibit a higher incidence of AD and increased symptom severity, we explored chemokine and cytokine concentrations in post-mortem brain tissue from the BA21 region of African Americans and Caucasians with AD using multiplex assays. IL-1β, MIG, TRAIL, and FADD levels were significantly increased in African Americans while levels of IL-3 and IL-8 were significantly decreased. Those effects did not interact with gender; however, overall levels of CCL25, CCL26 and CX3CL1 were significantly decreased in women. The NLRP3 inflammasome is thought to be critically involved in AD. Increased activation of this inflammasome in African Americans is consistent with the current results.

BACKGROUND

Eosinophilic oesophagitis is an inflammatory condition characterized by a dense eosinophilic infiltrate. The migration of eosinophils into the oesophagus is influenced by cytokines such as IL-5, IL-13 and eotaxin-3. The aim of this study was to evaluate changes in the cytokine expression profiles (IL-5, IL-13 and eotaxin-3/CCL26) in children after topical steroid treatment.

METHODS

a prospective case-control study was performed in 23 paediatric patients (age 5-16 years) with a histological diagnosis of eosinophilic oesophagitis. Histological evaluation and cytokine levels assay (IL-5, IL-13 and eotaxin-3/CCL26) in the proximal and distal oesophagus were performed before, and after 8 weeks of topical budesonide. Data were compared with a matched healthy control group.

RESULTS

quantitative expression levels of IL-5, IL-13 and eotaxin-3 were significantly higher in the eosinophilic oesophagitis group both compared to healthy subjects (p<0.0001). A significant reduction of the eosinophil infiltrate as well as of IL-5, IL-13 and eotaxin-3 mucosal profiles was observed after steroid treatment both at the proximal and distal oesophagus (p<0.0001).

CONCLUSIONS

IL-5, IL-13 and eotaxin-3/CCL26 are significantly over-expressed in the oesophageal epithelium of children with eosinophilic oesophagitis. Topical steroid treatment (inhaled and swallowed budesonide) can induce clinical response with partial mucosal remission.

New literature data and the results of own researches concerning the role of excessive body weight and the development of type 2 diabetes mellitus in humans are presented in the analytical review. Inaccordance with current insights, obesity and type 2 diabetes are considered diseases of inflammatory nature, characterized by systemic chronic low-grade inflammation, where different kinds of cytokines are cardinally involved. Unfavourable life style, i.e. excessive, high-energy, and irrational nutrition--an excessive consumption of animal fats and foods containing the high amount of glucose and starch with an insufficient use of high fiber vegetables, fish and vitamin D, and also sedentary, inactive life style leads to adipocyte hypertrophy and migration of M1 macrophages into the adipose tissue (AT). As a result, there is a low-grade inflammation accompanied by an increased production of proinflammatory cytokines (IL-1, IL-6, TNF-α, etc.), adipokines (leptin, resistin, visfatin etc.) and chemokines (CCL2, CCL5, CCL26 and CX3C). Under the influence of these cytokines, on the one hand, IR "is emerged", and on the other--there is apoptosis of the β-cells, that should be followed by the occurrence of clinically diagnosed type 2 diabetes. However, there is also the opposite system in humans, protecting the organism from the development of type 2 diabetes, and including an increase in the formation of M2 macrophages and the increased formation of secretion of antidiabetic cytokines (IL-4, IL-10, IL-13, etc.) and adiponectin.

The aim of this study was to identify for the first time single nucleotide polymorphisms (SNPs) associated with H. contortus resistance in Florida Native sheep, using a targeted sequencing approach. One hundred and fifty three lambs were evaluated in this study. At the start of the trial, phenotypic records for fecal egg count (FEC), FAMACHA score, body condition score (BCS), and weight were recorded and deworming of sheep with levamisole (18 mg per kg of body weight) was performed. Ten days post-deworming (baseline) and 28 days post baseline, a full hematogram of each sheep was obtained and FEC, FAMACHA score, BCS and weight were assessed. Average daily gain was calculated at the end of the trial. Out of 153 animals, 100 sheep were selected for genotyping using a targeted sequencing approach. Targeted sequencing panel included 100 candidate genes for immune response against H. contortus. SNPs were discarded if call rate < 95% and minor allele frequency ≤0.05. A mixed model was used to analyze the response variables and included the identity by state matrix to control for population structure. A contemporary group (age, group and sex) was included as fixed effect. Bonferroni correction was used to control for multiple testing. Eighteen SNPs on chromosomes 1, 2, 3, 4, 6, 7, 11, 15, 18, 20, 24 and 26 were significant for different traits. Our results suggest that loci related to Th17, Treg and Th2 responses play an important role in the expression of resistant phenotypes. Several genes including ITGA4, MUC15, TLR3, PCDH7, CFI, CXCL10, TNF, CCL26, STAT3, GPX2, IL2RB and STAT6 were identified as potential markers for resistance to natural H. contortus exposure. This is the first study that evaluates potential genetic markers for H. contortus resistance in Florida Native sheep.

Eosinophilic esophagitis (EoE) is a relatively new condition described as an allergic-mediated disease of the esophagus. Clinically, it is characterized by dysphagia, food impaction, and reflux-like symptoms. Multiple genome-wide association studies (GWAS) have been conducted to identify genetic loci associated with EoE. The integration of numerous studies investigating the genetic polymorphisms in EoE and the Mendelian diseases associated with EoE are discussed to provide insights into the genetic risk of EoE, notably focusing on CCL26 and CAPN14. We focus on the genetic loci investigated thus far, and their classification according to whether the function near the loci is known. The pathophysiology of EoE is described by separately presenting the known function of each cell and molecule, with the major contributors being eosinophils, Th2 cells, thymic stromal lymphopoietin (TSLP), transforming growth factor (TGF)-β1, and interleukin (IL)-13. This review aims to provide detailed descriptions of the genetics and the comprehensive pathophysiology of EoE.

Keywords: genetic susceptibility; pathophysiology; polymorphism.

Background: Neuropsychiatric disorders have been linked to immune mechanisms. Altered peripheral levels of eotaxin-1/CCL11; a cytokine implicated in allergic reactions and aging process; have been reported in bipolar disorder (BD). Several brain areas, especially the temporal lobe, seem to display volume loss and accelerated aging in BD. This study aimed at exploring potential associations between eotaxins and brain volumes in patients with BD compared to controls.

Methods: Twenty-two euthymic patients with BD and 22 controls were enrolled in this study. Serum levels of eotaxin-1/CCL11, eotaxin-2/CCL24 and eotaxin-3/CCL26 were determined alongside brain volumes.

Results: There were no differences in the levels of eotaxins between patients and controls. A negative correlation was found between eotaxin-1/CCL11 levels and left-hemisphere's superior-temporal volume only in BD patients, which persisted with covariate adjusted model.

Conclusion: This study corroborates the emerging evidence of association between inflammation and brain volumes in BD. Our preliminary results also support the hypothesis of a possible role of eotaxin-1/CCL11 in accelerated brain aging in BD.

Keywords: Accelerated aging; Bipolar disorder; CCL-11; Eotaxin; Superior-temporal volume.

BACKGROUND

Allergic asthma is characterized by eosinophilic inflammation and airway obstruction. There is also an increased risk of pulmonary infection caused by Streptococcus pneumoniae, in particular during severe asthma where high levels of the glycoprotein, osteopontin (OPN), are present in the airways. Eosinophils can be recruited by chemokines activating the receptor CCR3 including eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, and MEC/CCL28. In addition to inducing chemotaxis, several of these molecules have defensin-like antibacterial properties. This study set out to elucidate the functional consequences of OPN binding to eosinophil-recruiting chemokines.

METHODS

Antibacterial activities of the chemokines were investigated using viable count assays and electron microscopy. Binding studies were performed by means of surface plasmon resonance. The potential interference of OPN with antibacterial, receptor-activating, and lipopolysaccharide-neutralizing abilities of these chemokines was investigated.

RESULTS

We found that OPN bound all eosinophil-recruiting chemokines with high affinity except for CCL5. The eosinophil-recruiting chemokines all displayed bactericidal activity against S. pneumoniae, but only CCL26 and CCL28 retained high antibacterial activity in the presence of sodium chloride at physiologic concentrations. Preincubation of the chemokines with OPN strongly inhibited their antibacterial activity against S. pneumoniae but did not affect their ability to activate CCR3. All chemokines investigated showed LPS-neutralizing activity that was impaired by OPN only in the case of CCL24.

CONCLUSIONS

The data suggest that OPN may impair host defense activities of the chemokines without affecting their eosinophil-recruiting properties. This could be one mechanism explaining the increased vulnerability to acquire pneumococcal infection in parallel with sustained allergic inflammation in asthma.

BACKGROUND

Much attention on the pathophysiology of nasal polyp (NP) has focused on eosinophils. Interleukin (IL)-4 and eotaxin-3 (C-C motif chemokine ligand 26, or CCL26) levels have been reported to be increased in eosinophilic nasal polyps. The aim of this study was to characterize CCL26 posttranscriptional regulation by the RNA-binding protein HuR in primary human nasal polyp-derived epithelial cells (hNPDECs) challenged with IL-4.

METHODS

A prospective, observational study was conducted. Nasal polyp tissues were obtained from eosinophilic (n = 12) and non-eosinophilic (n = 10) NP patients, and inferior turbinate (IT) tissues were taken from control subjects (n = 9) and cultured into hNPDECs. Expression of HuR and CCL26 were measured by immunohistochemistry, Western blot analysis, enzyme-linked immunoassay, and real-time polymerase chain reaction (PCR). The nucleocytoplasmic shuttling of HuR in hNPDECs was detected by immunofluorescence. Posttranscriptional regulation of CCL26 by HuR was tested by ribonucleoprotein immunoprecipitation assay (RIP) and dual-luciferase reporter assay. CCL26 mRNA stabilization was measured by quatititative PCR after treatment with actinomycin D. Student's t test and one-way analysis of variance were used.

RESULTS

Immunohistochemical data show that both HuR and CCL26 were highly expressed in NP tissues, especially eosinophilic NP tissues (p < 0.05). IL-4 stimulation increased CCL26 mRNA stability, and overexpression and knockdown of HuR affected CCL26 expression. Immunofluorescence data indicate that IL-4 altered the subcellular distribution of HuR. The RIP and dual-luciferase reporter assay results supply strong evidence for HuR binding to CCL26.

CONCLUSIONS

Our results provide strong support for the hypothesis that IL-4-induced expression of CCL26 in hNPDECs relies partly on CCL26 mRNA stabilization mediated by the interaction of HuR with CCL26 3'UTR.

Background: The role of chemokines in anaphylaxis is unclear.

Methods: We prospectively recruited 49 patients presenting to the emergency department with an acute episode of anaphylaxis and 28 healthy subjects. We measured serum levels of the chemokines CCL2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL17, CCL21, CCL22, CCL24, and CCL26, tryptase, the absolute number of circulating basophils, monocytes, lymphocytes, and PMNs, and whole blood FCER1A, CPA3 and HDC gene expression at two time points: during the anaphylactic episode and in convalescent samples collected approximately 3 months later. We then investigated the in vitro chemotactic activity of chemokines induced during anaphylaxis for the in vitro migration of the corresponding cells.

Results: Only CCL2 chemokine levels were significantly increased in anaphylaxis samples (median 514 pg/ml) compared to convalescent samples (284 pg/ml, P < 0.0001) and healthy subjects (279 pg/ml, P < 0.0001); there was no significant difference in any of the other chemokines. There was a significant positive correlation between the rates of increase of serum CCL2 (median [range]: 106.0% [- 44.7% to 557.4%]) and tryptase (133.8% [- 6.6% to 893.4%]; r = 0.68, P < 0.0001) and between the acute concentration of serum CCL2 and the acute concentration of serum tryptase (r = 0.77, P < 0.0001). The number of circulating basophils, but not other blood cells, significantly decreased during anaphylaxis (median 5.0 vs. 19.1 cells/µl in convalescent samples; P < 0.0001); a decrease in whole-blood gene expression of basophil markers (P ≤ 0.0018) confirmed these changes. Anaphylactic serum enhances the in vitro migration of basophils via CCL2-dependent chemotactic activity; in contrast, no CCL2-dependent chemotactic activity was observed for convalescent samples.

Conclusions: Our findings imply an important and specific role for CCL2-mediated chemotactic activity in the pathophysiology of human anaphylaxis.

Keywords: Anaphylaxis; Basophils; CCL2; Chemokines; Chemotaxis; Migration; Tryptase.

Background: Chronic rhinosinusitis (CRS) without nasal polyps (CRSsNP) is a common disease and is characterized by multiple inflammatory endotypes. However, molecular mechanisms in CRSsNP are poorly understood compared to polypoid CRS.

Objective: To identify mechanisms and biomarkers associated with inflammatory endotypes underpinning CRSsNP.

Methods: Ethmoid tissues and nasal lavage fluids (NLF) were obtained from control patients and patients with CRS. The gene expression profiles were determined by microarray as well as quantitative RT-PCR and expression of proteins was measured by ELISA and Luminex.

Results: Microarray found that 126, 241, and 545 genes were >3-fold and significantly elevated in CRSsNP with type (T) 1 endotype (T1 CRSsNP), T2 CRSsNP, and T3 CRSsNP, respectively, compared to control tissue. Selected identified genes were confirmed by RT-PCR. Gene set enrichment analysis suggested that T1 CRSsNP was associated with IFN-γ-signaling and anti-viral immunity controlled by T cells (Th1 and CD8+), NK cells and antigen presenting cells (APC); T2 CRSsNP was associated with STAT6-signaling and IgE-mediated activation controlled by eosinophils, mast cells, Th2 cells, ILC2 and APC; and T3 CRSsNP was associated with IL-17-signaling, acute-inflammatory response, complement-mediated inflammation and infection controlled by neutrophils, Th17 cells, B cells and APC. Results suggest that T1 (CXCL9 and CXCL10), T2 (eosinophilic proteins and CCL26) and T3 (CSF3) endotypic biomarkers in NLF may be able to distinguish tissue endotypes in CRSsNP.

Conclusions: Inflammatory endotypes in CRSsNP were controlled by different molecular mechanisms. NLF biomarker assays may allow for more precise and personalized medical treatments in CRS.

Keywords: Biomarker; Chronic rhinosinusitis without nasal polyps; Endotype; Transcriptome.

Acute ischemic stroke (AIS) is the most common type of stroke. Recent studies have found that AIS is closely involved in the immune regulation function of regulatory T cells (Tregs). C-C motif chemokine ligand 26 (CCL26) is a member of the chemokine family that plays an essential role in cell activation, cell differentiation, lymphocyte homing, and inflammatory and immune responses. The present study aimed to investigate the role of CCL26 in the regulation of Tregs in AIS. Peripheral blood mononuclear cells (PBMCs) were incubated with a CCL26-neutralizing antibody. The proportion of cluster of differentiation (CD)4⁺CD25⁺ forkhead box P3 (FOXP3)⁺ Tregs was increased, and the expression of FOXP3, phosphorylated signal transducer and activator of transcription 5 (p-STAT5), and that of the immunosuppressive factors, interleukin (IL)-10 and transforming growth factor (TGF)-β1, was upregulated. Conversely, the expression of immune-promoting factors, such as tumor necrosis factor (TNF)-α and IL-6 was significantly downregulated. Further experiments using CCL26 recombinant protein-treated PBMCs revealed a decreased proportion of CD4⁺CD25⁺FOXP3⁺ Tregs and the downregulated expression of FOXP3, p-STAT5, TGF-β1 and IL-10. Moreover, the expression of immunostimulatory factors, such as CX3C chemokine receptor 1, TNF-α and IL-6 was significantly upregulated. On the whole, these results demonstrate that CCL26 regulates the proportion of CD4⁺CD25⁺FOXP3⁺ Tregs and the production of inflammatory factors in PBMCs following AIS via the STAT5 pathway.

Keywords: C-C motif chemokine ligand 26; acute ischemic stroke; regulatory T cells; signal transducer and activator of transcription 5.