Chemokines are a large family of chemotactic cytokines that play critical roles in inflammation, development, and diseases. Chemokine expression is highly regulated during development and in response to environmental stimuli. The 3' untranslated regions (3'-UTRs) of mRNA are believed to be important in the control of chemokine gene expression. However, the regulatory effects of most chemokine 3'-UTRs have not been characterized previously. In this work, we systematically studied the effects of 43 CC and CXC chemokine 3'-UTRs on gene expression in eight human cell lines and two types of human primary cells. We found that chemokine 3'-UTRs had a wide spectrum of regulatory effects on mRNA abundance and protein production that were tightly correlated with the effects on mRNA stability. In general, 3'-UTRs had remarkably similar effects across all cell types studied. The presence of AU-rich elements, microRNA targets, and Pumilio binding sites were associated with chemokine 3'-UTR activity but did not fully account for all 3'-UTR activity detected using the reporter assay. Mutational analysis illustrated how specific cis-regulatory elements contributed to the regulatory effect of chemokine 3'-UTRs. These findings bring new insights into the mechanisms by which chemokine expression is regulated by 3'-UTRs.

Eosinophilic esophagitis (EoE) is a recently described disease in which exposure to specific foods and allergens leads to type 2 inflammation, epithelial barrier dysfunction, and difficulty in swallowing. In the current issue of the JCI, Wen and colleagues investigate tissue T cell heterogeneity in patients with EoE using single-cell RNA sequencing (scRNA-seq). Esophageal epithelium from individuals with EoE convtained a prominent population of Th2 cells not seen in controls. The short-chain fatty acid (SCFA) receptor FFAR3 was found to be highly expressed in EoE Th2 cells. Experiments presented here provide evidence that SCFAs may promote type 2 inflammation in allergic diseases such as EoE and asthma. This study provides an early example of scRNA-seq for identifying relevant cell populations and mechanisms underlying allergic diseases.

The clinical manifestations of asthma are caused by obstruction of the conducting airways of the lung. Two airway cell types are critical for asthma pathogenesis: epithelial cells and smooth muscle cells. Airway epithelial cells, which are the first line o

The development of pathologic mucus, which is not readily cleared from the airways, is an important contributor to the morbidity and mortality associated with asthma. It is not clear how the major airway mucins MUC5AC and MUC5B are organized within the mucus gel or how this gel contributes to airway obstruction in asthma. Here, we demonstrated that mucus plugs from individuals with fatal asthma are heterogeneous gels with distinct MUC5AC- and MUC5B-containing domains. Stimulation of cultured human bronchial epithelial cells with IL-13, a key mediator in asthma, induced the formation of heterogeneous mucus gels and dramatically impaired mucociliary transport. Impaired transport was not associated with defects in ciliary function but instead was related to tethering of MUC5AC-containing mucus gel domains to mucus-producing cells in the epithelium. Replacement of tethered mucus with untethered mucus restored mucociliary transport. Together, our results indicate that tethering of MUC5AC-containing domains to the epithelium causes mucostasis and likely represents a major cause of mucus plugging in asthma.

Chitin is produced in large amounts by fungi, insects, and other organisms and has been implicated in the pathogenesis of asthma. Airway epithelial cells are in direct contact with environmental particles and serve as the first line of defense against inhaled allergens and pathogens. The potential contributions of airway epithelial cells to chitin-induced asthma remain poorly understood. We hypothesized that chitin directly stimulates airway epithelial cells to release cytokines that promote type 2 immune responses and to induce expression of molecules which are important in innate immune responses. We found that chitin exposure rapidly induced the expression of three key type 2-promoting cytokines, IL-25, IL-33 and TSLP, in BEAS-2B transformed human bronchial epithelial cells and in A549 and H292 lung carcinoma cells. Chitin also induced the expression of the key pattern recognition receptors TLR2 and TLR4. Chitin induced the expression of miR-155, miR-146a and miR-21, each of which is known to up-regulate the expression of pro-inflammatory cytokines. Also the expression of SOCS1 and SHIP1 which are known targets of miR-155 was repressed by chitin treatment. The monoterpene phenol carvacrol (Car) and its isomer thymol (Thy) are found in herbal essential oils and have been shown to inhibit allergic inflammation in asthma models. We found that Car/Thy inhibited the effects of chitin on type 2-promoting cytokine release and on the expression of TLRs, SOCS1, SHIP1, and miRNAs. Car/Thy could also efficiently reduce the protein levels of TLR4, inhibit the increase in TLR2 protein levels in chitin plus Car/Thy-treated cells and increase the protein levels of SHIP1 and SOCS1, which are negative regulators of TLR-mediated inflammatory responses. We conclude that direct effects of chitin on airway epithelial cells are likely to contribute to allergic airway diseases like asthma, and that Car/Thy directly inhibits epithelial cell pro-inflammatory responses to chitin.

Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins alpha4beta7, alphaLbeta2, and alphaMbeta2 were heterologously expressed in K562 cells. alpha4beta7 initiated adhesion under flow and localized to microvilli, whereas beta2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the alpha4beta7 cytoplasmic and/or transmembrane domains with the homologous domains of alphaLbeta2 or alphaMbeta2. Unexpectedly, these chimeras efficiently mediated adhesion to the alpha4beta7 ligand mucosal addressin cell adhesion molecule-1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of alpha4 and beta2 integrins do not account for differences in ability to support attachment under flow or in membrane localization. Integrins alpha4beta1, alpha5beta1, alpha6Abeta1, alphavbeta3, and alphaEbeta7 also localized to microvilli. Transmembrane proteins known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans and CD47, were concentrated on microvilli as well. These findings suggest that interactions between the extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli.

Functional characterization of noncoding sequences is crucial for understanding the human genome and learning how genetic variation contributes to disease. 3'2 untranslated regions (UTRs) are an important class of noncoding sequences, but their functions r

Thymic dendritic cells (tDCs) play an important role in central tolerance by eliminating self-reactive thymocytes or differentiating them to regulatory T (Treg) cells. However, the molecular and cellular mechanisms underlying these functions are not completely understood. We found that mouse tDCs undergo maturation following cognate interaction with self-reactive CD4+ thymocytes and that this maturation is dependent on CD40 signaling. Ablation of CD40 expression in tDCs resulted in a significant reduction in the number of Treg cells in association with a significant reduction in the number of mature tDCs. In addition, CD40-deficient DCs failed to fully mature upon cognate interaction with CD4+ thymocytes in vitro and failed to differentiate them into Treg cells to a sufficient number. These findings suggest that tDCs mature and potentiate Treg cell development in feedback response to self-reactive CD4+ thymocytes.

AU-rich elements (AREs) are 3' UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3' UTR sequences that conform to these motifs affect their function. Here, we use functional annotation of sequences from 3' UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3' UTR sequence fragments from 4653 transcripts on gene expression and mRNA stability in Jurkat and Beas2B cells. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Finally, to investigate the advantages of our general experimental design we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3' UTRs.