Emodin suppresses mast cell migration via modulating the JAK2/STAT3/JMJD3/CXCR3 signaling to prevent cystitis.

AIMS: This study aimed to determine the preventive effects of emodin on cyclophosphamide (CYP)-induced cystitis and to explore the molecular mechanism. METHODS: In vivo, mice were modeled by CYP. Before a half hour of CYP treatment, Jumonji domain-containing protein-3 (JMJD3) inhibitors (GSK-J4) and emodin were used to treat CYP model mice. Bladder samples were stained for hematoxylin-eosin and toluidine blue. Next, JMJD3 was quantified by immunofluorescence staining, RT-PCR, and Western blot. CXCR3 was quantified by Western blot and ELISA. In vitro, before stimulated by lipopolysaccharide (LPS), human bladder smooth muscle cells (hBSMCs) were transfected with pcDNA3.1-JMJD3 plasmids, shRNA-JMJD3 plasmids or pretreated with emodin. Collected cells to detect JMJD3 and CXCR3 ligands again; collected supernatant of culture for Transwell assay. Finally, as the JAK2 inhibitor, AG490 was used to pretreat LPS-induced hBSMCs. Western blot was performed to quantify proteins. RESULTS: Emodin inhibited mast cell migration and suppressed the expression of JMJD3, CXCR3, and CXCR3 ligands, not only in vivo but also in vitro. The pharmacological effects of emodin were similar to GSK-J4 or JMJD3 inhibition. In addition, emodin significantly downregulated the phosphorylation of JAK2 and STAT3, and inhibited JMJD3/CXCR3 axis transduction like AG490. CONCLUSION: Emodin has a preventive effect on cystitis by inhibiting mast cell migration through inhibition of the JAK2/STAT3/JMJD3/CXCR3 signaling pathway.

Effect of IL-35 on apoptosis, adhesion, migration, and activation of eosinophils in allergic rhinitis.

BACKGROUND: Eosinophils play critical roles in the development of allergic rhinitis (AR) by releasing toxic substance. Interleukin-35 (IL-35), a newly identified anti-inflammatory cytokine, had potent inhibitive role for eosinophil infiltration in allergic disease. However, the direct effect of IL-35 on eosinophil was not clear. METHODS: Twenty AR children and sixteen controls were recruited. The correlation between IL-35 protein expression and blood eosinophil counts and activation was analyzed. The effect of IL-35 on eosinophil apoptosis and adhesion was analyzed by flow cytometry. Transwell system was used for the migration assay. The eosinophil cationic protein (ECP) from supernatant of eosinophils after IL-35 stimulation was detected by enzyme-linked immunosorbent assay kits. RESULTS: The IL-35 protein levels were negatively correlated with eosinophil counts (p < .01) and ECP concentration (p < .01) in AR children. IL-35 promotes apoptosis and inhibits adhesion, migration, and activation of eosinophils. Moreover, the mRNA expression of IL-12 receptor ß2 and glycoprotein 130 were significantly enhanced by eosinophils after IL-35 stimulation. The apoptosis induced by IL-35 was mediated by phosphoinositide 3-kinase (PI3K) pathway. IL-35 inhibits adhesion of eosinophils through extracellular regulated protein kinases (ERK) and PI3K pathways. The eosinophil chemotaxis and activation affected by IL-35 were mediated by PI3K and p38 mitogen-activated protein kinase (MAPK) pathways. CONCLUSION: Our results confirmed that IL-35 played inhibitive roles in apoptosis, adhesion, migration, and activation of eosinophils in AR, implying that IL-35 may be used as treatment target in future.

Genetic Variations in Nucleotide Excision Repair Pathway Genes and Risk of Allergic Rhinitis.

Background: Allergic rhinitis (AR) is the most frequent inflammatory disorder in the nasal mucosa that remains unclear etiology. Mounting studies suggested that genetic instability could trigger and worsen the inflammatory response. The nucleotide excision repair (NER) system is an important pathway in maintaining the stability of the genome. Therefore, the genetic variations in NER pathway genes may have potential effects on AR risk. Methods: We evaluated the correlation between 19 candidate single nucleotide polymorphisms (SNPs) in NER pathway genes and AR susceptibility by a case-control study in a Chinese population, which contains 508 AR cases and 526 controls. Results: Three independent SNPs were identified as significantly associated with AR susceptibility, including ERCC1 rs2298881 C > A (recessive model: adjusted odds ratios (OR) = 0.30, 95%confidence interval (CI) = 0.18-0.50, P < 0.0001), ERCC1 rs11615 G > A (dominant model: adjusted OR = 1.44, 95%CI = 1.04-2.01, P = 0.030), and XPC rs2228001 A > C (dominant model: adjusted OR = 0.68, 95%CI = 0.49-0.95, P = 0.024). Stratified analysis showed that ERCC1 rs2298881 AA genotype was correlated with a lower risk of AR among all the subgroups compared with rs2298881 CC/CA genotype. XPC rs2228001 AC/CC genotype reduced AR risk among the following subgroups: age > 60 months, clinical stage I and III. Conclusion: Our finding showed that genetic variations in NER pathway genes: ERCC1 and XPC may affect the risk of AR, which will provide new insights into the genetics of AR from the perspective of DNA damage repair.

Compliance, efficacy, and safety of subcutaneous and sublingual immunotherapy in children with allergic rhinitis.

BACKGROUND: Although previous studies had confirmed the effectiveness and safety of subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT), respectively, direct head-to-head comparison of SCIT vs SLIT is sparse. We aimed to compare the efficacy, safety, and compliance of SCIT and SLIT in allergic rhinitis (AR) children. METHODS: This study is a prospective, open-label, and single-center study performed between June 2017 and June 2018. A total of 325 children were grouped into SLIT, Alutard (SCIT1), and NovoHelisen Depot (NHD) (SCIT2) according to the parents' wishes. The adherence and reasons for dropout were recorded. The efficacy of SLIT and SCIT was evaluated by a combined symptom medication score. Adverse events (AEs) were recorded and graded during the whole treatment. RESULTS: The compliance rate was higher in the SCIT group compared with the SLIT group (P < .05). The total nasal symptom score (TNSS), rescue medication score (RMS), and symptom medication score (SMS) after 6-month, 12-month, and 2-year treatment were lower in the SCIT group compared with the SLIT group (P < .05). But the scores between the Alutard and NHD groups were not significantly different. The occurrence of AEs in the SCIT group was significantly higher compared with the SLIT group (P < .05). CONCLUSION: Our results suggested SCIT is more effective compared with SLIT to a certain degree, whereas SLIT had less AEs compared with SCIT. The AIT routes can be chosen according to personal specific conditions.

Inhibited interleukin 35 expression and interleukin 35-induced regulatory T cells promote type II innate lymphoid cell response in allergic rhinitis.

BACKGROUND: Interleukin (IL)-35 and IL-35-producing regulatory T cells (iTr35) have been reported to inhibit TH2 response in allergic rhinitis (AR). However, its effects on type II innate lymphoid cells (ILC2) are not well characterized. OBJECTIVE: To investigate the effect of IL-35 on ILC2 in AR. METHODS: A total of 25 patients with AR and 20 controls were recruited. The expression and regulation of IL-35 receptor in ILC2 were analyzed by real-time polymerase chain reaction. The effect of IL-35 on ILC2 differentiation and cytokine production was analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, iTr35 were cocultured with ILC2 to explore the effect of iTr35 on ILC2. The AR mice models were also established to confirm the role of IL-35 in vivo. RESULTS: The patients with AR had decreased IL-35 expression and iTr35 proportion and increased ILC2 and type II cytokines compared with the controls. Notably, IL-35 inhibited ILC2 differentiation and type II cytokine production by regulating IL-12Rß2 and gp130. IL-35 promoted the inducible costimulatory molecule expression by iTr35 and the inducible costimulatory molecule ligand expression by ILC2. IL-35-treated mice with AR presented decreased frequency and function of nasal ILC2. CONCLUSION: IL-35 inhibited ILC2 responses directly or through mutual contact between iTr35 and ILC2 in AR, suggesting that IL-35 may be used as a potential treatment target in AR.

The Effect of IL-35 on the Expression of Nasal Epithelial-Derived Proinflammatory Cytokines.

BACKGROUND: Airway epithelium plays an important role during the development of allergic rhinitis (AR), which is characterized by production of thymic stromal lymphopoietin (TSLP), interleukin 33 (IL-33), and interleukin 25 (IL-25). IL-35, mainly expressed by Treg cells, have negative regulation in Th2, Th17, and ILC2 inflammation. However, the effect of IL-35 on human nasal epithelial cells (HNECs) especially the secretion of nasal epithelial-derived proinflammatory cytokines as well as the possible mechanism is still unclear. METHODS: HNECs were cultured and stimulated by various stimulators. The expression of IL-33, IL-25, TSLP, eotaxin-1, eotaxin-2, and eotaxin-3 from supernatant was measured using real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). AR mice were developed to verify the effect of IL-35 on nasal epithelial cells in vivo. RESULTS: After Poly I:C stimulation, IL-35 inhibited the production of IL-25, and TSLP from HNECs increased significantly compared with baseline levels (P < 0.05). After Dermatophagoides pteronyssinus or Aspergillus fumigatus stimulation, IL-35 inhibited the production of IL-25, IL-33, and TSLP from HNECs increased significantly compared with baseline levels (P < 0.05). After Dermatophagoides pteronyssinus, IL-35 inhibited the production of eotaxin-1, eotaxin-2, and eotaxin-3 released from HNECs increased significantly compared with baseline levels (P < 0.05). Similarly, IL-35-treated AR mice presented with decreased expression of IL-33, IL-25, TSLP, eotaxin-1, eotaxin-2, and eotaxin-3 in nasal lavage fluid. CONCLUSION: IL-35 suppressed both type 2 inflammation-inducing cytokines and eosinophil chemotactic factor from HNECs, suggesting the important role of IL-35 during the development of AR.

Efficacy of Sublingual Immunotherapy in Allergic Rhinitis Children during Coronavirus Disease 2019.

BACKGROUND: Sublingual immunotherapy (SLIT) had good effectiveness for children with allergic rhinitis (AR). However, no studies explored the effect of persistent allergen exposure on SLIT treatment. Coronavirus disease 2019 (COVID-19) restricts outdoor activities of children significantly. We aimed to evaluate the effectiveness of SLIT during this special period. METHODS: A total of 335 AR children who sensitize to house dust mite (HDM) undergoing SLIT were recruited in this study. The clinical effectiveness and safety were evaluated at different time points using symptom and medication scores. The serum total IgE and specific IgE (sIgE) at different time points were detected by using the Unicap system. RESULTS: The total nasal symptoms score (TNSS) and total medication score (TMS) during the epidemic of COVID-19 increased significantly compared with the same period last year (p < 0.05), despite that they were still significantly lower than baseline levels (p < 0.05). The occurrence of adverse reactions at different time points had no significant differences. We also found that the family of the good response group had more frequent bedding cleaning. Both the tIgE and sIgE levels had no significant changes during SLIT treatment. CONCLUSION: Our results suggest that continuous HDM exposure reduced the effectiveness of SLIT, whereas effective reduction of HDM levels by frequent bed cleaning will be helpful during the SLIT treatment.

Risk Factors for Safety of Allergen-Specific Sublingual Immunotherapy in Children with Allergic Rhinitis.

A good compliance often attributes to good efficacy and safety of sublingual immunotherapy (SLIT). However, few studies have been conducted on the safety of SLIT treatment in children. We aimed to confirm the pretreatment parameters to predict the safety in children who underwent SLIT. 601 children with allergic rhinitis (AR) treated with SLIT were enrolled in this study. Baseline clinical information and laboratory parameters were collected. The clinical response and adverse events (AEs) were recorded and evaluated. A multivariate logistic regression model was established to confirm the predictors for AEs. The AEs were reported in 75 children (13.8%). The serum-specific IgE (s-IgE) level was significantly correlated with the occurrence of AEs by multivariate logistic regression analysis. Our receiver operating characteristic (ROC) analysis of the serum s-IgE levels >21.6 IU/mL had the best sensitivity (83.7%) and specificity (76.7%) to predict safety. The serum s-IgE level was significantly correlated with safety of SLIT in children, which may be helpful for patient selection before SLIT.

Leptin Regulated ILC2 Cell through the PI3K/AKT Pathway in Allergic Rhinitis.

BACKGROUND: Recent studies suggest that leptin is involved in Th2 response in allergic rhinitis (AR). However, the effect of leptin on type II innate lymphoid cells (ILC2s) in AR is not well characterized. METHODS: Twenty-six AR patients and 20 healthy controls were enrolled. Serum leptin levels were measured, and their correlation with ILC2 and type II cytokines were analyzed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. ILC2 differentiation and cytokine production stimulated by human recombinant leptin were analyzed by real-time polymerase chain reaction (PCR) and ELISA. AR mouse models were also established to verify the effect of leptin on ILC2 cell regulation. RESULTS: Our results showed that elevated serum leptin in AR patients was correlated with the percentage of ILC2 and the expression of type II cytokines. The recombinant leptin enhanced the expression of ILC2 cell transcription factors and type II cytokine through the PI3K/AKT pathway. The AR mice treated with leptin showed as stronger ILC2 inflammation and symptoms compared with control mice. CONCLUSIONS: Our data provide evidence that upregulation of leptin promotes ILC2 responses in AR and this process was achieved through the PI3K/AKT pathway.

Leptin Promoted IL-17 Production from ILC2s in Allergic Rhinitis.

BACKGROUND: Interleukin-17 plays important roles in allergic diseases. Several studies proved that leptin promoted Th17 immune responses by inducing RORγt transcription. ILC2 is an important member of the early stage of immune response. Therefore, we aimed to explore the effect of leptin on the IL-17 production by ILC2 in AR in this study. METHODS: Fifteen AR patients and fifteen healthy controls were enrolled. Serum leptin levels were measured, and their correlation with the frequency of IL-17+ ILC2 cells was analyzed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. ILC2 was stimulated by leptin, and the expression of IL-17, IL-5, and IL-13 was detected by ELISA. The correlated pathways were confirmed by real-time PCR. RESULTS: We found that serum leptin and the frequency of IL-17-producing ILC2s in AR were significantly higher compared with those in controls. After being incubated with leptin, the frequency of IL-17+ ILC2 cells and IL-17 production from ILC2 was upregulated compared with that in controls. We also found that leptin induced RORγt and Ahr expression by ILC2. Moreover, leptin-induced IL-17-producing ILC2 concomitantly expressed IL-5 and IL-13. CONCLUSIONS: Our data provide preliminary evidence that leptin-induced IL-17 production from ILC2 cells is dependent on RORγt and Ahr expression and the blockade of leptin may be a promising target for the treatment of AR.

Different KChIPs compete for heteromultimeric assembly with pore-forming Kv4 subunits.

Auxiliary Kv channel-interacting proteins 1-4 (KChIPs1-4) coassemble with pore-forming Kv4 α-subunits to form channel complexes underlying somatodendritic subthreshold A-type current that regulates neuronal excitability. It has been hypothesized that different KChIPs can competitively bind to Kv4 α-subunit to form variable channel complexes that can exhibit distinct biophysical properties for modulation of neural function. In this study, we use single-molecule subunit counting by total internal reflection fluorescence microscopy in combinations with electrophysiology and biochemistry to investigate whether different isoforms of auxiliary KChIPs, KChIP4a, and KChIP4bl, can compete for binding of Kv4.3 to coassemble heteromultimeric channel complexes for modulation of channel function. To count the number of photobleaching steps solely from cell membrane, we take advantage of a membrane tethered k-ras-CAAX peptide that anchors cytosolic KChIP4 proteins to the surface for reduction of background noise. Single-molecule subunit counting reveals that the number of KChIP4 isoforms in Kv4.3-KChIP4 complexes can vary depending on the KChIP4 expression level. Increasing the amount of KChIP4bl gradually reduces bleaching steps of KChIP4a isoform proteins, and vice versa. Further analysis of channel gating kinetics from different Kv4-KChIP4 subunit compositions confirms that both KChIP4a and KChIP4bl can modulate the channel complex function upon coassembly. Taken together, our findings show that auxiliary KChIPs can heteroassemble with Kv4 in a competitive manner to form heteromultimeric Kv4-KChIP4 channel complexes that are biophysically distinct and regulated under physiological or pathological conditions.

Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.

Olmsted syndrome (OS) is a rare congenital disorder characterized by palmoplantar and periorificial keratoderma, alopecia in most cases, and severe itching. The genetic basis for OS remained unidentified. Using whole-exome sequencing of case-parents trios, we have identified a de novo missense mutation in TRPV3 that produces p.Gly573Ser in an individual with OS. Nucleotide sequencing of five additional affected individuals also revealed missense mutations in TRPV3 (which produced p.Gly573Ser in three cases and p.Gly573Cys and p.Trp692Gly in one case each). Encoding a transient receptor potential vanilloid-3 cation channel, TRPV3 is primarily expressed in the skin, hair follicles, brain, and spinal cord. In transfected HEK293 cells expressing TRPV3 mutants, much larger inward currents were recorded, probably because of the constitutive opening of the mutants. These gain-of-function mutations might lead to elevated apoptosis of keratinocytes and consequent skin hyperkeratosis in the affected individuals. Our findings suggest that TRPV3 plays essential roles in skin keratinization, hair growth, and possibly itching sensation in humans and selectively targeting TRPV3 could provide therapeutic potential for keratinization or itching-related skin disorders.

The tetramerization domain potentiates Kv4 channel function by suppressing closed-state inactivation.

A-type Kv4 potassium channels undergo a conformational change toward a nonconductive state at negative membrane potentials, a dynamic process known as pre-open closed states or closed-state inactivation (CSI). CSI causes inhibition of channel activity without the prerequisite of channel opening, thus providing a dynamic regulation of neuronal excitability, dendritic signal integration, and synaptic plasticity at resting. However, the structural determinants underlying Kv4 CSI remain largely unknown. We recently showed that the auxiliary KChIP4a subunit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts with Kv4.3 channels to enhance CSI. In this study, we utilized the KChIP4a KID to probe key structural elements underlying Kv4 CSI. Using fluorescence resonance energy transfer two-hybrid mapping and bimolecular fluorescence complementation-based screening combined with electrophysiology, we identified the intracellular tetramerization (T1) domain that functions to suppress CSI and serves as a receptor for the binding of KID. Disrupting the Kv4.3 T1-T1 interaction interface by mutating C110A within the C3H1 motif of T1 domain facilitated CSI and ablated the KID-mediated enhancement of CSI. Furthermore, replacing the Kv4.3 T1 domain with the T1 domain from Kv1.4 (without the C3H1 motif) or Kv2.1 (with the C3H1 motif) resulted in channels functioning with enhanced or suppressed CSI, respectively. Taken together, our findings reveal a novel (to our knowledge) role of the T1 domain in suppressing Kv4 CSI, and that KChIP4a KID directly interacts with the T1 domain to facilitate Kv4.3 CSI, thus leading to inhibition of channel function.

Auxiliary KChIP4a suppresses A-type K+ current through endoplasmic reticulum (ER) retention and promoting closed-state inactivation of Kv4 channels.

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K(+) channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12-17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.

A TRPV4 channel C-terminal folding recognition domain critical for trafficking and function.

The Ca(2+)-permeable transient receptor potential vanilloid subtype 4 (TRPV4) channel mediates crucial physiological functions, such as calcium signaling, temperature sensing, and maintaining cell volume and energy homeostasis. Noticeably, most disease-causing genetic mutations are concentrated in the cytoplasmic domains. In the present study, we focused on the role of the TRPV4 C terminus in modulating protein folding, trafficking, and activity. By examining a series of C-terminal deletions, we identified a 20-amino acid distal region covering residues 838-857 that is critical for channel folding, maturation, and trafficking. Surface biotinylation, confocal imaging, and fluorescence-based calcium influx assay demonstrated that mutant proteins missing this region were trapped in the endoplasmic reticulum and unglycosylated, leading to accelerated degradation and loss of channel activity. Rosetta de novo structural modeling indicated that residues 838-857 assume a defined conformation, with Gly(849) and Pro(851) located at critical positions. Patch clamp recordings confirmed that lowering the temperature from 37 to 30 °C rescued channel activity of folding-defective mutants. Moreover, biochemical tests demonstrated that, in addition to participating in C-C interaction, the C terminus also interacts with the N terminus. Taken together, our findings indicate that the C-terminal region of TRPV4 is critical for channel protein folding and maturation, and the short distal segment plays an essential role in this process. Therefore, selectively disrupting the folding-sensitive region may present therapeutic potential for treating overactive TRPV4-mediated diseases, such as pain and skeletal dysplasias.

Baseline Severity and Disease Duration Can Predict the Response to Allergen-specific Immunotherapy in Allergic Rhinitis.

Allergen-specific immunotherapy (AIT) has confirmed its efficacy in improving the symptoms of allergic rhinitis. However, no reliable biomarkers have been identified to predict the efficacy of AIT were found. We aimed to find clinical and immunological markers to predict efficacy in children after 2 years of sublingual immunotherapy (SLIT). A total of 285 children diagnosed with allergic rhinitis were recruited. The clinical efficacy was evaluated by comparing endpoint and baseline symptom and medication scores (SMS). Baseline clinical and immunological markers (serum total and specific immunoglobulin [Ig]E) and their correlation with clinical efficacy were analyzed. Of the 285 children recruited, 249 completed the 2-year SLIT program. After 2 years of SLIT, 68.3% of the children showed a significant response. Children in the Remarkable Response Group had the highest baseline SMS and most extended disease duration, followed by the Effective Relief and Unresponsive Group. Correlation analysis demonstrated that SMS improvement was positively correlated with baseline SMS (r=0.67) and disease duration (r=0.35). SMS improvement was not correlated with age, body mass index, total or specific IgE levels, or their ratios. Our results show that baseline SMS and disease duration can predict the efficacy of SLIT. Our study can guide the selection of suitable candidates for SLIT.

Mild pretreatment with Brønsted acidic deep eutectic solvents for fractionating ß-O-4 linkage-rich lignin with high sunscreen performance and evaluation of enzymatic saccharification synergism.

Herein, a mild fractionation method by employing polyol-based Brønsted acidic DESs (BDESs) was proposed to extract lignin with well-preserved ß-O-4 substructures and to enhance fermentable sugar yields simultaneously. For ethylene glycol-oxalic acid (EG-OA), more than 53 % of lignin was obtained and superb carbohydrate digestibility (i.e., glucose and xylose yields were reached to 94.6 % and 87.7 %, respectively) was achieved after pretreatment. Remarkably, detailed structural studies revealed that the polyol was incorporated into lignin, which stabilized reactive carbocation intermediates formed during BDESs treatment and prevented undesired recondensation reactions. This lignin protection mechanism was shown to play a key role in enzymatic hydrolysis enhancement and lignin valorization. The resultant ß-O-4 linkage-rich lignin fractions were attractive for natural sunscreen applications due to their lighter color and excellent UV-blocking performance. Overall, this work proposed a sustainable and economically practical lignin-first biorefinery approach that is beneficial for achieving comprehensive valorization of lignocellulose.

Overexpression of KCNJ2 enhances maturation of human-induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Although human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel Kir2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking. METHODS: iPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs). RESULTS: We showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of Kir2.1 and current density of Kir2.1-encoded IK1. The larger IK1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by IK1 blocker BaCl2. KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca2+ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction. CONCLUSIONS: Overexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca2+ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine.

Association between the genetic variants of base excision repair pathway genes and allergic rhinitis susceptibility in Chinese children.

Background: Allergic rhinitis (AR) is a frequent inflammatory disorder of the upper respiratory tract, which has complex patterns of inheritance. Accumulating evidence has shown the key roles of DNA damage in inflammatory diseases, and the base excision repair (BER) is the primary pathway responsible for DNA repair during inflammation. Methods: Here, we performed a case-control study to investigate the associations between 20 potentially functional single nucleotide polymorphisms (SNPs) in 6 BER pathway genes (PARP1, hOGG1, FEN1, APEX1, LIG3, and XRCC1) and AR susceptibility in 508 AR cases and 526 controls which originated in China. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for evaluating the association strength. Results: We found that hOGG1 rs1052133 G > C and XRCC1 rs2682585 G > A polymorphisms were associated with decreased AR risk (adjusted OR = 0.67, 95% CI = 0.47-0.94, P = 0.022; and adjusted OR = 0.21, 95% CI = 0.06-0.79, P = 0.022, respectively). Stratification analysis suggested that: hOGG1 rs1052133 GC/CC genotype reduced AR risk in subjects among following subgroups: age ≤60 months, females, and moderate AR; XRCC1 rs2682585 GG genotype decreased AR risk in subjects age >60 months, and LIG3 rs1052536 TT genotype increased AR risk in subjects of severe AR. Conclusion: Our findings indicated that the genetic variants of hOGG1, XRCC1, and LIG3 genes might affect AR susceptibility in the Chinese population, which will provide novel insight into the genetic underpinnings of AR from the DNA damage level.

High density lipoprotein inhibited group II innate lymphoid cells proliferation and function in allergic rhinitis.

BACKGROUND: More and more studies had suggested that dyslipidemia was closely related to allergic diseases. High density lipoprotein (HDL) often plays anti-inflammatory and anti-oxidative roles by suppressing immune cell chemotaxis and activation. We aimed to explore the role of HDL in the regulation of group II innate lymphoid cells (ILC2) in allergic rhinitis (AR). METHODS: The blood lipid levels and their correlation with symptom scores of 20 AR subjects and 20 controls were analyzed. Purified ILC2 were stimulated by HDL and cytokines production were examined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The mRNA levels of GATA binding protein 3(GATA3) and retinoid-related orphan receptor α (RORα) expressed by ILC2 were detected using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: HDL level was significantly lower in AR than controls and correlated with the symptom scores. The serum HDL levels were negatively related to the increased number of ILC2, IL-5+ ILC2, and IL-13+ ILC2 in AR patients. HDL decreased the number of ILC2 and type II cytokines levels significantly by inhibiting expression of GATA3 and RORα. CONCLUSIONS: Our data provide preliminary evidence that HDL may play a negative role in ILC2 inflammation in AR, suggesting that HDL may serve as promising treatment target in AR.