IL-31-dependent neurogenic inflammation restrains cutaneous type 2 immune response in allergic dermatitis.

Despite robust literature associating IL-31 with pruritic inflammatory skin diseases, its influence on cutaneous inflammation and the interplay between inflammatory and neurosensory pathways remain unmapped. Here, we examined the consequences of disrupting Il31 and its receptor Il31ra in a mouse model of house dust mite (HDM)-induced allergic dermatitis. Il31-deficient mice displayed a deficit in HDM dermatitis-associated scratching, consistent with its well-established role as a pruritogen. In contrast, Il31 deficiency increased the number and proportion of cutaneous type 2 cytokine-producing CD4+ T cells and serum IgE in response to HDM. Furthermore, Il4ra+ monocytes and macrophages capable of fueling a feedforward type 2 inflammatory loop were selectively enriched in Il31ra-deficient HDM dermatitis skin. Thus, IL-31 is not strictly a proinflammatory cytokine but rather an immunoregulatory factor that limits the magnitude of type 2 inflammatory responses in skin. Our data support a model wherein IL-31 activation of IL31RA+ pruritoceptors triggers release of calcitonin gene-related protein (CGRP), which can mediate neurogenic inflammation, inhibit CD4+ T cell proliferation, and reduce T cell production of the type 2 cytokine IL-13. Together, these results illustrate a previously unrecognized neuroimmune pathway that constrains type 2 tissue inflammation in the setting of chronic cutaneous allergen exposure and may explain paradoxical dermatitis flares in atopic patients treated with anti-IL31RA therapy.

MARCH1 Controls an Exhaustion-like Program of Effector CD4+ T Cells Promoting Allergic Airway Inflammation.

Persistent antigenic signaling leads to T cell exhaustion, a dysfunctional state arising in many chronic infections and cancers. Little is known concerning mechanisms limiting exhaustion in immune-stimulatory diseases such as asthma. We report that membrane-associated RING-CH1 (MARCH1), the ubiquitin ligase that mediates surface turnover of MHC class II (MHCII) and CD86 in professional APCs, plays an essential role in restraining an exhaustion-like program of effector CD4+ T cells in a mouse model of asthma. Mice lacking MARCH1 or the ubiquitin acceptor sites of MHCII and CD86 exhibited increased MHCII and CD86 surface expression on lung APCs, and this increase promoted enhanced expression of immune-inhibitory receptors by effector CD4+ T cells and inhibited their proliferation. Remarkably, ablation of MARCH1 in mice with established asthma reduced airway infiltration of eosinophils and Th2 cells. Thus, MARCH1 controls an exhaustion-like program of effector CD4+ T cells during allergic airway inflammation and may serve as a therapeutic target for asthma.

Lymph node-resident dendritic cells drive TH2 cell development involving MARCH1.

Type 2 T helper (TH2) cells are protective against parasitic worm infections but also aggravate allergic inflammation. Although the role of dendritic cells (DCs) in TH2 cell differentiation is well established, the underlying mechanisms are largely unknown. Here, we show that DC induction of TH2 cells depends on membrane-associated RING-CH-1 (MARCH1) ubiquitin ligase. The pro-TH2 effect of MARCH1 relied on lymph node (LN)­resident DCs, which triggered T cell receptor (TCR) signaling and induced GATA-3 expression from naïve CD4+ T cells independent of tissue-driven migratory DCs. Mice with mutations in the ubiquitin acceptor sites of MHCII and CD86, the two substrates of MARCH1, failed to develop TH2 cells. These findings suggest that TH2 cell development depends on ubiquitin-mediated clearance of antigen-presenting and costimulatory molecules by LN-resident DCs and consequent control of TCR signaling.

A Defect in Thymic Tolerance Causes T Cell-Mediated Autoimmunity in a Murine Model of COPA Syndrome.

COPA syndrome is a recently described Mendelian autoimmune disorder caused by missense mutations in the coatomer protein complex subunit α (COPA) gene. Patients with COPA syndrome develop arthritis and lung disease that presents as pulmonary hemorrhage or interstitial lung disease (ILD). Immunosuppressive medications can stabilize the disease, but many patients develop progressive pulmonary fibrosis, which requires life-saving measures, such as lung transplantation. Because very little is understood about the pathogenesis of COPA syndrome, it has been difficult to devise effective treatments for patients. To date, it remains unknown which cell types are critical for mediating the disease as well as the mechanisms that lead to autoimmunity. To explore these issues, we generated a CopaE241K/+ germline knock-in mouse bearing one of the same Copa missense mutations in patients. Mutant mice spontaneously developed ILD that mirrors lung pathology in patients, as well as elevations of activated cytokine-secreting T cells. In this study, we show that mutant Copa in epithelial cells of the thymus impairs the thymic selection of T cells and results in both an increase in autoreactive T cells and decrease in regulatory T cells in peripheral tissues. We demonstrate that T cells from CopaE241K/+ mice are pathogenic and cause ILD through adoptive transfer experiments. In conclusion, to our knowledge, we establish a new mouse model of COPA syndrome to identify a previously unknown function for Copa in thymocyte selection and demonstrate that a defect in central tolerance is a putative mechanism by which COPA mutations lead to autoimmunity in patients.

March1-dependent modulation of donor MHC II on CD103+ dendritic cells mitigates alloimmunity.

In transplantation, donor dendritic cells (do-DCs) initiate the alloimmune response either by direct interaction with host T cells or by transferring intact donor MHC to host DCs. However, how do-DCs can be targeted for improving allograft survival is still unclear. Here we show CD103+ DCs are the major do-DC subset involved in the acute rejection of murine skin transplants. In the absence of CD103+ do-DCs, less donor MHC-II is carried to host lymph nodes, fewer allogenic T cells are primed and allograft survival is prolonged. Incubation of skin grafts with the anti-inflammatory mycobacterial protein DnaK reduces donor MHC-II on CD103+DCs and prolongs graft survival. This effect is mediated through IL-10-induced March1, which ubiquitinates and decreases MHC-II levels. Importantly, in vitro pre-treatment of human DCs with DnaK reduces their ability to prime alloreactive T cells. Our findings demonstrate a novel therapeutic approach to dampen alloimmunity by targeting donor MHC-II on CD103+DCs.

MARCH1 protects the lipid raft and tetraspanin web from MHCII proteotoxicity in dendritic cells.

Dendritic cells (DCs) produce major histocompatibility complex II (MHCII) in large amounts to function as professional antigen presenting cells. Paradoxically, DCs also ubiquitinate and degrade MHCII in a constitutive manner. Mice deficient in the MHCII-ubiquitinating enzyme membrane-anchored RING-CH1, or the ubiquitin-acceptor lysine of MHCII, exhibit a substantial reduction in the number of regulatory T (Treg) cells, but the underlying mechanism was unclear. Here we report that ubiquitin-dependent MHCII turnover is critical to maintain homeostasis of lipid rafts and the tetraspanin web in DCs. Lack of MHCII ubiquitination results in the accumulation of excessive quantities of MHCII in the plasma membrane, and the resulting disruption to lipid rafts and the tetraspanin web leads to significant impairment in the ability of DCs to engage and activate thymocytes for Treg cell differentiation. Thus, ubiquitin-dependent MHCII turnover represents a novel quality-control mechanism by which DCs maintain homeostasis of membrane domains that support DC's Treg cell-selecting function.

Prognostic impact of left ventricular mass change in patients with ST-elevation myocardial infarction.

Prognostic significance between progression of left ventricular hypertrophy (LVH) and clinical outcomes in patients with ST-elevation myocardial infarction (STEMI) is uncertain. The objective of this study was to investigate prognostic impact of progression of LV mass index (LVMI) in patients with STEMI.We analyzed the data and clinical outcomes of patients with STEMI who received successful coronary intervention. A total of 200 patients who had echocardiographic follow-up between 12 and 36 months were finally enrolled. According to change in LVMI compared to baseline LVMI, patients were classified into progression group and nonprogression group. Progression of LVMI was defined when increment of LMVI was greater than 10% compared to baseline LVMI. End points were major adverse cardiac events within 5 years, including death, recurrent MI, target vessel revascularization, and hospitalization due to heart failure.Progression of LVMI occurred in 55 patients. In the progression group, rate of recurrent MI was higher (13 vs 2%, P = .026) and the event-free survival of recurrent MI was significantly worse (log-rank P < .001) than that in the nonprogression group. Adjusted hazard ratio of progression of LVMI for recurrent MI was 10.253 (95% confidence intervals 2.019-52.061, P = .005).Increased LVMI was an independent predictor for adverse events, especially for recurrent MI, in patients with STEMI.

CD40 Mediates Maturation of Thymic Dendritic Cells Driven by Self-Reactive CD4+ Thymocytes and Supports Development of Natural Regulatory T Cells.

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.

Ubiquitin-mediated fluctuations in MHC class II facilitate efficient germinal center B cell responses.

Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and selection. Selection involves B cells competing for T cell help based on the amount of antigen they capture and present on their MHC class II (MHCII) proteins. How GC B cells are able to rapidly and repeatedly transition between mutating their B cell receptor genes and then being selected shortly after is not known. We report that MHCII surface levels and degradation are dynamically regulated in GC B cells. Through ectopic expression of a photoconvertible MHCII-mKikGR chimeric gene, we found that individual GC B cells differed in the rates of MHCII protein turnover. Fluctuations in surface MHCII levels were dependent on ubiquitination and the E3 ligase March1. Increases in March1 expression in centroblasts correlated with decreases in surface MHCII levels, whereas CD83 expression in centrocytes helped to stabilize MHCII at that stage. Defects in MHCII ubiquitination caused GC B cells to accumulate greater amounts of a specific peptide-MHCII (pMHCII), suggesting that MHCII turnover facilitates the replacement of old complexes. We propose that pMHCII complexes are periodically targeted for degradation in centroblasts to favor the presentation of recently acquired antigens, thereby promoting the fidelity and efficiency of selection.

The Role of Dendritic Cells in Central Tolerance.

Dendritic cells (DCs) play a significant role in establishing self-tolerance through their ability to present self-antigens to developing T cells in the thymus. DCs are predominantly localized in the medullary region of thymus and present a broad range of self-antigens, which include tissue-restricted antigens expressed and transferred from medullary thymic epithelial cells, circulating antigens directly captured by thymic DCs through coticomedullary junction blood vessels, and peripheral tissue antigens captured and transported by peripheral tissue DCs homing to the thymus. When antigen-presenting DCs make a high affinity interaction with antigen-specific thymocytes, this interaction drives the interacting thymocytes to death, a process often referred to as negative selection, which fundamentally blocks the self-reactive thymocytes from differentiating into mature T cells. Alternatively, the interacting thymocytes differentiate into the regulatory T (Treg) cells, a distinct T cell subset with potent immune suppressive activities. The specific mechanisms by which thymic DCs differentiate Treg cells have been proposed by several laboratories. Here, we review the literatures that elucidate the contribution of thymic DCs to negative selection and Treg cell differentiation, and discusses its potential mechanisms and future directions.

Molecular mechanism and cellular function of MHCII ubiquitination.

The major histocompatibility complex class II (MHCII) is ubiquitinated via the evolutionarily conserved lysine in the cytoplasmic tail of the ß chain in dendritic cells (DCs) and B cells. The ubiquitination is mediated by the membrane-associated RING-CH1 (MARCH1) ubiquitin ligase although it can be also mediated by the homologous ligase MARCH8 in model cell lines. The ubiquitination promotes MHCII endocytosis and lysosomal sorting that results in a reduction in the level of MHCII at cell surface. Functionally, MHCII ubiquitination serves as a means by which DCs suppress MHCII expression and reduce antigen presentation in response to the immune regulatory cytokine interleukin-10 (IL-10) and regulatory T cells. Recently, additional roles of MHCII ubiquitination have emerged. MHCII ubiquitination promoted DC production of inflammatory cytokines in response to the Toll-like receptor ligands. It also potentiated DC ability to activate antigen-specific naive CD4(+) T cells while limiting the amount of antigens presented at cell surface. Similarly, MHCII ubiquitination promoted DC activation of CD4(+) thymocytes supporting regulatory T-cell development independent of its effect of limiting antigen presentation. Thus, ubiquitination appears to confer MHCII a function independent of presenting antigens by a mechanism yet to be identified.

The relationship between intravascular ultrasound-derived percent total atheroma volume and fractional flow reserve in the intermediate stenosis of proximal or middle left anterior descending coronary artery.

BACKGROUND: It remains undefined whether the atherosclerotic disease extent of the conductive vessel (expressed as intravascular ultrasound [IVUS]-derived percent total atheroma volume [%TAV]), correlates with functional severity of intermediate stenosis of left anterior descending artery (LAD). METHODS: An IVUS study and fractional flow reserve (FFR) measurements performed in 130 patients with coronary angiographic intermediate stenosis of proximal or middle LAD. %TAV was calculated as the percentage of total vessel volume occupied by total atheroma volume on IVUS. RESULTS: A significant correlation was observed between %TAV and FFR (r=-0.71, p<0.001). Minimal lumen area (MLA) correlated moderately with FFR (r=0.54, p<0.001). The independent predictors of FFR<0.8 were %TAV (odds ratio [OR]: 1.29, 95% confidence interval [CI]=1.18-1.40, p<0.001) and MLA (OR: 0.37, 95% CI=0.16-0.85, p=0.019). A receiver-operating characteristic curve suggested %TAV ≥ 39.0% (sensitivity 85%, specificity 83% and area under curve [AUC]=0.90) and MLA ≤ 2.6mm(2) (sensitivity 72%, specificity 70% and AUC=0.75) as the best cut-off values for FFR<0.8. Forty-eight point five (48.5%) of total studied lesions (63/130) showed %TAV ≥ 39.0%. Eighty-four point four (84.4%) of lesions (38/45) with %TAV ≥ 39.0% and MLA ≤ 2.6mm(2), and 72.2% of lesions (13/18) with %TAV ≥ 39.0% and MLA>2.6mm(2), FFR was less than 0.8. CONCLUSIONS: Volumetric quantification of the atherosclerotic disease extent of the coronary artery, expressed as IVUS-derived %TAV, showed a strong correlation with FFR. Not only the segmental luminal narrowing but also the total plaque burden of conductive artery are major determinants for the presence of myocardial ischemia in intermediate stenosis of LAD.

The role of FcεRI expressed in dendritic cells and monocytes.

Early studies regarding the function of FcεRI in dendritic cells (DCs) and monocytes have focused on its role in mediating inflammatory signaling and enhancing T cell immunity. It has been the case in part because FcεRI is the major receptor that mediates allergic inflammatory signaling in mast cells and basophils and because DCs and monocytes are antigen presenting cells capable of activating naïve and/or effector T cells. These studies have led to the general belief that FcεRI-mediated DC signaling and antigen presentation promote development and activation of Th2 cells and contribute to allergic inflammatory diseases. However, this belief has long suffered from a lack of evidence. Recently, studies have emerged that provide evidence supporting an opposing role: that FcεRI on DCs instead promotes immune homeostasis and regulation. In this review, we will update the current status of our understanding of FcεRI biology and function, with a specific focus on DCs and monocytes.

Point-of-care measurements of platelet inhibition after clopidogrel loading in patients with acute coronary syndrome: comparison of generic and branded clopidogrel bisulfate.

PURPOSE: Platelet-function suppression with antiplatelet therapy is effective in preventing and treating cardiovascular disease. Clopidogrel is a thienopyridine derivative that blocks platelet activation by adenosine diphosphate receptor binding. This study demonstrates the effects of generic clopidogrel bisulfate in comparison to branded clopidogrel bisulfate in patients with acute coronary syndromes. METHODS: This prospective, 2-arm, single-center, open-label trial used 1:1 randomization to assign patients to receive generic or branded clopidogrel bisulfate. Patients with unstable angina or non-ST-segment elevation myocardial infarction and scheduled to undergo coronary angiography were enrolled. Platelet function was measured with a P2Y12 assay and reported in P2Y12 reaction units (PRU) and aspirin reaction units (ARU) after randomization. Platelet function was measured at 2, 4, 8, and 24 hours after 600-mg clopidogrel loading. The clinical outcome was checked at 1 month after coronary angiography. FINDINGS: Ninety-five patients were enrolled and randomized to the generic or branded group. Ninety patients (62 men [69%], 28 women [31%]; mean age, 58 years) completed the study protocol. The clinical characteristics were similar between the 2 groups. The difference in the baseline PRU measurements between the generic and branded groups was not significant (274.8 [59.7] vs 285.4 [62.4], respectively; P = 0.414). There were significant differences in 2-hour PRU (231.1 [71.3] vs 266.9 [67.4]; P = 0.017) and 4-hour PRU (227.3 [80.4] vs 265.7 [71.0]; P = 0.020); however, 24-hour PRU (200.5 [82.1] vs 220.6 [75.8]; P = 0.253) was similar. No death, myocardial infarction, target lesion revascularization, stent thrombosis, or Thrombolysis in Myocardial Infarction-defined major bleeding complications were reported during in-hospital stay or 1-month follow-up. IMPLICATION: In patients with ACS, loading of generic clopidogrel bisulfate was associated with an antiplatelet effect comparable to that of branded clopidogrel bisulfate. ClinicalTrials.gov identifier: NCT02060786.

Accumulation of BDCA1⁺ dendritic cells in interstitial fibrotic lung diseases and Th2-high asthma.

Dendritic cells (DCs) significantly contribute to the pathology of several mouse lung disease models. However, little is known of the contribution of DCs to human lung diseases. In this study, we examined infiltration with BDCA1⁺ DCs of human lungs in patients with interstitial lung diseases or asthma. Using flow cytometry, we found that these DCs increased by 5∼6 fold in the lungs of patients with idiopathic pulmonary fibrosis or hypersensitivity pneumonitis, which are both characterized by extensive fibrosis in parenchyma. The same DC subset also significantly increased in the lung parenchyma of patients with chronic obstructive pulmonary disease, although the degree of increase was relatively modest. By employing immunofluorescence microscopy using FcεRI and MHCII as the specific markers for BDCA1⁺ DCs, we found that the numbers of BDCA1⁺ DCs also significantly increased in the airway epithelium of Th2 inflammation-associated asthma. These findings suggest a potential contribution of BDCA1⁺ DCs in human lung diseases associated with interstitial fibrosis or Th2 airway inflammation.

Impact of female gender on bleeding complications after transradial coronary intervention (from the Korean Transradial Coronary Intervention registry).

Besides poor clinical outcomes, female gender has been known as a high-risk factor for bleeding complications. This study aimed to investigate the impact of gender on clinical outcomes and bleeding complications after transradial coronary intervention (TRI). The Korean TRI registry is a retrospective multicenter registry with 4,890 patients who underwent percutaneous coronary intervention in 2009 at 12 centers. To compare clinical outcomes and bleeding complications between the male and female groups, we performed a propensity score matching in patients who received TRI. A total of 1,194 patients (597 in each group) were studied. The primary outcome was 1-year major adverse cardiac events, including all-cause mortality, myocardial infarction, target vessel revascularization, and stroke. The secondary outcome was major bleeding (composite of bleeding requiring transfusion of ≥2 units of packed cells or bleeding that was fatal). The proportion of major adverse cardiac events was similar between the 2 groups (6.2% vs 4.7%, p = 0.308). The female group had a greater incidence of major bleeding (0.3% vs 3.2%, p <0.001). On multivariate analysis, female gender (odds ratio [OR] 7.748, 95% confidence interval [CI] 1.767 to 13.399), age ≥75 years (OR 5.824, 95% CI 2.085 to 16.274), and chronic kidney disease (OR 7.264, 95% CI 2.369 to 12.276) were independent predictors of major bleeding. In conclusion, the female gender had a tendency for more bleeding complications than male gender after TRI without difference in the clinical outcome.

Antigen-conjugated human IgE induces antigen-specific T cell tolerance in a humanized mouse model.

Dendritic cells (DCs) play an important role in immune homeostasis through their ability to present Ags at steady state and mediate T cell tolerance. This characteristic renders DCs an attractive therapeutic target for the induction of tolerance against auto-antigens or allergens. Accordingly, Ag-conjugated DC-specific Abs have been proposed to be an excellent vehicle to deliver Ags to DCs for presentation and tolerance induction. However, this approach requires laborious reagent generation procedures and entails unpredictable side effects resulting from Ab-induced crosslinking of DC surface molecules. In this study, we examined whether IgE, a high-affinity, non-cross-linking natural ligand of FcεRI, could be used to target Ags to DCs and to induce Ag-specific T cell tolerance. We found that Ag-conjugated human IgE Fc domain (Fcε) effectively delivered Ags to DCs and enhanced Ag presentation by 1000- to 2500-fold in human FcεRIα-transgenic mice. Importantly, this presentation resulted in a systemic deletion of Ag-specific T cells and prevented these mice from developing delayed-type hypersensitivity, which is critically dependent on Ag-specific T cell immunity. Thus, targeting FcεRI on DCs via Ag-Fcε fusion protein may serve an alternative method to induce Ag-specific T cell tolerance in humans.

Accelerated dissociation of IgE-FcεRI complexes by disruptive inhibitors actively desensitizes allergic effector cells.

BACKGROUND: The remarkably stable interaction of IgE with its high-affinity receptor FcεRI on basophils and mast cells is critical for the induction of allergic hypersensitivity reactions. Because of the exceptionally slow dissociation rate of IgE-FcεRI complexes, such allergic effector cells permanently display allergen-specific IgE on their surface and immediately respond to allergen challenge by releasing inflammatory mediators. We have recently described a novel macromolecular inhibitor that actively promotes the dissociation of IgE from FcεRI through a molecular mechanism termed facilitated dissociation. OBJECTIVE: Here we assessed the therapeutic potential of this non-immunoglobulin-based IgE inhibitor E2_79, a designed ankyrin repeat protein (DARPin), as well as a novel engineered biparatopic DARPin bi53_79, and directly compared them with the established anti-IgE antibody omalizumab. METHODS: IgE-FcεRI complex dissociation was analyzed in vitro by using recombinant proteins in ELISA and surface plasmon resonance, ex vivo by using human primary basophils with flow cytometry, and in vivo by using human FcεRI α-chain transgenic mice in a functional passive cutaneous anaphylaxis test. RESULTS: We show that E2_79-mediated removal of IgE from primary human basophils fully abrogates IgE-dependent cell activation and release of proinflammatory mediators ex vivo. Furthermore, we report that omalizumab also accelerates the dissociation of IgE from FcεRI, although much less efficiently than E2_79. Using the biparatopic IgE targeting approach, we further improved the disruptive potency of E2_79 by approximately 100-fold and show that disruptive IgE inhibitors efficiently prevent passive cutaneous anaphylaxis in mice expressing the human FcεRI α-chain. CONCLUSION: Our findings highlight the potential of such novel IgE inhibitors as important diagnostic and therapeutic tools for management of allergic diseases.