High IFN-γ and low SLPI mark severe asthma in mice and humans

Severe asthma (SA) is a challenge to control, as patients are not responsive to high doses of systemic corticosteroids (CS). In contrast, mild-moderate asthma (MMA) is responsive to low doses of inhaled CS, indicating that Th2 cells, which are dominant in MMA, do not solely orchestrate SA development. Here, we analyzed broncholalveolar lavage cells isolated from MMA and SA patients and determined that IFN-γ (Th1) immune responses are exacerbated in the airways of individuals with SA, with reduced Th2 and IL-17 responses. We developed a protocol that recapitulates the complex immune response of human SA, including the poor response to CS, in a murine model. Compared with WT animals, Ifng^–/– mice subjected to this SA model failed to mount airway hyperresponsiveness (AHR) without appreciable effect on airway inflammation. Conversely, AHR was not reduced in Il17ra^–/– mice, although airway inflammation was lower. Computer-assisted pathway analysis tools linked IFN-γ to secretory leukocyte protease inhibitor (SLPI), which is expressed by airway epithelial cells, and IFN-γ inversely correlated with SLPI expression in SA patients and the mouse model. In mice subjected to our SA model, forced SLPI expression decreased AHR in the absence of CS, and it was further reduced when SLPI was combined with CS. Our study identifies a distinct immune response in SA characterized by a dysregulated IFN-γ/SLPI axis that affects lung function.     

Elevated levels of placental growth factor represent an adaptive host response in sepsis

Recently, we demonstrated that circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are increased in sepsis (Yano, K., P.C. Liaw, J.M. Mullington, S.C. Shih, H. Okada, N. Bodyak, P.M. Kang, L. Toltl, B. Belikoff, J. Buras, et al. 2006. J. Exp. Med. 203:1447–1458). Moreover, enhanced VEGF/Flk-1 signaling was shown to contribute to sepsis morbidity and mortality. We tested the hypothesis that PlGF also contributes to sepsis outcome. In mouse models of endotoxemia and cecal ligation puncture, the genetic absence of PlGF or the systemic administration of neutralizing anti-PlGF antibodies resulted in higher mortality compared with wild-type or immunoglobulin G–injected controls, respectively. The increased mortality associated with genetic deficiency of PlGF was reversed by adenovirus (Ad)-mediated overexpression of PlGF. In the endotoxemia model, PlGF deficiency was associated with elevated circulating levels of VEGF, induction of VEGF expression in the liver, impaired cardiac function, and organ-specific accentuation of barrier dysfunction and inflammation. Mortality of endotoxemic PlGF-deficient mice was increased by Ad-mediated overexpression of VEGF and was blocked by expression of soluble Flt-1. Collectively, these data suggest that up-regulation of PlGF in sepsis is an adaptive host response that exerts its benefit, at least in part, by attenuating VEGF signaling.     

Recurrent panniculitis in a man with asthma receiving treatment with leukotriene-modifying agents

Leukotriene-modifying drugs are novel agents introduced recently to treat asthma. Both 5-lipoxygenase inhibitors, such as zileuton, and leukotriene receptor antagonists, such as zafirlukast and montelukast, have proved effective in the treatment of asthma. To our knowledge, there have been no detailed reports regarding dermatologic manifestations of this class of drugs. This article describes an unusual case of erythema nodosum in a 46-year-old asthmatic man who received 2 different leukotriene modifiers.     

Helicobacter urease–induced activation of the TLR2/NLRP3/IL-18 axis protects against asthma

Inflammasome activation and caspase-1–dependent (CASP1-dependent) processing and secretion of IL-1β and IL-18 are critical events at the interface of the bacterial pathogen Helicobacter pylori with its host. Whereas IL-1β promotes Th1 and Th17 responses and gastric immunopathology, IL-18 is required for Treg differentiation, H. pylori persistence, and protection against allergic asthma, which is a hallmark of H. pylori–infected mice and humans. Here, we show that inflammasome activation in DCs requires the cytoplasmic sensor NLRP3 as well as induction of TLR2 signaling by H. pylori. Screening of an H. pylori transposon mutant library revealed that pro–IL-1β expression is induced by LPS from H. pylori, while the urease B subunit (UreB) is required for NLRP3 inflammasome licensing. UreB activates the TLR2-dependent expression of NLRP3, which represents a rate-limiting step in NLRP3 inflammasome assembly. ureB-deficient H. pylori mutants were defective for CASP1 activation in murine bone marrow–derived DCs, splenic DCs, and human blood-derived DCs. Despite colonizing the murine stomach, ureB mutants failed to induce IL-1β and IL-18 secretion and to promote Treg responses. Unlike WT H. pylori, ureB mutants were incapable of conferring protection against allergen-induced asthma in murine models. Together, these results indicate that the TLR2/NLRP3/CASP1/IL-18 axis is critical to H. pylori–specific immune regulation.     

CCR6 is required for IL-23–induced psoriasis-like inflammation in mice

Psoriasis is a common immune-mediated chronic inflammatory skin disorder, but the mechanisms of pathogenesis are still poorly understood. IL-23 is expressed in psoriatic skin, and IL-23 injection produces IL-22–dependent psoriasiform changes in mouse skin. Th17 cells produce IL-22 and display CCR6, the CCL20 receptor; CCR6⁺ T cells and CCL20 are abundant in psoriatic skin. We investigated a possible role for CCR6 in recruiting Th17 cells and producing psoriasiform pathology by injecting IL-23 into the skin of WT and Ccr6^–/– mice. Unlike for WT mice, IL-23–injected ears of Ccr6^–/– mice showed neither substantial epidermal/dermal changes nor increased Il22 mRNA expression. However, injection of IL-22 yielded equivalent psoriasiform changes in WT and Ccr6^–/– mice. Surprisingly, IL-23–injected ears of WT and Ccr6^–/– mice contained similar numbers of Th cells able to make IL-17A and/or IL-22. Furthermore, in ears of Rag1^–/– mice, IL-23 initially induced skin changes and levels of Il22 mRNA that were indistinguishable from WT mice, revealing at least one non–T cell source for IL-22. We conclude that CCR6 is essential in a model of IL-23–induced, IL-22–mediated dermatitis, which develops in sequential T cell–independent and T cell–dependent phases. These findings reveal an expanded role for CCR6 in IL-23–related responses and identify CCR6 as a potential therapeutic target in psoriasis.     

Inhibition of cyclooxygenase-2 in hematopoietic cells results in salt-sensitive hypertension

Inhibition of prostaglandin (PG) production with either nonselective or selective inhibitors of cyclooxygenase-2 (COX-2) activity can induce or exacerbate salt-sensitive hypertension. This effect has been previously attributed to inhibition of intrinsic renal COX-2 activity and subsequent increase in sodium retention by the kidney. Here, we found that macrophages isolated from kidneys of high-salt–treated WT mice have increased levels of COX-2 and microsomal PGE synthase–1 (mPGES-1). Furthermore, BM transplantation (BMT) from either COX-2–deficient or mPGES-1–deficient mice into WT mice or macrophage-specific deletion of the PGE₂ type 4 (EP₄) receptor induced salt-sensitive hypertension and increased phosphorylation of the renal sodium chloride cotransporter (NCC). Kidneys from high-salt–treated WT mice transplanted with Cox2^–/– BM had increased macrophage and T cell infiltration and increased M1- and Th1-associated markers and cytokines. Skin macrophages from high-salt–treated mice with either genetic or pharmacologic inhibition of the COX-2 pathway expressed decreased M2 markers and VEGF-C production and exhibited aberrant lymphangiogenesis. Together, these studies demonstrate that COX-2–derived PGE₂ in hematopoietic cells plays an important role in both kidney and skin in maintaining homeostasis in response to chronically increased dietary salt. Moreover, these results indicate that inhibiting COX-2 expression or activity in hematopoietic cells can result in a predisposition to salt-sensitive hypertension.     

TLR9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through DC-derived Notch ligand delta-like 4

TLR9 activation is important for the maintenance of mycobacteria-elicited pulmonary granulomatous responses, hallmarks of protective immune responses following mycobacterial infection. However, the mechanism or mechanisms underlying this effect of TLR9 are not clear. Here, we show that Tlr9-deficient mice challenged with a Mycobacterium antigen display an altered Th17 cytokine profile, decreased accumulation of granuloma-associated myeloid DCs, and profoundly impaired delta-like 4 (dll4) Notch ligand expression. Mechanistic analysis revealed that WT bone marrow–derived DCs but not macrophages promoted the differentiation of Th17 cells from bacillus Calmette-Guérin–challenged (BCG-challenged) lung CD4⁺ T cells. Both lung and bone marrow DCs isolated from Tlr9-deficient mice inoculated with Mycobacterium antigen expressed lower levels of dll4 Notch ligand than the same cells isolated from WT mice. Passively immunizing WT mice with neutralizing antibodies specific for dll4 during granuloma formation resulted in larger granulomas and lower levels of Th17-related cytokines. In addition, dll4 specifically regulated Th17 activation in vitro. Together, these results suggest dll4 plays an important role in promoting Th17 effector activity during a mycobacterial challenge. Furthermore, TLR9 seems to be required for optimal dll4 expression and the regulation of Mycobacterium antigen–elicited granuloma formation in mice.     

Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis

Topical application of coal tar is one of the oldest therapies for atopic dermatitis (AD), a T helper 2 (Th2) lymphocyte–mediated skin disease associated with loss-of-function mutations in the skin barrier gene, filaggrin (FLG). Despite its longstanding clinical use and efficacy, the molecular mechanism of coal tar therapy is unknown. Using organotypic skin models with primary keratinocytes from AD patients and controls, we found that coal tar activated the aryl hydrocarbon receptor (AHR), resulting in induction of epidermal differentiation. AHR knockdown by siRNA completely abrogated this effect. Coal tar restored filaggrin expression in FLG-haploinsufficient keratinocytes to wild-type levels, and counteracted Th2 cytokine–mediated downregulation of skin barrier proteins. In AD patients, coal tar completely restored expression of major skin barrier proteins, including filaggrin. Using organotypic skin models stimulated with Th2 cytokines IL-4 and IL-13, we found coal tar to diminish spongiosis, apoptosis, and CCL26 expression, all AD hallmarks. Coal tar interfered with Th2 cytokine signaling via dephosphorylation of STAT6, most likely due to AHR-regulated activation of the NRF2 antioxidative stress pathway. The therapeutic effect of AHR activation herein described opens a new avenue to reconsider AHR as a pharmacological target and could lead to the development of mechanism-based drugs for AD.     

A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to IL-25 and contributes to airway hyperreactivity

Airway hypersensitive reaction (AHR) is an animal model for asthma, which is caused or enhanced by environmental factors such as allergen exposure. However, the precise mechanisms that drive AHR remain unclear. We identified a novel subset of natural killer T (NKT) cells that expresses the interleukin 17 receptor B (IL-17RB) for IL-25 (also known as IL-17E) and is essential for the induction of AHR. IL-17RB is preferentially expressed on a fraction of CD4⁺ NKT cells but not on other splenic leukocyte populations tested. IL-17RB⁺ CD4⁺ NKT cells produce predominantly IL-13 and Th2 chemokines upon stimulation with IL-25 in vitro. IL-17RB⁺ NKT cells were detected in the lung, and depletion of IL-17RB⁺ NKT cells by IL-17RB–specific monoclonal antibodies or NKT cell–deficient Jα18^−/− mice failed to develop IL-25–dependent AHR. Cell transfer of IL-17RB⁺ but not IL-17RB⁻ NKT cells into Jα18^−/− mice also successfully reconstituted AHR induction. These results strongly suggest that IL-17RB⁺ CD4⁺ NKT cells play a crucial role in the pathogenesis of asthma.     

Increased mitochondrial arginine metabolism supports bioenergetics in asthma

High levels of arginine metabolizing enzymes, including inducible nitric oxide synthase (iNOS) and arginase (ARG), are typical in asthmatic airway epithelium; however, little is known about the metabolic effects of enhanced arginine flux in asthma. Here, we demonstrated that increased metabolism sustains arginine availability in asthmatic airway epithelium with consequences for bioenergetics and inflammation. Expression of iNOS, ARG2, arginine synthetic enzymes, and mitochondrial respiratory complexes III and IV was elevated in asthmatic lung samples compared with healthy controls. ARG2 overexpression in a human bronchial epithelial cell line accelerated oxidative bioenergetic pathways and suppressed hypoxia-inducible factors (HIFs) and phosphorylation of the signal transducer for atopic Th2 inflammation STAT6 (pSTAT6), both of which are implicated in asthma etiology. Arg2-deficient mice had lower mitochondrial membrane potential and greater HIF-2α than WT animals. In an allergen-induced asthma model, mice lacking Arg2 had greater Th2 inflammation than WT mice, as indicated by higher levels of pSTAT6, IL-13, IL-17, eotaxin, and eosinophils and more mucus metaplasia. Bone marrow transplants from Arg2-deficient mice did not affect airway inflammation in recipient mice, supporting resident lung cells as the drivers of elevated Th2 inflammation. These data demonstrate that arginine flux preserves cellular respiration and suppresses pathological signaling events that promote inflammation in asthma.     

Neutrophil AKT2 regulates heterotypic cell-cell interactions during vascular inflammation

Interactions between platelets, leukocytes, and activated endothelial cells are important during microvascular occlusion; however, the regulatory mechanisms of these heterotypic cell-cell interactions remain unclear. Here, using intravital microscopy to evaluate mice lacking specific isoforms of the serine/threonine kinase AKT and bone marrow chimeras, we found that hematopoietic cell–associated AKT2 is important for neutrophil adhesion and crawling and neutrophil-platelet interactions on activated endothelial cells during TNF-α–induced venular inflammation. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under shear conditions. In particular, neutrophil AKT2 was critical for membrane translocation of αMβ2 integrin, β2-talin1 interaction, and intracellular Ca²⁺ mobilization. We found that the basal phosphorylation levels of AKT isoforms were markedly increased in neutrophils and platelets isolated from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow rates. Our results provide evidence that neutrophil AKT2 regulates αMβ2 integrin function and suggest that AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions under thromboinflammatory conditions such as SCD.     

Treatment of experimental asthma by long-term gene therapy directed against IL-4 and IL-13

The clinical manifestations of allergic asthma are believed to result from a dysregulated, T helper 2 lymphocyte (Th2)-biased response to antigen. Although asthma symptoms can be controlled acutely, there is a need for a therapy that will address the underlying immune dysfunction and provide continuous control of chronic airway inflammation. The Th2-type cytokines, IL-13 and IL-4, have been demonstrated to play a crucial role in asthma pathogenesis and their selective neutralization results in the alleviation of asthmatic symptoms in mouse models. The activity of both of these cytokines can be inhibited by a mutant IL-4 protein, IL-4 receptor antagonist (IL-4RA), and thus, continual IL-4RA therapy might be beneficial in treatment of chronic asthma. To explore the potential utility of long-term gene therapy for the treatment of asthma we used a recombinant adeno-associated virus (AAV) vector to deliver and provide sustained expression of IL-4RA in vivo. We show that AAV-mediated delivery of IL-4RA to the airways of mice reduces airway hyperresponsiveness (AHR) and airway eosinophilia triggered by either IL-13 or IL-4. Furthermore, AAV-delivered IL-4RA, expressed either systemically or in the airways of mice following allergen sensitization, significantly inhibited development of airway eosinophilia and mucus production and reduced the levels of asthma-associated Th2 cytokines and AHR in the experimental mouse model of allergic asthma. Thus, gene therapy can be a potential therapeutic option to treat and control chronic airway inflammation and asthmatic symptoms.     

IL-21R is essential for epicutaneous sensitization and allergic skin inflammation in humans and mice

Atopic dermatitis (AD) is a common allergic inflammatory skin disease caused by a combination of intense pruritus, scratching, and epicutaneous (e.c.) sensitization with allergens. To explore the roles of IL-21 and IL-21 receptor (IL-21R) in AD, we examined skin lesions from patients with AD and used a mouse model of allergic skin inflammation. IL-21 and IL-21R expression was upregulated in acute skin lesions of AD patients and in mouse skin subjected to tape stripping, a surrogate for scratching. The importance of this finding was highlighted by the fact that both Il21r^–/– mice and WT mice treated with soluble IL-21R–IgG2aFc fusion protein failed to develop skin inflammation after e.c. sensitization of tape-stripped skin. Adoptively transferred OVA-specific WT CD4⁺ T cells accumulated poorly in draining LNs (DLNs) of e.c. sensitized Il21r^–/– mice. This was likely caused by both DC-intrinsic and nonintrinsic effects, because trafficking of skin DCs to DLNs was defective in Il21r^–/– mice and, to a lesser extent, in WT mice reconstituted with Il21r^–/– BM. More insight into this defect was provided by the observation that skin DCs from tape-stripped WT mice, but not Il21r^–/– mice, upregulated CCR7 and migrated toward CCR7 ligands. Treatment of epidermal and dermal cells with IL-21 activated MMP2, which has been implicated in trafficking of skin DCs. These results suggest an important role for IL-21R in the mobilization of skin DCs to DLNs and the subsequent allergic response to e.c. introduced antigen.     

Agonistic induction of PPARγ reverses cigarette smoke–induced emphysema

The development of emphysema in humans and mice exposed to cigarette smoke is promoted by activation of an adaptive immune response. Lung myeloid dendritic cells (mDCs) derived from cigarette smokers activate autoreactive Th1 and Th17 cells. mDC-dependent activation of T cell subsets requires expression of the SPP1 gene, which encodes osteopontin (OPN), a pleiotropic cytokine implicated in autoimmune responses. The upstream molecular events that promote SPP1 expression and activate mDCs in response to smoke remain unknown. Here, we show that peroxisome proliferator–activated receptor γ (PPARG/Pparg) expression was downregulated in mDCs of smokers with emphysema and mice exposed to chronic smoke. Conditional knockout of PPARγ in APCs using Cd11c-Cre Pparg^flox/flox mice led to spontaneous lung inflammation and emphysema that resembled the phenotype of smoke-exposed mice. The inflammatory phenotype of Cd11c-Cre Pparg^flox/flox mice required OPN, suggesting an antiinflammatory mechanism in which PPARγ negatively regulates Spp1 expression in the lung. A 2-month treatment with a PPARγ agonist reversed emphysema in WT mice despite continual smoke exposure. Furthermore, endogenous PPARγ agonists were reduced in the plasma of smokers with emphysema. These findings reveal a proinflammatory pathway, in which reduced PPARγ activity promotes emphysema, and suggest that targeting this pathway in smokers could prevent and reverse emphysema.     

MALT1 in asthma children: A potential biomarker for monitoring exacerbation risk and Th1/Th2 imbalance‐mediated inflammation

BackgroundMucosa‐associated lymphoid tissue lymphoma translocation protein 1 (MALT1) participates in the immune‐related allergic response and inflammation flare, while its clinical role in asthma children is still unknown. Herein, this study aimed to investigate MALT1 expression, and its correlation with exacerbation risk, T helper (Th)1, Th2 cells (and their secreted cytokines), as well as inflammatory cytokines in asthma children.MethodsSixty children with asthma exacerbation and 60 children with remission asthma were enrolled in this study; then their blood MALT1, Th1, Th2 cells, tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), interferon‐gamma (IFN‐γ), and interleukin‐4 (IL‐4) were detected. Besides, blood MALT1 in another 20 health controls was also determined.ResultsMucosa‐associated lymphoid tissue lymphoma translocation protein 1 was highest in children with asthma exacerbation, followed by children with remission asthma, and lowest in health controls (p < 0.001). MALT1 could distinguish children with asthma exacerbation from children with remission asthma (area under the curve (AUC): 0.757, 95% CI: 0.670–0.843). In children with asthma exacerbation, MALT1 was negatively linked with IFN‐γ (p = 0.002) and Th1 cells (p = 0.050), but positively related to Th2 cells (p = 0.027) and exhibited a positive correlation trend (without statistical significance) with IL‐4 (p = 0.066); meanwhile, MALT1 was positively correlated with exacerbation severity (p = 0.010) and TNF‐α (p = 0.003), but not linked with IL‐6 (p = 0.096). In children with remission asthma, MALT1 only was negatively associated with Th1 cells (p = 0.023), but positively linked with TNF‐α (p = 0.023).ConclusionMucosa‐associated lymphoid tissue lymphoma translocation protein 1 serves as a potential biomarker for monitoring exacerbation risk and Th1/Th2 imbalance‐mediated inflammation of asthma children.     

Med12 gain-of-function mutation causes leiomyomas and genomic instability

Uterine leiomyomas are benign tumors that can cause pain, bleeding, and infertility in some women. Mediator complex subunit 12 (MED12) exon 2 variants are associated with uterine leiomyomas; however, the causality of MED12 variants, their genetic mode of action, and their role in genomic instability have not been established. Here, we generated a mouse model that conditionally expresses a Med12 missense variant (c.131G>A) in the uterus and demonstrated that this alteration alone promotes uterine leiomyoma formation and hyperplasia in both WT mice and animals harboring a uterine mesenchymal cell–specific Med12 deletion. Compared with WT animals, expression of Med12 c.131G>A in conditional Med12–KO mice resulted in earlier onset of leiomyoma lesions that were also greater in size. Moreover, leiomyomatous, Med12 c.131G>A variant–expressing uteri developed chromosomal rearrangements. Together, our results show that the common human leiomyoma–associated MED12 variant can cause leiomyomas in mice via a gain of function that drives genomic instability, which is frequently observed in human leiomyomas.