Treatment of acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA) results in terminal differentiation of leukemic cells toward neutrophil granulocytes. Administration of ATRA leads to massive changes in gene expression, including down-regulation of cell proliferation-related genes and induction of genes involved in immune function. One of the most induced genes in APL NB4 cells is transglutaminase 2 (TG2). RNA interference-mediated stable silencing of TG2 in NB4 cells (TG2-KD NB4) coupled with whole genome microarray analysis revealed that TG2 is involved in the expression of a large number of ATRA-regulated genes. The affected genes participate in granulocyte functions, and their silencing lead to reduced adhesive, migratory, and phagocytic capacity of neutrophils and less superoxide production. The expression of genes related to cell-cycle control also changed, suggesting that TG2 regulates myeloid cell differentiation. CC chemokines CCL2, CCL3, CCL22, CCL24, and cytokines IL1B and IL8 involved in the development of differentiation syndrome are expressed at significantly lower level in TG2-KD NB4 than in wild-type NB4 cells upon ATRA treatment. Based on our results, we propose that reduced expression of TG2 in differentiating APL cells may suppress effector functions of neutrophil granulocytes and attenuate the ATRA-induced inflammatory phenotype of differentiation syndrome.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Although the pathogenesis of AD is not fully understood, we and others have shown that IL-4 plays a key role. In this study we aimed to identify keratinocyte genes regulated by IL-4 that may play important roles in the pathophysiology of AD. HaCat cells were treated with IL-4 at various concentrations for 24h, and PCR gene array on inflammation/autoimmunity was performed three times for analysis of differential gene expression. Of all the 370 genes examined, 32 and 53 genes are up- and down-regulated, respectively. Specifically related to AD, chemokines CCL3L1, CCL8, CCL24, CCL25, CCL26, CXCL6 and CXCL16 are up-regulated by IL-4. Pro-inflammatory factors, such as IL-19, IL-20, IL-1α, IL-12Rβ2, IL-25, IL-31RA, OSMR and nitric oxide synthase 2, are also up-regulated. In addition, IL-4 up-regulates VEGFA, a pro-angiogenic factor. In contrast, antimicrobial peptides (AMPs) or factors involved in APM production, such as IFN-κ, S100s, Toll-like receptors, and several chemokines are down-regulated. Similarly IL-4 also down-regulates TNF-α, lymphotoxin-β, an IgE suppressor, TNFSF18, a T-cells function regulator, and the glucocorticoid receptor. On the in vivo level, real-time RT-PCR on the selected genes confirmed that IL-4 up-regulates chemokines, proinflammatory cytokines while it suppresses AMP production related genes in the skin obtained from IL-4 Tg mice. Detailed examination of these genes will delineate their specific roles in chemotaxis, inflammation, angiogenesis and AMP production, all of which may contribute to the development and progression of AD.

In this study, we evaluated the anti-inflammatory response and the mechanism by which dehydroepiandrosterone modulates immunity in ovalbumin-sensitized asthmatic mice. Female BALB/c mice were sensitized and challenged with ovalbumin and then treated with oral administration of dehydroepiandrosterone on days 21 to 27. The results showed dehydroepiandrosterone could suppress airway hyperresponsiveness and decrease eosinophil infiltration of the lungs in ovalbumin-sensitized mice. Moreover, dehydroepiandrosterone inhibited chemokines, including CCL11/eotaxin-1 and CCL24/eotaxin-2, and Th2-associated cytokine levels in bronchoalveolar lavage fluid. After the inflammatory human bronchial epithelial cell line BEAS-2B was treated with dehydroepiandrosterone, levels of proinflammatory cytokines and chemokines were inhibited, including IL-6, IL-8, CCL11, and CCL24. We suggested that dehydroepiandrosterone inhibited inflammation in bronchial epithelial cells as indicated by the suppression of Th2-associated cytokines and chemokines. Dehydroepiandrosterone also suppressed eosinophil migration and infiltration into the lung to improve the symptoms of asthma in ovalbumin-sensitized mice.

BACKGROUND

Mast cells (MCs) are abundant in the inflammatory infiltrate in eosinophilic oesophagitis (EoE), but decrease with disease remission. However, their phenotype, role in the pathophysiology of the disease, and modulation after effective dietary therapy are still unclear.

OBJECTIVE

To define the phenotype of oesophageal MCs, their modulation through dietary therapy, and their association with clinical manifestations of EoE.

METHODS

Oesophageal mucosal samples from 10 adult patients with EoE obtained before and after effective six-food elimination diet (SFED) therapy, as well as from 10 control subjects were analysed. Eosinophil and MC density were quantified. Gene expression of chemoattractants for eosinophils (CCL11, CCL24, and CCL26), MCs (SCF), and their receptors (CCR3 and SCFR, respectively) were assessed by means of qPCR. Gene and protein expression of specific MC proteases (CPA3, CMA, and TPSB2) were evaluated with qPCR and immunofluorescence. Clinical manifestations and atopic background were recorded.

RESULTS

MC density was significantly increased in EoE compared with controls, decreasing after dietary treatment (18.6 to 1.44 cells/hpf, respectively; P < 0.001). The MCTC subtype predominated in the oesophageal mucosa (90%) in both patients with EoE and controls. Gene expression of MC-related proteases, eotaxins, and SCF were up-regulated in patients with EoE, but significantly decreased after therapy, regardless of atopic background. Epithelial peaks of MCs and eosinophils were significantly associated (ρ = 0.80) in EoE and correlated with the symptom score (ρ = 0.78). Gene expression of MC proteases and eotaxins also correlated with the symptom score (P < 0.05).

CONCLUSIONS

MC and its proteases seem to play a relevant role in the pathophysiology and symptoms of EoE, which can be reversed after effective dietary treatment.

Rhinovirus (RV) is responsible for the majority of virus-induced asthma exacerbations. We showed previously that RV infection of ovalbumin-sensitized and -challenged BALB/c mice induces production of type 2 cytokines from M2-polarized macrophages. In the present study, we sought to determine the mechanism of RV-induced cytokine expression. We infected bone marrow-derived macrophages (BMMs) from BALB/c mice with RV serotype 1B, a minor group virus that infects mouse cells. Selected cultures were pretreated with IL-4, a type 2 cytokine increased in allergic asthma. RV infection of untreated cells increased messenger RNA and protein expression of the M1 cytokines TNF-α, CXCL1, and IL-6 but failed to induce expression of the M2 cytokines CCL22 and CCL24. Cells pretreated with IL-4 showed decreased expression of M1 cytokines but increased expression of Ym-1, Arg-1 (M2 markers), CCL22, and CCL24. Infection with ultraviolet (UV)-irradiated, replication-deficient RV elicited similar cytokine responses, suggesting that the outcome is replication independent. Consistent with this, viral RNA copy number did not increase in RV-treated BMMs or bronchoalveolar macrophages. RV-induced cytokine expression was not affected when cells were pretreated with cytochalasin D, suggesting that viral endocytosis is not required for the response. Finally, RV-induced cytokine expression and viral attachment were abolished in BMMs from myeloid differentiation factor 88 and Toll-like receptor (TLR)2 KO mice, suggesting a specific requirement of TLR2. We conclude that RV elicits a proinflammatory cytokine response in BMMs through a cell-surface-mediated, TLR2-dependent mechanism that does not require viral endocytosis or replication.

Human colonic epithelial cells express T helper type 1 (Th1)-associated chemoattractants, yet little is known about the production of Th2-associated chemoattractants. CCL11/eotaxin-1, CCL24/eotaxin-2 and CCL26/eotaxin-3 are known to attract CCR3-expressing, Th2-polarized lymphocytes. We studied constitutive and inflammation-induced expression and production of CCR3 together with its ligands in the colon and peripheral blood of patients with inflammatory bowel disease (IBD) by flow cytometry, reverse transcription–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA). We further defined the regulated expression of these chemokines by RT–PCR and ELISA using cultured human epithelial cell lines. A higher fraction of peripheral T lymphocytes were found to be positive for CCR3 in patients with ulcerative colitis (UC) compared to Crohn’s disease (CD), while almost no CCR3(+) T cells were found in normal controls (NC). Similarly, higher and more frequent expression of CCR3 was observed in colonic biopsies from patients with UC, regardless of the disease activity, when compared to CD or NCs. Serum CCL11/eotaxin-1 was increased significantly in UC (306 ± 87 pg/ml) and less so in CD (257 ± 43 pg/ml), whereas CCL24/eotaxin-2, and CCL26/eotaxin-3 were increased only in UC. Colonic expression of the three chemokines was minimal in NCs but high in inflammatory bowel diseases (especially UC) and was independent of disease activity. Th2, and to a lesser extent Th1, cytokines were able to induce expression and production of all three eotaxins from colonic epithelial cells in culture. CCR3 and ligands over-expression would appear to be a characteristic of UC. The production of CCR3 ligands by human colonic epithelial cells suggests further that epithelium can play a role in modulating pathological T cell-mediated mucosal inflammation.

BACKGROUND

Eotaxin-2/CCL24 and eotaxin-3/CCL26 play an important role in eosinophil chemotaxis and activation in asthma. We previously demonstrated that eotaxin/CCL11 is profibrogenic for human lung fibroblasts. The effect of eotaxin-2/ CCL24 and eotaxin-3/CCL26 on lung fibroblasts has not yet been investigated.

OBJECTIVE

To evaluate whether eotaxin-2/CCL24 and eotaxin-3/CCL26 modulate fibrotic properties of lung fibroblasts.

METHODS

Fibroblast proliferation was evaluated by means of 3-hydroxythymidine incorporation. Collagen production was assessed by means of 3-hydroxyproline incorporation and biochemical staining. Chemotaxis was determined using Boyden chambers. Expression of alpha-smooth muscle actin was evaluated by means of immunostaining. Transforming growth factor beta1 release was assessed using enzyme-linked immunosorbent assay. Parametric analysis of variance, followed by the Tukey-Kramer multiple comparisons test, was used to calculate statistical significance.

RESULTS

Eotaxin-2/CCL24 but not eotaxin-3/CCL26 stimulated human lung fibroblast proliferation and collagen synthesis. In contrast, eotaxin-3/CCL26 but not eotaxin-2/CCL24 promoted fibroblast migration. Neither eotaxin-2/CCL24 nor eotaxin-3/ CCL26 induced the expression of alpha-smooth muscle actin or transforming growth factor beta1 from lung fibroblasts.

CONCLUSIONS

Eotaxin-2/CCL24 and eotaxin-3/CCL26 have differential profibrogenic effects on human lung fibroblasts. These CC chemokines may, therefore, contribute to airway remodeling in asthma.

House dust mite (HDM) challenge is commonly used in murine models of allergic asthma for preclinical pathophysiological studies. However, few studies define objective readouts or biomarkers in this model. In this study we characterized immune responses and defined molecular markers that are specifically altered after HDM challenge. In this murine model, we used repeated HDM challenge for two weeks which induced hallmarks of allergic asthma seen in humans, including airway hyper-responsiveness (AHR) and elevated levels of circulating total and HDM-specific IgE and IgG1. Kinetic studies showed that at least 24 h after last HDM challenge results in significant AHR along with eosinophil infiltration in the lungs. Histologic assessment of lung revealed increased epithelial thickness and goblet cell hyperplasia, in the absence of airway wall collagen deposition, suggesting ongoing tissue repair concomitant with acute allergic lung inflammation. Thus, this model may be suitable to delineate airway inflammation processes that precede airway remodeling and development of fixed airway obstruction. We observed that a panel of cytokines e.g. IFN-γ, IL-1β, IL-4, IL-5, IL-6, KC, TNF-α, IL-13, IL-33, MDC and TARC were elevated in lung tissue and bronchoalveolar fluid, indicating local lung inflammation. However, levels of these cytokines remained unchanged in serum, reflecting lack of systemic inflammation in this model. Based on these findings, we further monitored the expression of 84 selected genes in lung tissues by quantitative real-time PCR array, and identified 31 mRNAs that were significantly up-regulated in lung tissue from HDM-challenged mice. These included genes associated with human asthma (e.g. clca3, ear11, il-13, il-13ra2, il-10, il-21, arg1 and chia1) and leukocyte recruitment in the lungs (e.g. ccl11, ccl12 and ccl24). This study describes a biosignature to enable broad and systematic interrogation of molecular mechanisms and intervention strategies for airway inflammation pertinent to allergic asthma that precedes and possibly potentiates airway remodeling and fibrosis.

OBJECTIVE

The aim of our study was to measure CCL24 (eotaxin-2) levels in nasal lavage fluid of patients with different forms of sinonasal chronic eosinophilic inflammation to verify the relationship with nasal hypereosinophilia and symptoms.

METHODS

Patients with nasal hypereosinophilia were randomly recruited and grouped in persistent allergic rhinitis, non-allergic rhinitis with eosinophilia syndrome (NARES) and chronic rhinosinusitis with polyps. Non rhinitic volunteers were recruited as controls. CCL24 concentration was measured by `Quantikine Human CCL24 Immunoassay`. Differential cell counts were performed by microscopic cytological examination of nasal tissue scraped by inferior turbinate.

RESULTS

CCL24 levels measured in patient groups were significantly higher compared to control group with the highest levels in NARES patients. Eotaxin- 2 levels were significantly correlated to severity of symptoms and to the percentage of eosinophils in nasal tissue.

CONCLUSIONS

We revealed high levels of CCL24 in all patient groups showing a significant correlation with the degree of eosinophilia and clinical symptoms. A prolonged accumulation of CCL24 inside the nasal mucosa may sustain the process of unspecific self-perpetuating eosinophil recruitment pathognomonic of these patients.

Cardiac manifestations are a major cause of morbidity and mortality in patients with eosinophil-associated diseases. Eosinophils are thought to play a pathogenic role in myocarditis. We investigated the pathways that recruit eosinophils to the heart using a model of eosinophilic myocarditis, in which experimental autoimmune myocarditis (EAM) is induced in IFNγ-/- IL-17A-/- mice. Two conditions are necessary for efficient eosinophil trafficking to the heart: high eotaxin (CCL11, CCL24) expression in the heart and expression of the eotaxin receptor CCR3 by eosinophils. We identified cardiac fibroblasts as the source of CCL11 in the heart interstitium. CCL24 is produced by F4/80+ macrophages localized at inflammatory foci in the heart. Expression of CCL11 and CCL24 is controlled by Th2 cytokines, IL-4 and IL-13. To determine the relevance of this pathway in humans, we analyzed endomyocardial biopsy samples from myocarditis patients. Expression of CCL11 and CCL26 was significantly increased in eosinophilic myocarditis compared to chronic lymphocytic myocarditis and positively correlated with the number of eosinophils. Thus, eosinophil trafficking to the heart is dependent on the eotaxin-CCR3 pathway in a mouse model of EAM and associated with cardiac eotaxin expression in patients with eosinophilic myocarditis. Blocking this pathway may prevent eosinophil-mediated cardiac damage.

Eotaxins (CCL11, CCL24, CCL26) originating from airway epithelial cells and leukocytes have been detected in bronchoalveolar lavage of asthmatics. Although the alveolar epithelium is the destination of uncleared allergens and other inflammatory products, scanty information exists on their contribution to the generation and regulation of the eotaxins. We envisioned a state whereby alveolar type II cells, a known source of other inflammatory proteins, could be involved in both the production and regulation of CCL24 and CCL26. Herein, we demonstrated that all three eotaxins are constitutively expressed in A549 cells. IL-4 and IL-13 stimulated a concentration-dependent secretion of CCL24 and CCL26. The cytokines did not act synergistically. Cycloheximide and actinomycin D abrogated IL-4- and IL-13-dependent CCL26 but not CCL24 secretion. Both IL-13 and IL-4 stimulated CCL26 synthesis that was inhibited in a concentration-dependent manner by CCL26 but not CCL24. Only CCL26 reduced expression of CCR3 receptors by 30-40%. On the other hand, anti-CCR3 pretreatment reduced IL-4+IL-13-dependent CCL26 secretion, implying autoregulation. A CCR3-specific antagonist (SB-328437) significantly decreased IL-4-dependent synthesis and release of CCL26. Eosinophils treated with medium from IL-4-stimulated A549 cells preincubated with anti-CCL26 showed a marked decrease of superoxide anion production compared with anti-CCL24 treated. These results suggest that CCL26 is a major eotaxin synthesized and released by alveolar epithelial cells and is involved in autoregulation of CCR3 receptors and other eotaxins. This CCL26-CCR3 ligand-receptor system may be an attractive target for development of therapeutics that limits progress of inflammation in airway disease.

T helper type 2 (Th2) cells, type 2 innate lymphoid cells (ILC2s) and eosinophil progenitors have previously been described to produce interleukin-5 (IL-5) in the airways upon allergen provocation or by direct administration of IL-33. Eosinophilic airway inflammation is known to be associated with IL-5-dependent eosinophil development in the bone marrow, however, the source of IL-5 remains unclear. T helper cells, ILC2s and CD34+ progenitors have been proposed to be involved in this process, therefore, we investigated whether these cells are taking part in eosinophilopoiesis by producing IL-5 locally in the bone marrow in IL-33-driven inflammation. Airway exposure with IL-33 led to eosinophil infiltration in airways and elevated eotaxin-2/CCL24. Importantly, IL-5 production as well as expression of the IL-33 receptor increased in ILC2s in the bone marrow under this treatment. A small but significant induction of IL-5 was also found in CD34+ progenitors but not in T helper cells. Similar results were obtained by in vitro stimulation with IL-33 where ILC2s rapidly produced large amounts of IL-5, which coincided with the induction of eosinophil hematopoiesis. IL-33-mediated eosinophil production was indeed dependent on IL-5 as both airway and bone marrow eosinophils decreased in mice treated with anti-IL-5 in combination with IL-33. Interestingly, the responsiveness of ILC2s to IL-33 as well as IL-33-induced eotaxin-2/CCL24 were independent of the levels of IL-5. In summary, we demonstrate for the first time that IL-33 acts directly on bone marrow ILC2s, making them an early source of IL-5 and part of a process that is central in IL-33-driven eosinophilia.

Chemokines and chemokine receptors interaction have presented important role in leukocyte migration to specific immune reaction sites. Recently, it has been reported that chemokine receptors CXC (CXCR3) and CC (CCR5) were preferentially expressed on Th1 cells while CCR3 and CCR4 were preferentially expressed on Th2 cells. This study evaluated the mRNA expression of type 1 and type 2 chemokine and chemokine receptors in the cardiac tissue of Beagle dogs infected with distinct genetic groups of Trypanosoma cruzi (Y, Berenice-78 and ABC strains) during acute and chronic phases. To analyze the correlation between chemokine and chemokine receptors expression and the development of heart pathology, the chronic infected animals were divided into groups, according to the parasite strain and based on the degree of heart damage: cardiac and indeterminate form of Chagas disease. Our results indicated that cardiac type1/2 chemokines and their receptors were partially dependent on the genetic diversity of parasites as well as the polarization of clinical forms. Also, dogs presenting cardiac form showed lower heart tissue mRNA expression of CCL24 (type 2) and higher expression of CCL5, CCL4 and CXCR3 (type 1) when compared with those with indeterminate form of disease. Together, these data reinforce a close-relation between T. cruzi genetic population and the host specific type 1 immune response and, for the first time, we show the distribution of type 1/2 chemokines associated with the development of cardiac pathology using dogs, a well similar model to study human Chagas disease.

OBJECTIVE

We aimed to determine the plasma levels of cytokines in patients with obsessive-compulsive disorder (OCD) as compared with healthy controls and to investigate whether there is any association between their concentrations and OCD clinical and therapeutic features.

METHODS

Forty patients with OCD and 40 healthy controls had their plasmas assessed for a range of cytokines (tumor necrosis factor-α, or TNF-α), chemokines (CCL2, CCL3, CCL11, CCL24, CXCL8, CXCL9, CXCL10), and other mediators (TNF soluble receptors sTNFR1 and sTNFR2 and interleukin-1 receptor antagonist) by enzyme-linked immunosorbent assay. Patients with OCD were further examined with the Mini-International Neuropsychiatric Interview, the Obsessive-Compulsive Inventory-Revised, and the Beck Depression Inventory.

RESULTS

Compared with healthy controls, patients with OCD exhibited significantly increased plasma levels of CCL3, CXCL8, sTNFR1, and sTNFR2. Among patients with OCD, there was a positive correlation between relative antidepressant dose and sTNFr2 levels. Furthermore, although the levels of sTNFR1 correlated positively with the severity of washing symptoms, CCL24 levels correlated negatively with the severity of hoarding.

CONCLUSIONS

The levels of certain immune markers are increased in adult patients with OCD and seem to vary according to predominant symptoms dimensions. Other studies are required to establish whether our findings truly reflect immunologic dysfunction in OCD or are the result of other hidden confounding factors.

There is increasing evidence that inflammation plays a pivotal role in the pathogenesis of some forms of pulmonary hypertension (PH). We recently demonstrated that deficiency of adiponectin (APN) in a mouse model of PH induced by eosinophilic inflammation increases pulmonary arterial remodeling, pulmonary pressures, and the accumulation of eosinophils in the lung. Based on these data, we hypothesized that APN deficiency exacerbates PH indirectly by increasing eosinophil recruitment. Herein, we examined the role of eosinophils in the development of inflammation-induced PH. Elimination of eosinophils in APN-deficient mice by treatment with anti-interleukin-5 antibody attenuated pulmonary arterial muscularization and PH. In addition, we observed that transgenic mice that are devoid of eosinophils also do not develop pulmonary arterial muscularization in eosinophilic inflammation-induced PH. To investigate the mechanism by which APN deficiency increased eosinophil accumulation in response to an allergic inflammatory stimulus, we measured expression levels of the eosinophil-specific chemokines in alveolar macrophages isolated from the lungs of mice with eosinophilic inflammation-induced PH. In these experiments, the levels of CCL11 and CCL24 were higher in macrophages isolated from APN-deficient mice than in macrophages from wild-type mice. Finally, we demonstrate that the extracts of eosinophil granules promoted the proliferation of pulmonary arterial smooth muscle cells in vitro. These data suggest that APN deficiency may exacerbate PH, in part, by increasing eosinophil recruitment into the lung and that eosinophils could play an important role in the pathogenesis of inflammation-induced PH. These results may have implications for the pathogenesis and treatment of PH caused by vascular inflammation.

Despite considerable differences in primary structure, the chemokines eotaxin-1/CCL11, eotaxin-2/CCL24 and eotaxin-3/CCL26 signal via a single receptor, CCR3, but exhibit different potencies and efficacies. To examine receptor/ligand interactions in more detail, we performed alanine scanning mutagenesis of 21 charged residues within the extracellular loops (ECLs) of CCR3. Following transient expression in the L1.2 cell line, CCR3 mutants were assessed for their ability to be expressed at the cell surface, bind CCL11 and induce chemotactic responses to CCL11, CCL24 and CCL26. The majority of constructs were well expressed at the cell surface and bound CCL11 with low nanomolar affinity. Exceptions to this rule included the mutants E175A and E176A (ECL2) which were poorly expressed and responded weakly to all three ligands in chemotaxis assays. In contrast, the mutants K26 (amino-terminus) E179 and E180 (ECL2) responded in chemotaxis assays to CCL11 and CCL24, but not to CCL26. Mutation of residues in ECL3 was informative, with the D272A, K277A and D280A mutants exhibiting reduced chemotactic responses to two or more of the three ligands examined, despite being expressed on the cell surface at levels similar to WT CCR3. This suggests a major role for ECL3 in the recognition of all three eotaxins. In summary, distinct acidic and basic residues within CCR3 determine both receptor expression and activation by the eotaxins. Determining how these chemokines interact with their receptor at the molecular level should increase our understanding of the process of chemokine receptor activation.

BACKGROUND

Although several studies have claimed that mesenchymal stem cells (MSC) derived from human tissues can ameliorate allergic airway inflammation, the immunomodulatory mechanism of MSCs remains unclear.

OBJECTIVE

We aimed to determine the effects and the underlying mechanism of tonsil-derived MSCs (T-MSC) on allergic inflammation compared with adipose tissue-derived stem cells (ASC) in a mouse model of allergic rhinitis (AR).

METHODS

MSCs were isolated from human palatine tonsil (T-MSC) and the surface markers were analyzed. The effect of T-MSCs was evaluated in 24 BALB/c mice that were randomly divided into four groups (negative control group, positive control group, T-MSC group, and ASC group). MSCs were administered intravenously to ovalbumin (OVA) sensitized mice (T-MSC and ASC groups) on days 18 to 23, and subsequent OVA challenge was conducted daily from days 24 to 28. Several parameters of allergic inflammation were assessed.

RESULTS

T-MSC and ASC had similar characteristics in surface markers. Intravenous injection of T-MSC significantly reduced allergic symptoms, eosinophil infiltration, serum total, and OVA-specific immunoglobulin E (IgE), and the nasal and systemic T-helper (Th) 2 cytokine profile. Further analysis revealed that nasal innate cytokines, such as interleukin (IL) 25 and IL-33, and chemokines, such as CCL11, CCL24, induction was suppressed in T-MSCs injected groups, which explained their underlying mechanism. In addition, the T-MSC group had more inhibition of allergic inflammation than did the ASC group, which might be attributed to the more proliferative activity of T-MSC.

CONCLUSIONS

Administration of T-MSC effectively reduced allergic symptoms and inflammatory parameters in the mouse model of AR. T-MSC treatment reduced Th2 cytokines and OVA-specific IgE secretion from B cells. In addition, innate cytokine (IL-25 and IL-33) expression and eotaxin messenger RNA expression was inhibited in the nasal mucosa, which is suggestive of the mechanism of reduced allergic inflammation. Therefore, T-MSC treatment is potentially an alternative therapeutic modality in AR.

Group 2 innate lymphoid cells (ILC2) are an important source of the type 2 cytokines interleukin (IL)-5 and IL-13 that are critical to the allergic airway phenotype. Previous studies reported that histone deacetylase (HDAC) inhibition by trichostatin A (TSA) downregulated adaptive allergic immune responses; however, the effect of HDAC inhibition on the early innate allergic immune response is unknown. Therefore, we investigated the effect of TSA on innate airway inflammation mediated by ILC2 activation.

BALB/c mice were challenged intranasally with Alternaria extract, exogenous recombinant mouse IL-33 (rmIL-33) or the respective vehicles for four consecutive days following TSA or vehicle treatment. Bronchoalveolar lavage (BAL) fluids and lungs were harvested 24 h after the last challenge.

We found that TSA treatment significantly decreased the number of ILC2 expressing IL-5 and IL-13 in the lungs challenged with Alternaria extract or rmIL-33 compared with vehicle treatment (p<0.05). TSA treatment significantly decreased protein expression of IL-5, IL-13, CCL11 and CCL24 in the lung homogenates from Alternaria extract-challenged mice or rmIL-33-challenged mice compared with vehicle treatment (p<0.05). Further, TSA treatment significantly decreased the number of perivascular eosinophils and mucus production in the large airways that are critical components of the asthma phenotype (p<0.05). TSA did not change early IL-33 release in the BAL fluids; however, TSA decreased lung IL-33 expression from epithelial cells 24 h after last Alternaria extract challenge compared with vehicle treatment (p<0.05).

These results reveal that TSA reduces allergen-induced ILC2 activation and the early innate immune responses to an inhaled protease-containing aeroallergen.

BACKGROUND

Transforming growth factor (TGF)-beta is an important modulator of immune functions and cellular responses, such as differentiation, proliferation, migration and apoptosis. The Smad proteins, which are intracellular TGF-beta signal transducers, mediate most actions of TGF-beta.

OBJECTIVE

This study examines the role of Smad3 in a murine model of contact hypersensitivity (CHS).

METHODS

The CHS response to oxazolone was studied in Smad3-deficient mice. The ear swelling response was measured and skin biopsies from oxazolone-sensitized skin areas were obtained for RNA isolation, immunohistochemical analyses and histology. Ear draining lymph nodes were collected for RNA isolation and proliferation tests. Quantitative real-time polymerase chain reaction was used to quantify mRNA expression of cytokines, chemokines and transcription factors. Results The expression of proinflammatory [interleukin (IL)-1beta, tumour necrosis factor-alpha, IL-6], Th2 (IL-4) and Th17 type cytokines (IL-17), as well as regulatory components (TGF-beta, Foxp3) increased significantly at the mRNA level in the skin of oxazolone-treated Smad3-/- mice when compared with wild-type controls. The expression of the Th1 type cytokine IFN-gamma and the chemokines CXCL9 and CXCL10 was, however, unaffected by the lack of Smad3. The number of neutrophils and expression of the chemokines CCL3 and CXCL5, which are both involved in neutrophil recruitment, were increased in mice lacking Smad3. Also Th2 type chemokines CCL24, CCL3 and CXCL5 were increased in the skin of Smad3-/- mice compared with wild-type mice. In the lymph nodes, mRNA of IL-1beta and IL-17, but not IL-4, TGF-beta or Foxp3, was increased in Smad3-/- mice during the CHS response.

CONCLUSIONS

The lack of intact TGF-beta signalling via Smad3 results in an increased proinflammatory, Th2 and Th17 type response in the skin, as well as increased expression of regulatory elements such as TGF-beta and Foxp3. Understanding the role of Smad3 in the CHS response may offer treatment and prevention strategies in this often disabling disease.

The skin is a difficult to access tissue for efficient delivery of large and/or charged macromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. MiR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the nuclear factor (NF)-κB pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-κB pathway and the genes known to be activated by NF-κB, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.

The aim of the present study was to elucidate whether the culture of cells recovered from induced sputum may represent a suitable model to evaluate cytokine and chemokine production by airway inflammatory cells. Sputum induction was performed in 21 normal subjects and 30 asthmatic patients. A total of 21 out of the 30 asthmatic patients were taking inhaled corticosteroids, while the remaining nine were steroid-naive asthmatics. The steroid-naive group was evaluated before and after a 14-day treatment with oral prednisone (40 mg.day(-1)). The supernatant of lysed and centrifuged sputum and the supernatant of sputum cell culture were analysed. Tumour necrosis factor-alpha, interleukin (IL)-8 (CXCL8), IL-1beta, IL-13 and eotaxin-2 (CCL24) concentrations were determined by specific ELISA. Eotaxin-2 production by cell culture was higher in the asthma group (131+/-108 pg.mL(-1)) than in the control group (36+/-41 pg.mL(-1)) and treatment with oral corticosteroids eliminated this difference. In addition, reduction of eotaxin-2 levels by corticosteroid treatment was greater in cell culture (81.3% reduction) than in sputum (26.4%). There was correlation between the decrease in eotaxin-2 production and the decrease in blood eosinophil number and between eotaxin-2 and eosinophils in sputum. Eotaxin-2 may play an important role in asthma and the response to corticosteroid treatment suggests that analysis of sputum cell culture is relevant as an inflammatory parameter.

CC-chemokine receptor 3 (CCR3) is a chemokine receptor for which major ligands, CC-chemokine ligand (CCL) 11, CCL24, and CCL26, are known to be involved in chemotaxis for eosinophils. In the present study, we evaluated the effect of a low molecular weight CCR3-receptor antagonist, Ki19003 (4-[[5-(2,4-dichlorobenzylureido)pentyl][1-(4-chlorophenyl)ethyl]amino]butanoic acid), on airway remodeling in a mouse model of allergic asthma. BALB/c mice were sensitized twice by intraperitoneal injection of ovalbumin (OA) and exposed daily to 1% OA for 3 weeks. Twenty-four hours after the final antigen challenge, bronchoalveolar lavage and histological examinations were carried out. Ki19003 clearly inhibited antigen-induced increase in the number of eosinophils in bronchoalveolar lavage fluid (BALF), but did not affect the number of other cell types examined in this study. Ki19003 also inhibited the increased production of transforming growth factor-beta1 in BALF and the amount of hydroxyproline in the lungs in a dose-dependent manner. Furthermore, Ki19003 significantly attenuated allergen-induced subepithelial and peribronchial fibrosis. These findings indicate that CCR3 antagonism prevents not only the infiltration of eosinophils into the airways but also the development of allergen-induced subepithelial and peribronchial fibrosis. Therefore, a CCR3 antagonist may be useful in the treatment of airway remodeling, especially subepithelial and peribronchial fibrosis, in allergic asthma.

BACKGROUND

The function of eosinophils has been attributed to host defense, immunomodulation, and fibrosis. Although eosinophils are found among infiltrating cells in a broad spectrum of skin diseases, their pathogenic role remains uncertain. This study aimed to analyze the cytokine expression by eosinophils in different skin diseases.

METHODS

Skin specimens from different skin diseases [allergic/reactive, infectious, autoimmune, and tumors/lymphomas (LY)] were stained by antibodies directed to eosinophil cationic protein, cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-5, IL-6, IL-10, IL-11, IL-13, IL-17, IL-25, IL-33, interferon-γ, transforming growth factor (TGF)-β, and thymic stromal lymphopoietin], eotaxins (CCL11, CCL24, and CCL26), metalloproteinase (MMP)-9 as well as extracellular matrix proteins (tenascin-C and procollagen-3) and then analyzed by laser scanning microscopy.

RESULTS

The number of eosinophils varied considerably in and between disease groups and did not correlate with the numbers of accompanying inflammatory cells. The expression of IL-5, IL-6, IL-11, TGF-β, CCL24, and MMP-9 by eosinophils significantly differed between disease groups. Eosinophils in tumors/LY predominantly expressed IL-6, TGF-β, and CCL24, but not IL-11. On the other hand, in autoimmune diseases, eosinophils largely contributed to MMP-9 production. IL-5-generating eosinophils were particularly obvious in allergic and infectious diseases.

CONCLUSIONS

In skin diseases, eosinophil expresses a broad spectrum of cytokines. The different cytokine expression patterns suggest distinct functional roles of eosinophils in these diseases that might be related to host defense, immunomodulation, fibrosis, and/or tumor development.

Cognitive impairment and depressive symptoms are of great interest in Parkinson's disease (PD), since they are very common and lead to increased disability with poor quality of life. Inflammatory mechanisms have been implicated in PD and its nonmotor symptoms. In the current pilot study, we aimed to evaluate plasma levels of chemokines in PD patients and to analyze the putative association of chemokines with depressive symptoms and cognitive performance. We hypothesized that higher chemokines levels are associated with worse cognitive performance and increased depressive symptoms in PD. For this purpose, 40 PD patients and 25 age- and gender-matched controls were subjected to a clinical evaluation including cognitive and mood tests. Peripheral blood was drawn and plasma levels of CCL2/MCP-1, CCL11/eotaxin, CCL24/eotaxin-2, and CXCL10/IP-10 were measured by enzyme-linked immunosorbent assay. PD patients and control individuals presented comparable plasma concentrations of all the evaluated chemokines. In PD patients, CXCL10/IP-10 plasma levels correlated positively with Hoehn and Yahr staging scale. In addition, the higher CXCL10/IP-10 levels, the worse performance on cognitive tests. Although there was no significant difference between PD patients and control individuals regarding chemokines levels, our preliminary results showed that CXCL10/IP-10 may be associated with cognitive status in PD.