Background

Subepithelial fibrosis is one of the most critical structural changes affecting bronchial airway function during asthma. Eosinophils have been shown to contribute to the production of pro-fibrotic cytokines, TGF-β and IL-11, however, the mechanism regulating this process is not fully understood.

Objective

In this report, we investigated whether cytokines associated with inflammation during asthma may induce eosinophils to produce pro-fibrotic cytokines.

Methods

Eosinophils were isolated from peripheral blood of 10 asthmatics and 10 normal control subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and the production of TGF-β and IL-11 was determined using real time PCR and ELISA assays.

Results

The basal expression levels of eosinophil derived TGF-β and IL-11 cytokines were comparable between asthmatic and healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of pro-fibrotic cytokines. However, stimulating eosinophils with Th17 cytokines resulted in the enhancement of TGF-β and IL-11 expression in asthmatic but not healthy individuals. This effect of IL-17 on eosinophils was dependent on p38 MAPK activation as inhibiting the phosphorylation of p38 MAPK, but not other kinases, inhibited IL-17 induced pro-fibrotic cytokine release.

Conclusions

Th17 cytokines might contribute to airway fibrosis during asthma by enhancing production of eosinophil derived pro-fibrotic cytokines. Preventing the release of pro-fibrotic cytokines by blocking the effect of Th17 cytokines on eosinophils may prove to be beneficial in controlling fibrosis for disorders with IL-17 driven inflammation such as allergic and autoimmune diseases.

HIV-1 Nef protein down-regulates several cell surface receptors through its interference with the cell sorting and trafficking machinery. Here we demonstrate for the first time the ability of Nef to down-regulate cell surface expression of the negative immune modulator CTLA-4. Down-regulation of CTLA-4 required the Nef motifs DD175, EE155 and LL165, all known to be involved in vesicle trafficking. Disruption of the lysosomal functions by pH-neutralizing agents prevented CTLA-4 down-regulation by Nef, demonstrating the implication of the endosomal/lysosomal compartments in this process. Confocal microscopy experiments visualized the co-localization between Nef and CTLA-4 in the early and recycling endosomes but not at the cell surface. Overall, our results provide a novel mechanism by which HIV-1 Nef interferes with the surface expression of the negative regulator of T cell activation CTLA-4. Down-regulation of CTLA-4 may contribute to the mechanisms by which HIV-1 sustains T cell activation, a critical step in viral replication and dissemination.

Background

Acute asthma attacks remain a frequent cause of emergency department (ED) visits and hospital admission. Many factors encourage patients to seek asthma treatment at the emergency department. These factors may be related to the patient himself or to a health system that hinders asthma control. The aim of this study was to identify the main factors that lead to the frequent admission of asthmatic patients to the ED.

Methods

A cross-sectional survey of all the patients who visited the emergency room with bronchial asthma attacks over a 9-month period was undertaken at two major academic hospitals. The following data were collected: demographic data, asthma control in the preceding month, where and by whom the patients were treated, whether the patient received education about asthma or its medication and the patients’ reasons for visiting the ED.

Result

Four hundred fifty (N = 450) patients were recruited, 39.1% of whom were males with a mean age of 42.3 ± 16.7. The mean duration of asthma was 155.90 ± 127.13 weeks. Approximately half of the patients did not receive any information about bronchial asthma as a disease, and 40.7% did not receive any education regarding how to use asthma medication. Asthma was not controlled or partially controlled in the majority (97.7%) of the patients preceding the admission to ED. The majority of the patients visited the ED to receive a bronchodilator by nebuliser (86.7%) and to obtain oxygen (75.1%). Moreover, 20.9% of the patients believed that the ED managed them faster than the clinic, and 21.1% claimed that their symptoms were severe enough that they could not wait for a clinic visit. No education about asthma and uncontrolled asthma are the major factors leading to frequent ED visits (three or more visits/year), p-value = 0.0145 and p-value = 0.0003, respectively. Asthma control also exhibited a significant relationship with inhaled corticosteroid ICS use (p-value =0.0401) and education about asthma (p-value =0.0117).

Conclusion

This study demonstrates that many avoidable risk factors lead to uncontrolled asthma and frequent ED visits.

Background

Inhaled corticosteroids represent the most common treatment for asthma. Although most asthmatic patients respond well, a significant proportion of severe asthmatics require higher doses or even fail to respond to oral or inhaled corticosteroids. We previously reported that glucocorticoid receptor-beta is associated with corticosteroid resistance in airway epithelial cells from asthmatic patients and that Th-17 cytokines increase steroid insensitivity via a mechanism involving GR-beta upregulation. We aim to investigate whether IL-17A and F cytokines enhance steroid unresponsiveness in PBMCs from normal subjects and severe asthmatics via the upregulation of GR-beta isoform.

Methods

PBMCs were cultured for 48 hours in the presence or absence of IL-2, IL-4, IL-17A, IL-17F or IL-23 cytokines. Expression of GR-alpha, GR-beta, GILZ and IL-6 was determined using Q-RT-PCR and/or Western blotting. Response to Dexamethasone was determined on the inhibition of PHA-induced proliferation by Dexamethasone (IC50) using either 3H-thymidine or CFSE-labelled cells. Response of the cells to Dexamethasone-induced apoptosis was determined by Annexin-V staining.

Results

Treatment of PBMCs with IL-17A+IL-17F combined significantly decreased the mRNA expression of GR-alpha while that of GR-beta was significantly upregulated. IL-2+IL-4 in combination significantly decreased GR-alpha expression but had no effect on GR-beta receptor expression. IL17A+IL17F+IL23 combined induced the highest ratio of GR-beta/GR-alpha in PBMC from normal subjects. Either IL-17A+F or IL-2+IL-4 combinations significantly decreased the inhibitory effect of Dexamethasone on PBMC proliferation (IL-17A+F IC50 = 190 nM Dex; IL-2+4 IC50 = 1060 nM Dex), when compared to the control without cytokine stimulation. In the presence of Dexamethasone, IL-2+IL-4 but not IL-17A+IL-17F, inhibited the expression of the glucocorticoid-inducible leucine zipper gene (GILZ) in PBMCs from both normal (60%) and asthmatics (45–50%), which was correlated with significantly higher apoptosis in cells stimulated with IL-2+IL-4.

Conclusions

IL-17A, IL-17F, IL-2, and IL-4, which are known to be upregulated in the blood and lung tissue of asthmatics, contribute to steroid insensitivity of severe asthmatic patients by modulating the expression of GR-alpha and GR-beta receptors on peripheral blood PBMCs. GR-beta could protect PBMCs from Dex-induced apoptosis. Furthermore, the increased GR-beta/GR-alpha ratios by both IL-17A+F and IL-2+4 cytokines correlates with the decreased inhibitory effect of Dexamethasone on PHA-induced PBMC proliferation.

Background

B lymphocytes are known to be important cytokine sources in inflammation and play a pathogenic role by producing autoantibodies in a number of chronic immunological diseases. However, B cell depletion therapy induced an exacerbation of symptoms in some patients with autoimmune disorders, revealing that B cells play a critical anti-inflammatory role mediated by IL-10 release. We therefore investigated the human B cell regulatory subset producing IL-10 in response to stimulation.

Methods

Highly purified B cells were obtained from tonsils by using a multiple-step separation procedure which included rosette depletion, adherence depletion, CD3+ cell magnetic-activated depletion and CD19+ magnetic-activated positive cell selection. CD20+ purity was verified by flow cytometry. The CD19+CD20+ B cells were stimulated with CpG oligonucleotide, IL-4, IFN-gamma, anti-CD40, IL-17A and IL-17F, either alone or in combination. The expression of both IL-6 and IL-10 mRNA was analyzed by quantitative RT-PCR and by ELISA. B regulatory cell subsets expressing IL-10 and the markers CD5 and CD1d were quantified by FACS analysis. B cell proliferation was determined by 3H thymidine incorporation or CFSE labeling.

Results

Expression of IL-10 mRNA and protein in purified B cells from tonsils was weakly stimulated by anti-CD40 antibody, CpG oligonucleotide or with IL-17. When B cells were simultaneously stimulated with IL-17, anti-CD40 antibody and CpG oligonucleotide, the mRNA and protein expression of IL-10 was strongly increased (n = 3; P ≤ 0.001). B cells proliferation was also significantly increased. In contrast, stimulation with IL-4 alone or in combination with anti-CD40 antibody, decreased the expression of IL-10 (n = 3; P ≤ 0.001).

Conclusions

TLR9 receptor stimulation synergizes with CD40 and IL-17 receptors stimulation in the induced proliferation and potent release of IL-10 cytokine while decreasing IL-6 production in B cells. These novel findings provide evidence that B lymphocytes might be an important source of the anti-inflammatory IL-10 cytokine, and provide novel evidence that stimulation of B lymphocytes with IL-17 cytokine could be an important regulatory mechanism in immune responses.

Background

Asthma is a chronic inflammatory disorder of the lung airways that is associated with airway remodeling and hyperresponsiveness. Its is well documented that the smooth muscle mass in asthmatic airways is increased due to hypertrophy and hyperplasia of the ASM cells. Moreover, eosinophils have been proposed in different studies to play a major role in airway remodeling. Here, we hypothesized that eosinophils modulate the airways through enhancing ASM cell proliferation. The aim of this study is to examine the effect of eosinophils on ASM cell proliferation using eosinophils isolated from asthmatic and normal control subjects.

Methods

Eosinophils were isolated from peripheral blood of 6 mild asthmatics and 6 normal control subjects. ASM cells were incubated with eosinophils or eosinophil membranes and ASM proliferation was estimated using thymidine incorporation. The mRNA expression of extracellular matrix (ECM) in ASM cells was measured using quantitative real-time PCR. The effect of eosinophil-derived proliferative cytokines on ASM cells was determined using neutralizing antibodies. The role of eosinophil derived Cysteinyl Leukotrienes in enhancing ASM was also investigated.

Results

Co-culture with eosinophils significantly increased ASM cell proliferation. However, there was no significant difference in ASM proliferation following incubation with eosinophils from asthmatic versus normal control subjects. Co-culture with eosinophil membranes had no effect on ASM proliferation. Moreover, there was no significant change in the mRNA expression of ECM proteins in ASM cells following co-culture with eosinophils when compared with medium alone. Interestingly, blocking the activity of cysteinyl Leukotries using antagonists inhibited eosinophil-derived ASM proliferation.

Conclusions

Eosinophils enhances the proliferation of ASM cells. This role of eosinophil does not seem to depend on ASM derived ECM proteins nor on Eosinophil derived TGF-β or TNF-α. Eosinophil seems to induce ASM proliferation via the secretion of Cysteinyl Leukotrienes.

Herpes simplex encephalitis (HSE) is the most common sporadic viral encephalitis of childhood. Autosomal recessive (AR) UNC-93B and TLR3 deficiencies and autosomal dominant (AD) TLR3 and TRAF3 deficiencies underlie HSE in some children. We report here unrelated HSE children with AR or AD TRIF deficiency. The AR form of the disease was found to be due to a homozygous nonsense mutation that resulted in a complete absence of the TRIF protein. Both the TLR3- and the TRIF-dependent TLR4 signaling pathways were abolished. The AD form of disease was found to be due to a heterozygous missense mutation, resulting in a dysfunctional protein. In this form of the disease, the TLR3 signaling pathway was impaired, whereas the TRIF-dependent TLR4 pathway was unaffected. Both patients, however, showed reduced capacity to respond to stimulation of the DExD/H-box helicases pathway. To date, the TRIF-deficient patients with HSE described herein have suffered from no other infections. Moreover, as observed in patients with other genetic etiologies of HSE, clinical penetrance was found to be incomplete, as some HSV-1–infected TRIF-deficient relatives have not developed HSE. Our results provide what we believe to be the first description of human TRIF deficiency and a new genetic etiology for HSE. They suggest that the TRIF-dependent TLR4 and DExD/H-box helicase pathways are largely redundant in host defense. They further demonstrate the importance of TRIF for the TLR3-dependent production of antiviral IFNs in the CNS during primary infection with HSV-1 in childhood.

Lysyl-tRNA synthetase (LysRS) is packaged into human immunodeficiency virus type 1 (HIV-1) via its interaction with Gag, and this enzyme facilitates the selective packaging of tRNA3Lys, the primer for initiating reverse transcription, into HIV-1. The Gag/LysRS interaction is detected at detergent-resistant membrane but not in membrane-free cell compartments that contain Gag and LysRS. LysRS is found (i) in the nucleus, (ii) in a cytoplasmic high-molecular-weight aminoacyl-tRNA synthetase complex (HMW aaRS complex), (iii) in mitochondria, and (iv) associated with plasma membrane. The cytoplasmic form of LysRS lacking the mitochondrial import signal was previously shown to be efficiently packaged into virions, and in this report we also show that LysRS compartments in nuclei, in the HMW aaRS complex, and at the membrane are also not required as a primary source for viral LysRS. Exogenous mutant LysRS species unable to either enter the nucleus or bind to the cell membrane are still incorporated into virions. Many HMW aaRS components are not packaged into the virion along with LysRS, and the interaction of LysRS with p38, a protein that binds tightly to LysRS in the HMW aaRS complex, is not required for the incorporation of LysRS into virions. These data indicate that newly synthesized LysRS may interact rapidly with Gag before the enzyme has the opportunity to move to the above-mentioned cellular compartments. In confirmation of this idea, we found that newly synthesized LysRS is associated with Gag after a 10-min pulse with [35S]cysteine/methionine. This observation is also supported by previous work indicating that the incorporation of LysRS into HIV-1 is very sensitive to the inhibition of new synthesis of LysRS.

During human immunodeficiency virus type 1 (HIV-1) assembly in HIV-1-transfected COS7 cells, almost all steady-state Gag/Gag and Gag/GagPol complexes are membrane bound. However, exposure to 1% Triton X-100 gives results indicating that while all Gag/GagPol complexes remain associated with the detergent-resistant membrane (DRM), only 30% of Gag/Gag complexes are associated with the DRM. Analysis of the localization of newly synthesized Gag/Gag and Gag/GagPol to the membrane indicates that after a 10-min pulse with radioactive [35S]Cys-[35S]Met, all newly synthesized Gag/GagPol is found at the DRM. Only 30% of newly synthesized Gag/Gag moves to the membrane, and at 0 min of chase, only 38% of this membrane-bound Gag/Gag is associated with the DRM. During the first 30 min of chase, most membrane-bound Gag/Gag moves to the DRM, while between 30 and 60 min of chase, there is a significant decrease in membrane-bound Gag/Gag and Gag/GagPol. Since the localization of newly synthesized Gag/Gag to the DRM and the interaction of GagPol with Gag both depend upon Gag multimerization, the rapid localization of GagPol to the DRM probably reflects the interaction of all newly synthesized GagPol with the first newly synthesized polymeric Gag to associate with the DRM.

We have examined the influence of RNA upon the interaction of Gag-Pol with Gag during human immunodeficiency virus type 1 (HIV-1) assembly. COS7 cells were transfected with protease-negative HIV-1 proviral DNA, and Gag/Gag-Pol complexes were detected by coimmunoprecipitation with anti-integrase. In COS7 cells, Gag/Gag-Pol is found almost entirely in pelletable, membrane-bound complexes. Exposure of cells to 1% Triton X-100 releases Gag/Gag-Pol from bulk membrane, but the complexes remain pelletable. The role of RNA in facilitating the interaction between Gag and Gag-Pol was examined in these bulk membrane-free, pelletable complexes. The specific presence of viral genomic RNA is not required to maintain the Gag/Gag-Pol interaction, but some type of RNA is, since exposure to RNase destabilized the Gag/Gag-Pol complex. When present only in Gag, the nucleocapsid mutation R7R10K11S, which inhibits Gag binding to RNA, inhibits the formation of both Gag and Gag/Gag-Pol complexes. When present only in Gag-Pol, this mutation has no effect upon complex formation. This result indicates that Gag-Pol may not interact directly with RNA but rather requires RNA-facilitated Gag multimerization for its interaction with Gag.