Thymic Stromal Lymphopoietin Induces Migration in Human Airway Smooth Muscle Cells

Airway remodeling due to increased airway smooth muscle (ASM) mass, likely due to enhanced migration and proliferation, has been shown to be highly associated with decline in lung function in asthma. Thymic stromal lymphopoietin (TSLP) is an IL-7-like, pro-allergic cytokine that has been shown to be necessary and sufficient for the development of allergic asthma. Human ASM (HASM) cells express TSLP receptor (TSLPR), the activation of which leads to enhanced release of proinflammatory mediators such as IL-6, CCL11/eotaxin-1, and CXCL8/IL-8. We show here that TSLP induces HASM cell migration through STAT3 activation since lentiviral-shRNA inhibition of STAT3 abrogated the TSLP-induced cell migration. Moreover, TSLP induced multiple cytoskeleton changes in HASM cells such as actin polymerization, cell polarization, and activation of small GTPase Rac1. Collectively, our data suggest a pro-migratory function of TSLP in ASM remodeling and provides better rationale for targeting TSLP/TSLPR pathway for therapeutic approaches in allergic asthma

IgE induces proliferation in human airway smooth muscle cells: role of MAPK and STAT3 pathways

Airway remodeling is not specifically targeted by current asthma medications, partly owing to the lack of understanding of remodeling mechanisms, altogether posing great challenges in asthma treatment. Increased airway smooth muscle (ASM) mass due to hyperplasia/hypertrophy contributes significantly to overall airway remodeling and correlates with decline in lung function. Recent evidence suggests that IgE sensitization can enhance the survival and mediator release in inflammatory cells. Human ASM (HASM) cells express both low affinity (FcεRII/CD23) and high affinity IgE Fc receptors (FcεRI), and IgE can modulate the contractile and synthetic function of HASM cells. IgE was recently shown to induce HASM cell proliferation but the detailed mechanisms remain unknown. We report here that IgE sensitization induces HASM cell proliferation, as measured by 3H-thymidine, EdU incorporation, and manual cell counting. As an upstream signature component of FcεRI signaling, inhibition of spleen tyrosine kinase (Syk) abrogated the IgE-induced HASM proliferation. Further analysis of IgE-induced signaling depicted an IgE-mediated activation of Erk 1/2, p38, JNK MAPK, and Akt kinases. Lastly, lentiviral-shRNA-mediated STAT3 silencing completely abolished the IgE-mediated HASM cell proliferation. Collectively, our data provide mechanisms of a novel function of IgE which may contribute, at least in part, to airway remodeling observed in allergic asthma by directly inducing HASM cell proliferation

Proinflammatory and Th2 Cytokines Regulate the High Affinity IgE Receptor (FcεRI) and IgE-Dependant Activation of Human Airway Smooth Muscle Cells

BACKGROUND:The high affinity IgE receptor (FcepsilonRI) is a crucial structure for IgE-mediated allergic reactions. We have previously demonstrated that human airway smooth muscle (ASM) cells express the tetrameric (alphabetagamma2) FcepsilonRI, and its activation leads to marked transient increases in intracellular Ca(2+) concentration, release of Th-2 cytokines and eotaxin-1/CCL11. Therefore, it was of utmost importance to delineate the factors regulating the expression of FcepsilonRI in human (ASM) cells. METHODOLOGY/PRINCIPAL FINDINGS:Incubation of human bronchial and tracheal smooth muscle (B/TSM) cells with TNF-alpha, IL-1beta or IL-4 resulted in a significant increase in FcepsilonRI-alpha chain mRNA expression (p<0.05); and TNF-alpha, IL-4 enhanced the FcepsilonRI-alpha protein expression compared to the unstimulated control at 24, 72 hrs after stimulation. Interestingly, among all other cytokines, only TNF-alpha upregulated the FcepsilonRI-gamma mRNA expression. FcepsilonRI-gamma protein expression remained unchanged despite the nature of stimulation. Of note, as a functional consequence of FcepsilonRI upregulation, TNF-alpha pre-sensitization of B/TSM potentially augmented the CC (eotaxin-1/CCL11 and RANTES/CCL5, but not TARC/CCL17) and CXC (IL-8/CXCL8, IP-10/CXCL10) chemokines release following IgE stimulation (p<0.05, n = 3). Furthermore, IgE sensitization of B/TSM cells significantly enhanced the transcription of selective CC and CXC chemokines at promoter level compared to control, which was abolished by Lentivirus-mediated silencing of Syk expression. CONCLUSIONS/SIGNIFICANCE:Our data depict a critical role of B/TSM in allergic airway inflammation via potentially novel mechanisms involving proinflammatory, Th2 cytokines and IgE/FcepsilonRI complex

Pentraxin 3 (PTX3) Expression in Allergic Asthmatic Airways: Role in Airway Smooth Muscle Migration and Chemokine Production

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 is produced by immune and structural cells. However, very little is known about the expression of PTX3 and its role in allergic asthma.We sought to determine the PTX3 expression in asthmatic airways and its function in human airway smooth muscle cells (HASMC). In vivo PTX3 expression in bronchial biopsies of mild, moderate and severe asthmatics was analyzed by immunohistochemistry. PTX3 mRNA and protein were measured by real-time RT-PCR and ELISA, respectively. Proliferation and migration were examined using (3)H-thymidine incorporation, cell count and Boyden chamber assays.PTX3 immunoreactivity was increased in bronchial tissues of allergic asthmatics compared to healthy controls, and mainly localized in the smooth muscle bundle. PTX3 protein was expressed constitutively by HASMC and was significantly up-regulated by TNF, and IL-1β but not by Th2 (IL-4, IL-9, IL-13), Th1 (IFN-γ), or Th-17 (IL-17) cytokines. In vitro, HASMC released significantly higher levels of PTX3 at the baseline and upon TNF stimulation compared to airway epithelial cells (EC). Moreover, PTX3 induced CCL11/eotaxin-1 release whilst inhibited the fibroblast growth factor-2 (FGF-2)-driven HASMC chemotactic activity.Our data provide the first evidence that PTX3 expression is increased in asthmatic airways. HASMC can both produce and respond to PTX3. PTX3 is a potent inhibitor of HASMC migration induced by FGF-2 and can upregulate CCL11/eotaxin-1 release. These results raise the possibility that PTX3 may play a dual role in allergic asthma

Use of Immunoglobulin G Avidity To Determine the Course of Disease in Visceral and Post-Kala-Azar Dermal Leishmaniasis Patients

In the present study, anti-Leishmania immunoglobulin G (IgG) avidity was used to estimate the approximate time of disease manifestation. Significant differences (P < 0.0001) were found between the levels of anti-rKE-16 IgG avidity in leishmaniasis patients with recent and chronic diseases. More than 76% of patients with an illness duration of less than 6 months had avidity of less than 70%, 94% of patients had less than 80% avidity, and all (100%) patients with illness of more than 6 months had avidity values higher than 70%. The study showed that avidity could successfully be used to pinpoint the duration of leishmaniasis

Kinesin Motor Domain of Leishmania donovani as a Future Vaccine Candidate▿

Visceral leishmaniasis (VL) is one of the important parasitic diseases, with approximately 350 million people at risk. Due to the nonavailability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. The present study was carried out to examine the immunological potential of kinesin protein from the microtubule locus of Leishmania donovani as a suitable vaccine candidate. In silico analysis of this region revealed clusters of major histocompatibility complex class I and II binding epitopes in its motor domain region. A recombinant protein was expressed from this region and named rLvacc. The antigenicity and immunogenicity studies of this protein by Western blot analysis revealed that rLvacc is strongly recognized by sera from acute VL patients. To evaluate its immunogenicity, peripheral blood mononuclear cells from cured VL patients were separated, and a lymphocyte proliferation assay was carried out in the presence of rLvacc. After lymphocyte proliferation, the pooled culture supernatant was assayed for anti-rLvacc antibody titers using an enzyme-linked immunosorbent assay. The results showed that immunoglobulin G2 (IgG2) subtype antibodies were predominant, while IgG1 subtype antibodies were produced in very low titers. On the basis of these ex vivo preliminary findings, its immunogenicity was studied in BALB/c mice. Vaccination with the DNA construct generated a good cellular immune response with significant increases in gamma interferon and interleukin-2 (IL-2) cytokine levels (Th1), but no increase in IL-4 levels (Th2). Taken together, our findings suggest the kinesin motor domain region of L. donovani as a potential vaccine candidate against visceral leishmaniasis

IgE Regulates the Expression of smMLCK in Human Airway Smooth Muscle Cells

<div><p>Previous studies have shown that enhanced accumulation of contractile proteins such as smooth muscle myosin light chain kinase (smMLCK) plays a major role in human airway smooth muscle cells (HASM) cell hypercontractility and hypertrophy. Furthermore, serum IgE levels play an important role in smooth muscle hyperreactivity. However, the effect of IgE on smMLCK expression has not been investigated. In this study, we demonstrate that IgE increases the expression of smMLCK at mRNA and protein levels. This effect was inhibited significantly with neutralizing abs directed against FcεRI but not with anti-FcεRII/CD23. Furthermore, Syk knock down and pharmacological inhibition of mitogen activated protein kinases (MAPK) (ERK1/2, p38, and JNK) and phosphatidylinositol 3-kinase (PI3K) significantly diminished the IgE-mediated upregulation of smMLCK expression in HASM cells. Taken together, our data suggest a role of IgE in regulating smMLCK in HASM cells. Therefore, targeting the FcεRI activation on HASM cells may offer a novel approach in controlling the bronchomotor tone in allergic asthma.</p></div

IgE enhances smMLCK expression in HASM cells through FcεRI.

<p>(<b>A</b>) HASM cells were serum-deprived for 48 h and then stimulated with IgE 5 μg/ml. Shown is fold increase in smMLCK mRNA level in IgE-stimulated HASM cells compared to unstimulated cells at corresponding time points. (<b>B</b>) HASM cells were stimulated with IgE 5 μg/ml for 48 and smMLCK protein expression was assessed by western blotting. The intensity of smMLCK band was normalized with that of β-actin. The shown blot is representative of three different experiments. (<b>C</b>) Cells were pretreated with anti-FcεRI mAb15/1 for 1 h before stimulation with IgE. MOPC21 was used as an isotype control. Western blot is a representative of three different experiments showing smMLCK protein content in different treatment groups. Fold change in the level of smMLCK upon different treatment as compared to untreated control is shown in the graphs. One way ANOVA was performed to determine the significance of data. <i>P</i><0.05 (*), (n>3).</p

IgE-mediated smMLCK protein expression is not affected by Lyn knock-down.

<p>Lyn knock-down was induced in HASM cells by transduction with lentiviral vector expressing Lyn specific shRNA. shRNA against unrelated scramble sequence was used as control. (<b>A</b>) Lentiviral transduction efficiency was found to be more than 90% for both scramble and Lyn specific shRNA as determined by measuring GFP content. Lentivirus-induced Lyn knock-down in HASM cells was shown by western blotting. (<b>B</b>) Protein extracts were prepared from Lyn silenced and scramble HASM cells, stimulated with IgE 5 μg/ml for 48 h after serum deprivation and smMLCK protein content was assessed by western blotting (n>3). One way ANOVA was performed to determine the significance of data. <i>P</i><0.05 (*).</p