PlexinD1 Deficiency in Lung Interstitial Macrophages Exacerbates House Dust Mite-Induced Allergic Asthma.

Interstitial macrophages (IMs) are key regulators of allergic inflammation. We previously showed that the absence of semaphorin 3E (Sema3E) exacerbates asthma features in both acute and chronic asthma models. However, it has not been studied whether Sema3E, via its receptor plexinD1, regulates IM function in allergic asthma. Therefore, we investigated the role of plexinD1 deficiency on IMs in allergic asthma. We found that the absence of plexinD1 in IMs increased airway hyperresponsiveness, airway leukocyte numbers, allergen-specific IgE, goblet cell hyperplasia, and Th2/Th17 cytokine response in the house dust mite (HDM)-induced allergic asthma model. Muc5ac, Muc5b, and α-SMA genes were increased in mice with Plxnd1-deficient IMs compared with wild-type mice. Furthermore, plexinD1-deficient bone marrow-derived macrophages displayed reduced IL-10 mRNA expression, at both the baseline and following HDM challenge, compared with their wild-type counterpart mice. Our data suggest that Sema3E/plexinD1 signaling in IMs is a critical pathway that modulates airway inflammation, airway resistance, and tissue remodeling in the HDM murine model of allergic asthma. Reduced IL-10 expression by plexinD1-deficient macrophages may account for these enhanced allergic asthma features.

Semaphorin3E/plexinD1 Axis in Asthma: What We Know So Far!

Semaphorin3E belongs to the large family of semaphorin proteins. Semaphorin3E was initially identified as axon guidance cues in the neural system. It is universally expressed beyond the nervous system and contributes to regulating essential cell functions such as cell migration, proliferation, and adhesion. Binding of semaphorin3E to its receptor, plexinD1, triggers diverse signaling pathways involved in the pathogenesis of various diseases from cancer to autoimmune and allergic disorders. Here, we highlight the novel findings on the role of semaphorin3E in airway biology. In particular, we highlight our recent findings on the function and potential mechanisms by which semaphorin3E and its receptor, plexinD1, impact airway inflammation, airway hyperresponsiveness, and remodeling in the context of asthma.

Semaphorin 3E Inhibits House Dust Mite-Induced Angiogenesis in a Mouse Model of Allergic Asthma.

Increased angiogenesis is a characteristic feature of remodeling in asthmatic airways and stems from the imbalance between pro-angiogenic and anti-angiogenic factors. Surprisingly, the factors regulating this process in allergic asthma are poorly defined. Previously, we showed an important role of semaphorins 3E (Sema3E) in growth factor-induced airway smooth muscle proliferation and migration in vitro, and in down-regulating airway inflammation, T helper 2/T helper 17 cytokine response, mucus cell hyperplasia, and airway hyperresponsiveness in vivo. However, the role of Sema3E in airway angiogenesis is not fully understood. Here, we investigated the role of Sema3E in airway angiogenesis using a house dust mite (HDM) murine model of allergic asthma. Intranasal treatment with recombinant Sema3E significantly reduced the expression of angiogenesis markers within the airways of HDM-challenged mice compared with untreated mice. HDM-induced expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 protein were diminished substantially on Sema3E treatment. Interestingly, Sema3E-treated mice showed an enhanced expression of the negative regulator of angiogenesis, soluble VEGF receptor 1, compared with the untreated mice. These events were reversed in Sema3E-deficient mice at baseline or on HDM challenge. Taken together, this study provides the first evidence that Sema3E modulates angiogenesis in allergic asthmatic airways via modulating pro- and anti-angiogenic factors.

Targeting the Semaphorin3E-plexinD1 complex in allergic asthma.

Asthma is a heterogenous airway disease characterized by airway inflammation and remodeling. It affects more than 300 million people worldwide and poses a significant burden on society. Semaphorins, discovered initially as neural guidance molecules, are ubiquitously expressed in various organs and regulate multiple signaling pathways. Interestingly, Semaphorin3E is a critical molecule in lung pathophysiology through its role in both lung development and homeostasis. Semaphorin3E binds to plexinD1, mediating regulatory effects on cell migration, proliferation, and angiogenesis. Recent in vitro and in vivo studies have demonstrated that the Semaphorin3E-plexinD1 axis is implicated in asthma, impacting inflammatory and structural cells associated with airway inflammation, tissue remodeling, and airway hyperresponsiveness. This review details the Semaphorin3E-plexinD1 axis in various aspects of asthma and highlights future directions in research including its potential role as a therapeutic target in airway allergic diseases.

Thymic stromal lymphopoietin-induced human asthmatic airway epithelial cell proliferation through an IL-13-dependent pathway.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) plays a pivotal role in the initiation of allergic airway inflammation. This cytokine is produced by several cell types, including human epithelial cells. OBJECTIVE: We sought to determine the effect of TSLP on proliferation and repair of epithelial cells isolated from asthmatic patients and healthy subjects. METHODS: Expression of TSLP receptor (TSLPR) and its response to inhaled corticosteroids was evaluated on bronchial biopsy specimens of healthy control subjects and asthmatic patients by means of immunohistochemistry. TSLPR, TSLP, and IL-13 mRNA expression was determined by means of quantitative PCR, and protein expression was measured by means of ELISA and Western blotting in epithelial cells isolated from asthmatic subjects compared with those isolated from healthy control subjects. The effect of TSLP on cell proliferation and wound healing was performed. RESULTS: TSLPR is expressed by bronchial epithelial cells in bronchial biopsy specimens and in cultured cells, with no difference between asthmatic patients and healthy control subjects. Inhaled corticosteroids did not affect this expression. TSLP mRNA and protein levels were significantly higher in epithelial cells isolated from asthmatic patients compared with those from healthy control subjects. TSLP stimulated IL-13 production by bronchial epithelial cells. TSLP induced airway epithelial cell proliferation and enhanced epithelial injury repair. This effect was abrogated with IL-13 neutralization. CONCLUSIONS: Our data indicate that epithelial cells express TSLPR and that TSLP induces bronchial epithelial cell proliferation and increases injury repair through IL-13 production. This suggests that TSLP and IL-13 loops play a homeostatic role on epithelial cell proliferation and repair.

New Insights on the Role of pentraxin-3 in Allergic Asthma.

Pentraxins are soluble pattern recognition receptors that play a major role in regulating innate immune responses. Through their interaction with complement components, Fcγ receptors, and different microbial moieties, Pentraxins cause an amplification of the inflammatory response. Pentraxin-3 is of particular interest since it was identified as a biomarker for several immune-pathological diseases. In allergic asthma, pentraxin-3 is produced by immune and structural cells and is up-regulated by pro-asthmatic cytokines such as TNFα and IL-1ß. Strikingly, some recent experimental evidence demonstrated a protective role of pentraxin-3 in chronic airway inflammatory diseases such as allergic asthma. Indeed, reduced pentraxin-3 levels have been associated with neutrophilic inflammation, Th17 immune response, insensitivity to standard therapeutics and a severe form of the disease. In this review, we will summarize the current knowledge of the role of pentraxin-3 in innate immune response and discuss the protective role of pentraxin-3 in allergic asthma.

PTX3 Deficiency Promotes Enhanced Accumulation and Function of CD11c+CD11b+ DCs in a Murine Model of Allergic Inflammation.

PTX3 is a unique member of the long pentraxins family and plays an indispensable role in regulating the immune system. We previously showed that PTX3 deletion aggravates allergic inflammation via a Th17 -dominant phenotype and enhanced CD4 T cell survival using a murine model of ovalbumin (OVA) induced allergic inflammation. In this study, we identified that upon OVA exposure, increased infiltration of CD11c+CD11b+ dendritic cells (DCs) was observed in the lungs of PTX3-/- mice compared to wild type littermate. Further analysis showed that a short-term OVA exposure led to an increased number of bone marrow common myeloid progenitors (CMP) population concomitantly with increased Ly6Chigh CCR2high monocytes and CD11c+CD11b+ DCs in the lungs. Also, pulmonary CD11c+CD11b+ DCs from OVA-exposed PTX3-/- mice exhibited enhanced expression of maturation markers, chemokines receptors CCR2, and increased OVA uptake and processing compared to wild type controls. Taken together, our data suggest that PTX3 deficiency heightened lung CD11c+CD11b+DC numbers and function, hence exacerbating airway inflammatory response.

Semaphorin 3E deficiency dysregulates dendritic cell functions: In vitro and in vivo evidence.

Regulation of dendritic cell functions is a complex process in which several mediators play diverse roles as a network in a context-dependent manner. The precise mechanisms underlying dendritic cell functions have remained to be addressed. Semaphorins play crucial roles in regulation of various cell functions. We previously revealed that Semaphorin 3E (Sema3E) contributes to regulation of allergen-induced airway pathology partly mediated by controlling recruitment of conventional dendritic cell subsets in vivo, though the underlying mechanism remained elusive. In this study, we investigate the potential regulatory role of Sema3E in dendritic cells. We demonstrated that bone marrow-derived dendritic cells differentiated from Sema3e-/- progenitors have an enhanced migration capacity both at the baseline and in response to CCL21. The enhanced migration ability of Sema3E dendritic cells was associated with an overexpression of the chemokine receptor (CCR7), elevated Rac1 GTPase activity and F-actin polymerization. Using a mouse model of allergic airway sensitization, we observed that genetic deletion of Sema3E leads to a time dependent upregulation of CCR7 on CD11b+ conventional dendritic cells in the lungs and mediastinal lymph nodes. Furthermore, aeroallergen sensitization of Sema3e-/- mice lead to an enhanced expression of PD-L2 and IRF-4 as well as enhanced allergen uptake in pulmonary CD11b+ DC, compared to wild type littermates. Collectively, these data suggest that Sema3E implicates in regulation of dendritic cell functions which could be considered a basis for novel immunotherapeutic strategies for the diseases associated with defective dendritic cells in the future.

The regulatory role of semaphorin 3E in allergic asthma.

Semaphorins were originally discovered as essential mediators involved in regulation of axonal growth during development of the nervous system. Ubiquitously expressed on various organs, they control several cellular functions by regulating essential signaling pathways. Among them, semaphorin3E binds plexinD1 as the primary receptor and mediates regulatory effects on cell migration, proliferation, and angiogenesis considered major physiological and pathological features in health and disease. Recent in vitro and in vivo experimental evidence demonstrate a key regulator role of semaphorin3E on airway inflammation, hyperresponsivenss and remodeling in allergic asthma. Herein, we aim to provide a broad overview of the biology of semaphorin family and review the recently discovered regulatory role of semaphorin3E in modulating immune cells and structural cells function in the airways. These findings support the concept of semaphorin3E/plexinD1 axis as a therapeutic target in allergic asthma.

Glucocorticoids regulate pentraxin-3 expression in human airway smooth muscle cells.

Pentraxin-3 (PTX3) is a multifunctional protein involved in both innate and adaptive immunity. Glucocorticoid (GC) is the first-line therapy to mitigate airway inflammation in asthma. Previous pieces of evidence showed that GC has divergent effects on PTX3 production in various cell types. The molecular mechanisms controlling PTX3 expression in HASMC are, however, not yet characterized. In this study, we demonstrate that the synthetic GC, dexamethasone (DEX) increases the expression of PTX3 both at the protein and mRNA levels. We also found that such an effect of DEX was dependent on de novo protein synthesis and the GC receptor (GR). While DEX increases PTX3 mRNA stability, it did not affect its promoter activity. Interestingly, HASMC pre-treated with p42/p44 ERK inhibitor, but not with p38 or JNK-MAPK inhibitors, significantly interfered with DEX-induced PTX3 secretion. Taken together, our data suggest that GC regulates PTX3 expression in HASMC through transcriptional and post-transcriptional mechanisms in a GR and ERK-dependent manner.

Downregulation of semaphorin 3E promotes hallmarks of experimental chronic allergic asthma.

Guidance cues such as semaphorins are attractive novel therapeutic targets for allergic disorders. We have previously described an inhibitory effect of semaphorin 3E (Sema3E) on human airway smooth muscle cell function. We have further addressed a canonical role for Sema3E in acute model of allergic asthma in vivo. Considering the chronic nature of the disease, the potential implication of Sema3E to alleviate long-lasting deficits should be investigated. Expression of Sema3E in a chronic model of allergic asthma was assessed after exposure to house dust mite (HDM) as a clinically relevant allergen. Chronic features of allergic asthma including airway hyper-responsiveness (AHR), inflammation, and remodeling were studied in Sema3E-deficient mice. Additionally, the effect of exogenous Sema3E treatment was evaluated in prophylactic and therapeutic experimental models. We have demonstrated that expression of Sema3E is robustly suppressed in the airways upon chronic HDM exposure. Chronic allergic airway disease was significantly augmented in Sema3E-deficient mouse model which was associated with an increased AHR, remodeling, and Th2/Th17 inflammation. Intranasal Sema3E administration restored chronic deficits of allergic asthma in mice. Data from this study unveil a key regulatory role of Sema3E in chronic course of asthma via orchestration of impaired inflammatory and remodeling responses.

Human airway smooth muscle cell proliferation from asthmatics is negatively regulated by semaphorin3A.

Airway smooth muscle (ASM) hyperplasia is a key feature of airway remodeling in development of lung diseases such as asthma. Anomalous proliferation of ASM cells directly contributes to ASM hyperplasia. However, the molecular mechanisms controlling ASM cell proliferation are not completely understood. Semaphorins are versatile regulators of various cellular processes including cell growth and proliferation. The role of semaphorins in ASM cell proliferation has remained to be addressed. Here, we report that semaphorin 3A (Sema3A) receptor, neuropilin 1 (Nrp1), is expressed on human ASM cells (HASMC) isolated from healthy and asthmatic donors and treatment of these cells with exogenous Sema3A inhibits growth factor-induced proliferation. Sema3A inhibitory effect on HASMC proliferation is associated with decreased tyrosine phosphorylation of PDGFR, downregulation of Rac1 activation, STAT3 and GSK-3ß phosphorylation. Bronchial sections from severe asthmatics displayed immunoreactivity of Nrp1, suggestive of functional contribution of Sema3A-Nrp1 axis in airway remodeling. Together, our data suggest Sema3A-Nrp1 signaling as a novel regulatory pathway of ASM hyperplasia.

TNF up-regulates Pentraxin3 expression in human airway smooth muscle cells via JNK and ERK1/2 MAPK pathways.

BACKGROUND: Long pentraxin 3 (PTX3) is a novel candidate marker for inflammation in many chronic diseases. As a soluble pattern recognition receptor, PTX3 is involved in amplification of inflammatory reactions and regulation of innate immunity. Previously, we demonstrate that human airway smooth muscle cells (HASMC) express constitutively PTX3 and upon TNF stimulation. However, very little is known about the mechanism governing its expression in HASMC. We sought to investigate the mechanism governing TNF induced PTX3 expression in primary HASMC. METHODS: HASMC were stimulated with TNF in the presence of transcriptional inhibitor actinomycin D (ActD) or MAPKs pharmacological inhibitors. PTX3 mRNA and protein expression were analyzed by Real-time RT-PCR and ELISA, respectively. PTX3 promoter activity was determined using luciferase assay. RESULTS: PTX3 mRNA and protein are expressed constitutively by HASMC and significantly up-regulated by TNF. TNF-induced PTX3 mRNA and protein release in HASMC were inhibited by transcriptional inhibitor actinomycin D. TNF induced significantly PTX3 promoter activation in HASMC. MAPK JNK and ERK1/2 specific inhibitors (SP600125 and UO126), but not p38, significantly down regulates TNF induced PTX3 promoter activity and protein release in HASMC. Finally, TNF mediated PTX3 promoter activity in HASMC was abolished upon mutation of NF-κß and AP1 binding sites. CONCLUSIONS: Our data suggest that TNF induced PTX3 in HASMC at least via a transcriptional mechanism that involved MAPK (JNK and ERK1/2), NF-κß and AP1 pathways. These results rise the possibility that HASMC derived PTX3 may participate in immune regulation in the airways.

Pentraxin 3: an immuno-regulator in the lungs.

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor that is a humoral component of the innate immune system. It interacts with pathogenic moieties, infected and dying host cells and facilitates their removal through activation of appropriate innate and adaptive mechanisms. PTX3 is secreted by a diverse variety of cells, ranging from immune cells to structural cells, in response to Toll like receptor (TLR) engagement, inflammatory stimuli, and physical and chemical stress. Further, PTX3 plays an essential role in female fertility as it facilitates the organization of extracellular matrix in the cumulus oophorus. Such activity is also implicated in post-inflammation tissue repair. PTX3 is a multifunctional protein and plays a non-redundant role in providing immunity against potential immunological dangers. Thus, we assessed its role in lung immunity, as lungs are at a constant risk of infections and tissue damage that is attributable to perpetual exposure to foreign agents.

Pentraxin 3 (PTX3) expression in allergic asthmatic airways: role in airway smooth muscle migration and chemokine production.

BACKGROUND: 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. OBJECTIVES AND METHODS: 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. RESULTS: 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-1ß 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. CONCLUSIONS: 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.

IL-9 induces CCL11 expression via STAT3 signalling in human airway smooth muscle cells.

BACKGROUND: Previous findings support the concept that IL-9 may play a significant role in mediating both pro-inflammatory and changes in airway responsiveness that characterizes the atopic asthmatic state. We previously demonstrated that human airway smooth muscle (ASM) cells express a functional IL-9R that mediate CCL11 expression. However, the signaling pathway governing this effect is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we showed that IL-9 mediated CCL11 expression in ASM cells does not rely on STAT6 or STAT5 but on STAT3 pathway. IL-9 induced rapid STAT3 activation in primary ASM cells that was not observed in case of STAT6 or STAT5. STAT3 binding to CCL11 promoter was also observed in vivo upon IL-9 stimulation of ASM cells. Disruption of STAT3 activity with SH2 domain binding inhibitory peptide results in significant reduction of IL-9 mediated CCL11 promoter activity. DN STAT3beta over-expression in ASM cells, but not Ser 727 STAT3 or STAT6 DN, abolishes IL-9 mediated CCL11 promoter activity. Finally, STAT3 but not STAT6 silenced ASM cells showed significant reduction in IL-9 mediated CCL11 promoter activity and mRNA expression. CONCLUSION/SIGNIFICANCE: Taken together, our results indicate that IL-9 mediated CCL11 via STAT3 signalling pathway may play a crucial role in airway inflammatory responses.