Cutting Edge: Proinflammatory and Th2 cytokines synergize to induce thymic stromal lymphopoietin production by human skin keratinocytes

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that strongly activates dendritic cells (DC) and can initiate allergic inflammation. The factors inducing the production of human TSLP are not known. In this study, we show that proinflammatory (TNF-alpha or IL-1alpha) and Th2 (IL-4 or IL-13) cytokines synergized to induce the production of TSLP in human skin explants. TSLP production in situ was restricted to epidermal keratinocytes of the suprabasal layer. TSLP production could not be inhibited by factors regulating Th2 inflammation, such as IL-10, TGF-beta, or IFN-gamma. Cytokine-treated skin culture supernatants induced the maturation of blood CD11c(+) DC in a TSLP-dependent manner. Our data provide the first evidence of TSLP induction and subsequent DC activation in human skin. Blocking TSLP-inducing cytokines could represent a novel strategy for the treatment of allergic diseases.     

Activation of TRPV1 mediates thymic stromal lymphopoietin release via the Ca/NFAT pathway in airway epithelial cells

The airway epithelium is exposed to a range of irritants in the environment that are known to trigger inflammatory response such as asthma. Transient receptor potential vanilloid 1 (TRPV1) is a Ca²⁺-permeable cation channel critical for detecting noxious stimuli by sensory neurons. Recently increasing evidence suggests TRPV1 is also crucially involved in the pathophysiology of asthma on airway epithelium in human. Here we report that in airway epithelial cells TRPV1 activation potently induces allergic cytokine thymic stromal lymphopoietin (TSLP) release. TSLP induction by protease-activated receptor (PAR)-2 activation is also partially mediated by TRPV1 channels.     

Preparation of recombinant human thymic stromal lymphopoietin protein

Human thymic stromal lymphopoietin (hTSLP) protein plays a central role in inflammation. Characterizing properties of hTSLP requires a recombinant overexpression system that produces correctly folded, active hTSLP. In this report, an efficient overexpression system for the production of hTSLP was developed. We constructed expression plasmids of the full-length hTslp gene with or without the signal peptide and transformed the plasmids into Escherichia coli. The design of the recombinant proteins included an N-terminal His-tag, which facilitated purification. An affinity gradient elution method was used to improve recovery and concentration levels of denatured hTSLP, with 90% purity observed following affinity chromatography. Refolding of the denatured hTSLP was tested using four different protein refolding approaches. The optimal refolding conditions involved stepwise buffer exchanges to reduce the urea concentration from 4 to 0?M in 50?mM Tris (pH 8.0), 1?mM EDTA, 50?mM NaCl, 10% glycerol, 400?mM L-Arg, 0.2?mM oxidized glutathione, and 2?mM reduced glutathione. The activity of the refolded recombinant hTSLP protein was measured by an ELISA assay. Interestingly, the presence of N-terminal signal peptide inhibited the overexpression of hTSLP in E. coli. The amount of recombinant hTSLP protein purified reached a level of 2.52?×?10^?3?mg/L.     

Constitutive and inducible thymic stromal lymphopoietin expression in human airway smooth muscle cells: role in chronic obstructive pulmonary disease

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that triggers dendritic cell-mediated T helper (Th)-2 inflammatory responses. Previous studies have demonstrated that human airway smooth muscle cells (HASMC) play a critical role in initiating or perpetuating airway inflammation by producing chemokines and cytokines. In this study, we first evaluated the expression of TSLP in primary HASMC and investigated how proinflammatory cytokines (TNF-alpha and IL-1beta) and Th-2 cytokines (IL-4, IL-9) regulate TSLP production from HASMC. TSLP mRNA and protein were assessed by real-time RT-PCR, ELISA, and immunofluorescence from primary HASMC cultures. Primary HASMC express constitutive level of TSLP. Incubation of HASMC with IL-1 or TNF-alpha resulted in a significant increase of TSLP mRNA and protein release from HASMC. Furthermore, combination of IL-1beta and TNF-alpha has an additive effect on TSLP release by HASMC. Primary HASMC pretreated with inhibitors of p38 or p42/p44 ERK MAPK, but not phosphatidylinositol 3-kinase, showed a significant decrease in TSLP release on IL-1beta and TNF-alpha treatment. Furthermore, TSLP immunoreactivity was present in ASM bundle from chronic obstructive pulmonary disease (COPD) and to lesser degree in normal subjects. Taken together, our data provide the first evidence of IL-1beta- and TNF-alpha-induced TSLP expression in HASMC via (p38, p42/p44) MAPK signaling pathways. Our results raise the possibility that HASMC may play a role in COPD airway inflammation via TSLP-dependent pathway.     

Diesel exhaust particle-treated human bronchial epithelial cells upregulate Jagged-1 and OX40 ligand in myeloid dendritic cells via thymic stromal lymphopoietin

Ambient particulate matter, including diesel exhaust particles (DEP), promotes the development of allergic disorders. DEP increase oxidative stress and influence human bronchial epithelial cell (HBEC)-dendritic cell interactions via cytokines, including thymic stromal lymphopoietin (TSLP). Upregulation of TSLP results in Th2 responses. Using primary culture HBEC and human myeloid dendritic cell (mDC) cocultures, we show in this study that DEP upregulation of Th2 responses occurred via HBEC-dependent mechanisms that resulted from oxidative stress. Moreover, DEP-treated HBEC and ambient particulate matter-treated HBEC upregulated OX40 ligand (OX40L) and the Notch ligand Jagged-1 mRNA and expression on mDC. Upregulation of OX40L as well as Jagged-1 on mDC required HBEC and did not occur in the presence of N-acetylcysteine. Furthermore, OX40L and Jagged-1 upregulation was inhibited when HBEC expression of TSLP was silenced. Thus, DEP treatment of HBEC targeted two distinct pathways in mDC that were downstream of TSLP expression. Upregulation of OX40L and Jagged-1 by mDC resulted in mDC-driven Th2 responses. These studies expand our understanding of the mechanism by which ambient pollutants alter mucosal immunity and promote disorders such as asthma.     

Th2 cytokines down-regulate TLR expression and function in human intestinal epithelial cells

TLRs serve important immune and nonimmune functions in human intestinal epithelial cells (IECs). Proinflammatory Th1 cytokines have been shown to promote TLR expression and function in IECs, but the effect of key Th2 cytokines (IL-4, IL-5, IL-13) on TLR signaling in IECs has not been elucidated so far. We stimulated human model IECs with Th2 cytokines and examined TLR mRNA and protein expression by Northern blotting, RT-PCR, real-time RT-PCR, Western blotting, and flow cytometry. TLR function was determined by I-kappaBalpha phosphorylation assays, ELISA for IL-8 secretion after stimulation with TLR ligands and flow cytometry for LPS uptake. IL-4 and IL-13 significantly decreased TLR3 and TLR4 mRNA and protein expression including the requisite TLR4 coreceptor MD-2. TLR4/MD-2-mediated LPS uptake and TLR ligand-induced I-kappaBalpha phosphorylation and IL-8 secretion were significantly diminished in Th2 cytokine-primed IECs. The down-regulatory effect of Th2 cytokines on TLR expression and function in IECs also counteracted enhanced TLR signaling induced by stimulation with the hallmark Th1 cytokine IFN-gamma. In summary, Th2 cytokines appear to dampen TLR expression and function in resting and Th1 cytokine-primed human IECs. Diminished TLR function in IECs under the influence of Th2 cytokines may protect the host from excessive TLR signaling, but likely also impairs the host intestinal innate immune defense and increases IEC susceptibility to chronic inflammation in response to the intestinal microenvironment. Taken together, our data underscore the important role of Th2 cytokines in balancing TLR signaling in human IECs.     

Intradermal administration of thymic stromal lymphopoietin induces a T cell- and eosinophil-dependent systemic Th2 inflammatory response

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) is sufficient to induce asthma or atopic dermatitis-like phenotypes when selectively overexpressed in transgenic mice, or when driven by topical application of vitamin D3 or low-calcemic analogues. Although T and B cells have been reported to be dispensable for the TSLP-induced inflammation in these models, little is known about the downstream pathways or additional cell types involved in the inflammatory response driven by TSLP. To characterize the downstream effects of TSLP in vivo, we examined the effects of exogenous administration of TSLP protein to wild-type and genetically deficient mice. TSLP induced a systemic Th2 inflammatory response characterized by increased circulating IgE and IgG1 as well as increased draining lymph node size and cellularity, Th2 cytokine production in draining lymph node cultures, inflammatory cell infiltrates, epithelial hyperplasia, subcuticular fibrosis, and up-regulated Th2 cytokine and chemokine messages in the skin. Responses to TSLP in various genetically deficient mice demonstrated T cells and eosinophils were required, whereas mast cells and TNF-alpha were dispensable. TSLP-induced responses were significantly, but not completely reduced in IL-4- and IL-13-deficient mice. These results shed light on the pathways and cell types involved in TSLP-induced inflammation.     

Thymic stromal lymphopoietin enhances tight-junction barrier function of human nasal epithelial cells

Epithelial-derived thymic stromal lymphopoietin (TSLP) triggers dendritic cell (DC)-mediated Th2-type inflammatory responses and is a master switch for allergic inflammatory diseases. In the present study, the expression and induction of TSLP and the effects of TSLP on the tight-junctional barrier of human nasal epithelial cells (HNECs) have been investigated in order to elucidate the role of TSLP in allergic rhinitis. We have found high expression of TSLP in the epithelium from patients with allergic rhinitis with recruitment and infiltration of DCs. In vitro, TSLP is significantly produced in HNECs after treatment with a toll-like receptor 2 (TLR2) ligand, Pam₃Cys-Ser-(Lys)₄, and a mixture of interleukin-1β and tumor necrosis factor-α. Treatment with TSLP rapidly enhances the barrier function of cultured HNECs, together with an increase of tight-junction proteins claudin-1, -4, -7, and occludin. The nasal-epithelial-derived TSLP thus not only activates DCs but also preserves the epithelial barrier via the upregulation of tight-junction proteins, thereby regulating antigen sensitization during the early stage of allergic rhinitis.     

Reversal of thymic stromal lymphopoietin-induced airway inflammation through inhibition of Th2 responses

Lung-specific thymic stromal lymphopoietin (TSLP) expression is sufficient for the development of an asthma-like chronic airway inflammatory disease. However, the nature of the downstream pathways that regulate disease development are not known. In this study, we used IL-4- and Stat6-deficient mice to establish the role of Th2-type responses downstream of TSLP. IL-4 deficiency greatly reduced, but did not eliminate, TSLP-induced airway hyperresponsiveness, airway inflammation, eosinophilia, and goblet cell metaplasia, while Stat6 deficiency eliminated these asthma-like symptoms. We further demonstrate, using the chronic model of TSLP-mediated airway inflammation, that blockade of both IL-4 and IL-13 responses, through administration of an anti-IL-4R alpha mAb, reversed asthma-like symptoms, when given to mice with established disease. Collectively these data provide insight into the pathways engaged in TSLP-driven airway inflammation and demonstrate that simultaneous blockade of IL-4 and IL-13 can reverse established airway disease, suggesting that this may be an effective approach for the therapy of Th2-mediated inflammatory respiratory disease.     

Cutting edge: direct action of thymic stromal lymphopoietin on activated human CD4+ T cells

Thymic stromal lymphopoietin (TSLP) is a cytokine that promotes CD4(+) T cell homeostasis and contributes to allergic and inflammatory responses. TSLP can act directly on mouse CD4(+) T cells, but in humans, the available data have indicated that TSLP receptors are not expressed on CD4(+) T cells and that TSLP instead activates dendritic cells, which in turn promote the proliferation and differentiation of CD4(+) T cells. We now unexpectedly demonstrate the presence of TSLP receptors on activated human CD4(+) T cells. Strikingly, whereas freshly isolated peripheral blood human T cells show little if any response to TSLP, TCR stimulation allows a potent response to this cytokine. Moreover, TSLP increases the sensitivity of human CD4(+) T cells to low doses of IL-2, augmenting responsiveness of these cells to TCR engagement. Our results establish that human CD4(+) T cells are direct targets for TSLP.     

布地奈德对A549细胞胸腺基质淋巴细胞生成素表达作用的研究

目的:观察细胞因子刺激气道上皮细胞胸腺基质淋巴细胞生成素(TSLP)表达是否涉及核因子κB(NF-κB),并探讨糖皮质激素布地奈德对气道上皮细胞TSLP表达和NF-κB核转位的影响.方法:A549细胞与细胞因子白介素1β(IL-1β)、白介素4(IL-4)和布地奈德共同孵育,以不加任何细胞因子或布地奈德培养的A549细胞为对照组,采用RT-PCR方法测定TSLP mRNA表达,细胞免疫荧光方法检测TSLP和NF-κB的表达情况.结果:与对照组比较,IL-1β(10 ng/ml)及IL-4(10 ng/ml)显著刺激A549细胞TSLP mRNA表达,且NF-κB(p65)核转住增加(均P＜0.05).布地奈德干预后TSLP mRNA的表达和NF-κB(p65)的核转位显著减少(P＜0.05).结论:细胞因子促进气道上皮细胞诱导性表达TSLP与NF-κB激活有关,抑制TSLP表达和NF-κB激活可能是布地奈德治疗哮喘的重要机制.     

Human thymic epithelial cells inhibit IL-15- and IL-2-driven differentiation of NK cells from the early human thymic progenitors

T/NK progenitors are present in the thymus; however, the thymus predominantly promotes T cell development. In this study, we demonstrated that human thymic epithelial cells (TEC) inhibit NK cell development. Most ex vivo human thymocytes express CD1a, indicating that thymic progenitors are predominantly committed to the T cell lineage. In contrast, the CD1a(-)CD3(-)CD56(+) NK population comprises only 0.2% (n = 7) of thymocytes. However, we observed increases in the percentage (20- to 25-fold) and absolute number (13- to 71-fold) of NK cells when thymocytes were cultured with mixtures of either IL-2, IL-7, and stem cell factor or IL-15, IL-7, and stem cell factor. TEC, when present in the cultures, inhibited the increases in the percentage (3- to 10-fold) and absolute number (3- to 25-fold) of NK cells. Furthermore, we show that TEC-derived soluble factors inhibit generation of NK-CFU and inhibit IL15- or IL2-driven NK cell differentiation from thymic CD34(+) triple-negative thymocytes. The inhibitory activity was found to be associated with a 8,000- to 30,000 Da fraction. Thus, our data demonstrate that TEC inhibit NK cell development from T/NK CD34(+) triple negative progenitors via soluble factor(s), suggesting that the human thymic microenvironment not only actively promotes T cell maturation but also controls the development of non-T lineage cells such as the NK lineage.     

Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.     

TLR3 activation evokes IL-6 secretion, autocrine regulation of Stat3 signaling and TLR2 expression in human bronchial epithelial cells

Human bronchial epithelial cells exposed to synthetic double-stranded RNA (poly I:C) exhibited increased IL-6 and RANTES secretion and TLR2 expression that was inhibited following TLR3 silencing. Increased NF-κB and Stat3 phosphorylation were detected after poly I:C exposure and pretreatment with neutralizing antibody targeting IL-6 receptor α (IL-6Rα -nAb) or blocking Jak2 and Stat3 activity inhibited Stat3 phosphorylation. TLR2 up-regulation by poly I:C was also reduced by IL-6Rα-nAb and inhibitors of Jak2, Stat3 and NF-κB phosphorylation, whereas RANTES secretion was unaffected, but abolished following NF-κB inhibition. Treatment with exogenous IL-6 failed to increase TLR2. These findings demonstrate that TLR3 activation differentially regulates TLR expression through autocrine signaling involving IL-6 secretion, IL-6Rα activation and subsequent phosphorylation of Stat3. The results also indicate that NF-κB and Stat3 are required for TLR3-dependent up-regulation of TLR2 and that its delayed expression was due to a requirement for IL-6-dependent Stat3 activation.     

17beta-estradiol suppresses TLR3-induced cytokine and chemokine production in endometrial epithelial cells

Background  

The human endometrium is an important site for contact between the host and pathogens ascending the reproductive tract, and thus plays an important role in female reproductive tract immunity. Previous work in our laboratory has suggested that Toll-like receptors (TLRs) are involved in endometrial epithelial recognition of pathogens and that ligation of endometrial TLRs results in the production of cytokines and chemokines important for both immune and reproductive functions of the endometrium. We have also demonstrated cyclic regulation of TLR3 mRNA and protein expression in human endometrium, suggesting that steroid hormones might play a role in the expression and function of TLR3. In this study, the effects of 17beta-estradiol (E2) and progesterone (P) on TLR3 expression and function in endometrial cell lines were investigated.