Anti‐Asthma Simplified Herbal Medicine Intervention‐induced long‐lasting tolerance to allergen exposure in an asthma model is interferon‐γ, but not transforming growth factor‐β dependent

Background Chronic allergic asthma is the result of a T‐helper type 2 (Th2)‐biased immune status. Current asthma therapies control symptoms in some patients, but a long‐lasting therapy has not been established. Anti‐Asthma Simplified Herbal Medicine Intervention (ASHMI^?), a Chinese herbal formula improved symptoms and lung function, and reduced Th2 responses in a controlled trial of patients with persistent moderate to severe asthma. Objective We evaluated the persistence of ASHMI^? beneficial effects following therapy in a murine model of chronic asthma and the immunological mechanisms underlying such effects. Methods BALB/c mice sensitized intraperitoneally with ovalbumin (OVA) received 3 weekly intratracheal OVA challenges to induce airway hyper‐reactivity (AHR) and inflammation (OVA mice). Additionally, OVA mice were treated with ASHMI^? (OVA/ASHMI^?) or water (OVA/sham) for 4 weeks, and then challenged immediately and 8 weeks post‐therapy. In other experiments, OVA mice received ASHMI^? treatment with concomitant neutralization of IFN‐γ or TGF‐β. Effects on airway responses, cytokine‐ and OVA‐specific IgE levels were determined 8 weeks post‐therapy. Results Before treatment, OVA mice exhibited AHR and pulmonary eosinophilic inflammation following OVA challenge, which was almost completely resolved immediately after completing treatment with ASHMI^? and did not re‐occur following OVA re‐challenge up to 8 weeks post‐therapy. Decreased allergen‐specific IgE and Th2 cytokine levels, and increased IFN‐γ levels also persisted at least 8 weeks post‐therapy. ASHMI^? effects were eliminated by the neutralization of IFN‐γ, but not TGF‐β, during therapy. Conclusion ASHMI^? induced long‐lasting post‐therapy tolerance to antigen‐induced inflammation and AHR. IFN‐γ is a critical factor in ASHMI^? effects. Cite this as: K. Srivastava, T. Zhang, N. Yang, H. Sampson and X. M. Li, Clinical & Experimental Allergy, 2010 (40) 1678–1688.     

Regulatory effects of IFN‐β on production of osteopontin and IL‐17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4⁺ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4⁺ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4⁺ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN⁺ and IL‐17⁺ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.     

T‐cell‐derived,but not B‐cell‐derived,IL‐10 suppresses antigen‐specific T‐cell responses in Litomosoides sigmodontis‐infected mice

IL‐10, a cytokine with pleiotropic functions is produced by many different cells. Although IL‐10 may be crucial for initiating protective Th2 responses to helminth infection, it may also function as a suppressive cytokine preventing immune pathology or even contributing to helminth‐induced immune evasion. Here, we show that B cells and T cells produce IL‐10 during murine Litomosoides sigmodontis infection. IL‐10‐deficient mice produced increased amounts of L. sigmodontis‐specific IFN‐γ and IL‐13 suggesting a suppressive role for IL‐10 in the initiation of the T‐cell response to infection. Using cell type‐specific IL‐10‐deficient mice, we dissected different functions of T‐cell‐ and B‐cell‐derived IL‐10. Litomosoides sigmodontis‐specific IFN‐γ, IL‐5, and IL‐13 production increased in the absence of T‐cell‐derived IL‐10 at early and late time points of infection. In contrast, B‐cell‐specific IL‐10 deficiency did not lead to significant changes in L. sigmodontis‐specific cytokine production compared to WT mice. Our results suggest that the initiation of Ag‐specific cellular responses during L. sigmodontis infection is suppressed by T‐cell‐derived IL‐10 and not by B‐cell‐derived IL‐10.     

Myeloid‐derived suppressor cells in inflammation: Uncovering cell subsets with enhanced immunosuppressive functions

Although originally described in tumor‐bearing hosts, myeloid‐derived suppressor cells (MDSC) have been detected under numerous pathological situations that cause enhanced demand of myeloid cells. Thus, MDSC might be part of a conserved response to different endogenous and exogenous stress signals, including inflammation. Two processes are fundamental for MDSC biology: differentiation from myeloid progenitors and full activation of their immune regulatory program by factors released from activated T cells or present in the microenvironment conditioned by either tumor growth or inflammation. How these two processes are controlled and linked is still an open question. In this issue of the European Journal of Immunology, a paper demonstrates that a combination of the known inflammatory molecules, IFN‐γ and LPS, sustains MDSC expansion and activation while suppressing differentiation of DC from bone marrow precursors. Moreover, this paper contributes to defining the cell subsets that possess immunoregulatory properties within the broad population of CD11b⁺Gr‐1⁺ cells, often altogether referred to as MDSC.     

Increased expression and role of thymic stromal lymphopoietin in nasal polyposis

Purpose

Nasal polyposis is a chronic inflammatory disease of the upper airways often associated with asthma and characterized by markedly increased numbers of eosinophils, Th2 type lymphocytes, fibroblasts, goblet cells and mast cells. Previous studies have shown elevated levels of thymic stromal lymphopoietin (TSLP) in atopic diseases like asthma, atopic dermatitis and mainly in animal models of allergic rhinitis (AR). Here, we investigated the expression of TSLP in nasal polyps from atopics and non-atopics in comparison with the nasal mucosa and its potential role in nasal polyposis.

Methods

Messenger RNA expression for TSLP, thymus and activation-regulated chemokine (TARC) and macrophage derived chemokine (MDC) in nasal polyps and nasal mucosa of atopics and non-atopics was analyzed by real time PCR. Immunoreactivity for TSLP in nasal polyps and in the nasal mucosa of patients with AR and non-allergic rhinitis (NAR) was analyzed by immunohistochemistry. Eosinophil counts was analyzed by Wright-Giemsa staining and nasal polyp tissue IgE, by ELISA.

Results

Messenger RNA expression for TSLP,TARC and MDC was markedly higher in nasal polyps as compared to the allergic nasal mucosa. Immunoreactivity for TSLP was detected in epithelial cells, endothelial cells, fibroblasts and inflammatory cells of the nasal mucosa and nasal polyps. The number of TSLP+ cells was significantly greater in the nasal mucosa of AR than NAR patients. The number of TSLP+ cells in nasal polyps from atopics was significantly greater than that of non-atopics and that in the allergic nasal mucosa. The number of TSLP+ cells correlated well with the number of eosinophils and the levels of IgE in nasal polyps.

Conclusions

The high expression of TSLP in nasal polyps and its strong correlation to eosinophils and IgE suggest a potential role for TSLP in the pathogenesis of nasal polyps by regulating the Th2 type and eosinophilic inflammation.     

Microfluidic‐based,live‐cell analysis allows assessment of NK‐cell migration in response to crosstalk with dendritic cells

Migration and localization of NK cells into peripheral tissues are tightly regulated under normal and pathological conditions. The physiological importance of NK cell–DC crosstalk has been well documented. However, the ways in which DCs regulate the migratory properties of NK cells (such as chemotaxis, chemokinesis, chemo‐repulsion) are not fully defined in vitro. Here, we employed a microfluidic platform to examine, at the single‐cell level, C57BL/6 NK‐cell migrations in a stable chemical gradient. We observed that soluble factors released by the immature and LPS‐activated mature DCs induced a high level of chemotactic movement of IL‐2‐activated NK cells in vitro. We confirmed these findings in a standard trans‐well migration assay, and identified CXCR3 as a key receptor on the NK cells that mediated the migration. More interestingly, we revealed a novel function of granulocyte macrophage colony‐stimulating factor in repulsing NK‐cell migrations. The future uses of such microfluidic device in the systematic evaluations of NK‐cell migratory responses in NK cell–DC crosstalk will provide new insights into the development of DC‐based NK‐cell therapies against tumor and infections.     

DC‐SIGN expression on podocytes and its role in inflammatory immune response of lupus nephritis

Podocytes, the main target of immune complex, participate actively in the development of glomerular injury as immune cells. Dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin (DC‐SIGN) is an innate immune molecular that has an immune recognition function, and is involved in mediation of cell adhesion and immunoregulation. Here we explored the expression of DC‐SIGN on podocytes and its role in immune and inflammatory responses in lupus nephritis (LN). Expression of DC‐SIGN and immunoglobulin (Ig)G1 was observed in glomeruli of LN patients. DC‐SIGN was co‐expressed with nephrin on podocytes. Accompanied by increased proteinuria of LN mice, DC‐SIGN and IgG1 expressions were observed in the glomeruli from 20 weeks, and the renal function deteriorated up to 24 weeks. Mice with anti‐DC‐SIGN antibody showed reduced proteinuria and remission of renal function. After the podocytes were stimulated by serum of LN mice in vitro, the expression of DC‐SIGN, major histocompatibility complex (MHC) class II and CD80 was up‐regulated, stimulation of T cell proliferation was enhanced and the interferon (IFN)‐γ/interleukin (IL)‐4 ratio increased. However, anti‐DC‐SIGN antibody treatment reversed these events. These results suggested that podocytes in LN can exert DC‐like function through their expression of DC‐SIGN, which may be involved in immune and inflammatory responses of renal tissues. However, blockage of DC‐SIGN can inhibit immune functions of podocytes, which may have preventive and therapeutic effects.     

B7‐1/2, but not PD‐L1/2 molecules,are required on IL‐10‐treated tolerogenic DC and DC‐derived exosomes for in vivo function

Costimulatory molecules, such as B7‐1/2 and PD‐L1/2 play an important role in the function of APC. The regulation of the surface levels of costimulatory molecules is one mechanism by which APC maintain the balance between tolerance and immunity. We examined the contributions of B7‐1/2 and PD‐L1/2 to the function of IL‐10‐treated, immunosuppressive DC as well as therapeutic exosomes derived from these DC. IL‐10 treatment of DC significantly downregulated surface expression of MHC II, B7‐1, B7‐2, and decreased levels of MHC I and PD‐L2. IL‐10 treatment of DC resulted in a modified costimulatory profile of DC‐secreted exosomes with a reduction in B7‐1, PD‐L1 and PD‐L2. We further demonstrate that absence of B7‐1 or B7‐2 on donor DC results in a loss of ability of IL‐10‐treated DC and their exosomes to suppress the delayed‐type hypersensitivity response, whereas IL‐10‐treated DC deficient in PD‐L1/2 as well as their secreted exosomes retained the ability to suppress delayed‐type hypersensitivity responses. We conclude that B7‐1 and B7‐2, but not PD‐L1 and PD‐L2, on IL‐10‐treated DC and DC‐derived exosomes play a critical role in immunosuppressive functions of both DC and exosomes.     

Myeloid‐derived suppressor cell activation by combined LPS and IFN‐γ treatment impairs DC development

Myeloid‐derived suppressor cells (MDSC) and DC are major controllers of immune responses against tumors or infections. However, it remains unclear how DC development and MDSC suppressor activity both generated from myeloid precursor cells are regulated. Here, we show that the combined treatment of BM‐derived MDSC with LPS plus IFN‐γ inhibited the DC development but enhanced MDSC functions, such as NO release and T‐cell suppression. This was not observed by the single treatments in vitro. In the spleens of healthy mice, we identified two Gr‐1^lowCD11b^highLy‐6C^highSSC^lowMo‐MDSC and Gr‐1^highCD11b^lowPMN‐MDSC populations with suppressive potential, whereas Gr‐1^highCD11b^high neutrophils and Gr‐1^lowCD11b^highSSC^low eosinophils were not suppressive. Injections of LPS plus IFN‐γ expanded these populations within the spleen but not LN leading to the block of the proliferation of CD8⁺ T cells. At the same time, their capacity to develop into DC was impaired. Together, our data suggest that spleens of healthy mice contain two subsets of MDSC with suppressive potential. A two‐signal‐program through combined LPS and IFN‐γ treatment expands and fully activates MDSC in vitro and in vivo.     

Multifunctional role of osteopontin in directing intrafibrillar mineralization of collagen and activation of osteoclasts

Mineralized collagen composites are of interest because they have the potential to provide a bone-like scaffold that stimulates the natural processes of resorption and remodeling. Working towards this goal, our group has previously shown that the nanostructure of bone can be reproduced using a polymer-induced liquid-precursor (PILP) process, which enables intrafibrillar mineralization of collagen with hydroxyapatite to be achieved. This prior work used polyaspartic acid (pASP), a simple mimic for acidic non-collagenous proteins, to generate nanodroplets/nanoparticles of an amorphous mineral precursor which can infiltrate the interstices of type-I collagen fibrils. In this study we show that osteopontin (OPN) can similarly serve as a process-directing agent for the intrafibrillar mineralization of collagen, even though OPN is generally considered a mineralization inhibitor. We also found that inclusion of OPN in the mineralization process promotes the interaction of mouse marrow-derived osteoclasts with PILP-remineralized bone that was previously demineralized, as measured by actin ring formation. While osteoclast activation occurred when pASP was used as the process-directing agent, using OPN resulted in a dramatic effect on osteoclast activation, presumably because of the inherent arginine–glycine–aspartate acid ligands of OPN. By capitalizing on the multifunctionality of OPN, these studies may lead the way to producing biomimetic bone substitutes with the capability of tailorable bioresorption rates.     

The DC‐HIL/syndecan‐4 pathway inhibits human allogeneic T‐cell responses

T‐cell activation is regulated by binding of ligands on APC to corresponding receptors on T cells. In mice, we discovered that binding of DC‐HIL on APC to syndecan‐4 (SD‐4) on activated T cells potently inhibits T‐cell activation. In humans, we now show that DC‐HIL also binds to SD‐4 on activated T cells through recognition of its heparinase‐sensitive saccharide moiety. DC‐HIL blocks anti‐CD3‐induced T‐cell responses, reducing secretion of pro‐inflammatory cytokines and blocking entry into the S phase of the cell cycle. Binding of DC‐HIL phosphorylates SD‐4's intracellular tyrosine and serine residues. Anti‐SD‐4 Ab mimics the ability of DC‐HIL to attenuate anti‐CD3 response more potently than Ab directed against other inhibitory receptors (CTLA‐4 or programmed cell death‐1). Among leukocytes, DC‐HIL is expressed highest by CD14⁺ monocytes and this expression can be upregulated markedly by TGF‐β. Among APC, DC‐HIL is expressed highest by epidermal Langerhans cells, an immature type of dendritic cells. Finally, the level of DC‐HIL expression on CD14⁺ monocytes correlates inversely with allostimulatory capacity, such that treatment with TGF‐β reduced this capacity, whereas knocking down the DC‐HIL gene augmented it. Our findings indicate that the DC‐HIL/SD‐4 pathway can be manipulated to treat T‐cell‐driven disorders in humans.