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1.  Mast Cells Expedite Control of Pulmonary Murine Cytomegalovirus Infection by Enhancing the Recruitment of Protective CD8 T Cells to the Lungs 
PLoS Pathogens  2014;10(4):e1004100.
The lungs are a noted predilection site of acute, latent, and reactivated cytomegalovirus (CMV) infections. Interstitial pneumonia is the most dreaded manifestation of CMV disease in the immunocompromised host, whereas in the immunocompetent host lung-infiltrating CD8 T cells confine the infection in nodular inflammatory foci and prevent viral pathology. By using murine CMV infection as a model, we provide evidence for a critical role of mast cells (MC) in the recruitment of protective CD8 T cells to the lungs. Systemic infection triggered degranulation selectively in infected MC. The viral activation of MC was associated with a wave of CC chemokine ligand 5 (CCL5) in the serum of C57BL/6 mice that was MC-derived as verified by infection of MC-deficient KitW-sh/W-sh “sash” mutants. In these mutants, CD8 T cells were recruited less efficiently to the lungs, correlating with enhanced viral replication and delayed virus clearance. A causative role for MC was verified by MC reconstitution of “sash” mice restoring both, efficient CD8 T-cell recruitment and infection control. These results reveal a novel crosstalk axis between innate and adaptive immune defense against CMV, and identify MC as a hitherto unconsidered player in the immune surveillance at a relevant site of CMV disease.
Author Summary
Being strategically located beneath endothelial and epithelial surfaces, mast cells (MC) serve as sentinels for invading pathogens at host-environment boundaries as part of the innate defense against infection. Host genetic resistance against cytomegaloviruses (CMV) is largely determined by the innate immune response, but an implication of MC in the adaptive immune defense against CMV has not been considered so far and is almost impossible to address in human infection. Using murine CMV as a model that in the past has already pioneered the discovery of fundamental principles in CMV-host interactions, our data reveal MC as central part of a novel crosstalk-axis between the innate and adaptive immune response to CMV. We found that upon host infection MC become rapidly activated and promote the recruitment of protective CD8 T cells to the lungs, a noted critical site of CMV pathogenesis in humans as well as in the mouse model. Enhanced tissue infiltration of CD8 T cells results in a reduced peak viral load and a faster clearance of productive infection. Realizing the importance of MC in the control of pulmonary CMV infection may help to develop new strategies for preventing CMV pneumonia by MC supplementation in recipients of hematopoietic cell transplantation.
PMCID: PMC3999167  PMID: 24763809
2.  The tick salivary protein sialostatin L inhibits the Th9-derived production of the asthma-promoting cytokine interleukin-9 and is effective in the prevention of experimental asthma1 
Ticks developed a multitude of different immune evasion strategies in order to obtain a blood meal. Sialostatin L is an immunosuppressive cysteine protease inhibitor present in the saliva of the hard tick Ixodes scapularis. Herein we demonstrate that sialostatin L strongly inhibits the production of IL-9 by Th9 cells. Since we could show recently that Th9-derived IL-9 is essentially involved in the induction of asthma symptoms, sialostatin L was used for the treatment of experimental asthma. Application of sialostatin L in a model of experimental asthma almost completely abrogated airway hyperresponsiveness and eosinophilia. Our data suggest that sialostatin L can prevent experimental asthma, most likely by inhibiting the IL-9 production of Th9 cells. Thus, alternative to IL-9 neutralization sialostatin L provides the basis for the development of innovative therapeutic strategies to treat asthma.
PMCID: PMC3523721  PMID: 22327077
Th9 cells; Lung; Allergy; Parasites; Rodents
3.  Implementation strategies of internet-based asthma self-management support in usual care. Study protocol for the IMPASSE cluster randomized trial 
Internet-based self-management (IBSM) support cost-effectively improves asthma control, asthma related quality of life, number of symptom-free days, and lung function in patients with mild to moderate persistent asthma. The current challenge is to implement IBSM in clinical practice.
This study is a three-arm cluster randomized trial with a cluster pre-randomisation design and 12 months follow-up per practice comparing the following three IBSM implementation strategies: minimum strategy (MS): dissemination of the IBSM program; intermediate strategy (IS): MS + start-up support for professionals (i.e., support in selection of the appropriate population and training of professionals); and extended strategy (ES): IS + additional training and ongoing support for professionals. Because the implementation strategies (interventions) are primarily targeted at general practices, randomisation will occur at practice level.
In this study, we aim to evaluate 14 primary care practices per strategy in the Leiden-The Hague region, involving 140 patients per arm. Patients aged 18 to 50 years, with a physician diagnosis of asthma, prescription of inhaled corticosteroids, and/or montelukast for ≥3 months in the previous year are eligible to participate. Primary outcome measures are the proportion of referred patients that participate in IBSM, and the proportion of patients that have clinically relevant improvement in the asthma-related quality of life. The secondary effect measures are clinical outcomes (asthma control, lung function, usage of airway treatment, and presence of exacerbations); self-management related outcomes (health education impact, medication adherence, and illness perceptions); and patient utilities. Process measures are the proportion of practices that participate in IBSM and adherence of professionals to implementation strategies. Cost-effective measurements are medical costs and healthcare consumption. Follow-up is six months per patient.
This study provides insight in the amount of support that is required by general practices for cost-effective implementation of IBSM. Additionally, design and results can be beneficial for implementation of other self-management initiatives in clinical practice.
Trial registration
the Netherlands National Trial Register NTR2970
PMCID: PMC3514342  PMID: 23171672
Asthma; Self-management; Telemanagement; E-health; Self-management; Implementation; Chronic care
4.  Regulatory T cells and regulation of allergic airway disease 
Diseases like asthma have dramatically increased in the last decades. The reasons for the rising prevalence are still controversially discussed. Besides the genetic predisposition a number of different causes are thought to affect the increase of allergies. These include the hygiene hypothesis as well as changes in intestinal microbiota. Allergic airway inflammation is driven by T cells but it has become clear that tolerance and also suppression of allergic inflammation are mediated by so called regulatory T cells (Tregs). Indeed, naturally occurring Treg as well as induced Tregs have been shown to suppress allergic airway disease. In addition, the effectiveness of different therapeutic strategies (e.g. allergen immunotherapy) are mediated via Tregs. In addition, several Treg based approaches have been shown to effectively suppress allergic airway disease in different models. However, more research is needed to explore these potentially interesting approaches for the treatment of human disease.
PMCID: PMC3714190  PMID: 23885322
Allergy; asthma; inflammation; regulatory T cell; suppression
5.  Th17 cells mediate pulmonary collateral priming 
Our laboratory has shown earlier that inhalational sensitization to new antigens is facilitated through an ongoing Th2-polarized inflammation of the lung, a phenomenon we call “collateral priming”.
We were interested to analyze whether a Th1-polarized pulmonary inflammation also facilitates priming towards new antigens and which cytokine(s) would be involved.
Th1-polarized T cells were generated in vitro and transferred into congenic mice. Mice were challenged initially with cognate antigen and an unrelated antigen; consecutively they received cognate antigen or the secondary antigen. Airway inflammation, antigen-specific IgG2a and airway hyperresponsiveness were assessed to determine the inflammatory phenotype with antibody blocking studies used to determine cytokine requirements for Th1 collateral priming.
Our experiments revealed that an ongoing inflammation of the lung induced by the transfer of Th1-polarized cells also facilitates priming towards new antigens which results in a lymphocytic inflammation of the lung. Interestingely, blocking studies identified IL-17A as a major contributor to this pathology. Accordingly, we could demonstrate for the first time that Th17-polarized cells alone can facilitate priming towards new antigens, inducing lymphocytic airway inflammation and strong airway hyperresponsiveness. Flow cytometric analysis revealed priming of endogenous T cells for IL-17A secretion with a distinct memory/effector phenotype compared to Th1 cells, thus presenting an exciting model to further elucidate differentiation of Th17 cells.
We show that airway inflammation mediated by Th17 cells facilitates sensitization to new antigens and confers increased airway responsiveness in a mouse model of polysensitization suggesting a mechanism involving IL-17A behind the increased risk for allergic sensitization in polysensitized individuals.
PMCID: PMC3129446  PMID: 21459426
Asthma; IL-17A; T helper cell; polysensitization
6.  DC-derived IL-18 drives Treg differentiation, murine Helicobacter pylori–specific immune tolerance, and asthma protection  
The Journal of Clinical Investigation  2012;122(3):1082-1096.
Persistent colonization with the gastric bacterial pathogen Helicobacter pylori causes gastritis and predisposes infected individuals to gastric cancer. Conversely, it is also linked to protection from allergic, chronic inflammatory, and autoimmune diseases. We demonstrate here that H. pylori inhibits LPS-induced maturation of DCs and reprograms DCs toward a tolerance-promoting phenotype. Our results showed that DCs exposed to H. pylori in vitro or in vivo failed to induce T cell effector functions. Instead, they efficiently induced expression of the forkhead transcription factor FoxP3, the master regulator of Tregs, in naive T cells. Depletion of DCs in mice infected with H. pylori during the neonatal period was sufficient to break H. pylori–specific tolerance. DC depletion resulted in improved control of the infection but also aggravated T cell–driven immunopathology. Consistent with the mouse data, DCs infiltrating the gastric mucosa of human H. pylori carriers exhibited a semimature DC-SIGN+HLA–DRhiCD80loCD86lo phenotype. Mechanistically, the tolerogenic activity of H. pylori–experienced DCs was shown to require IL-18 in vitro and in vivo; DC-derived IL-18 acted directly on T cells to drive their conversion to Tregs. CD4+CD25+ Tregs from infected wild-type mice but not Il18–/– or Il18r1–/– mice prevented airway inflammation and hyperresponsiveness in an experimental model of asthma. Taken together, our results indicate that tolerogenic reprogramming of DCs ensures the persistence of H. pylori and protects against allergic asthma in a process that requires IL-18.
PMCID: PMC3287234  PMID: 22307326
7.  IL-22 Is Produced by Innate Lymphoid Cells and Limits Inflammation in Allergic Airway Disease 
PLoS ONE  2011;6(7):e21799.
Interleukin (IL)-22 is an effector cytokine, which acts primarily on epithelial cells in the skin, gut, liver and lung. Both pro- and anti-inflammatory properties have been reported for IL-22 depending on the tissue and disease model. In a murine model of allergic airway inflammation, we found that IL-22 is predominantly produced by innate lymphoid cells in the inflamed lungs, rather than TH cells. To determine the impact of IL-22 on airway inflammation, we used allergen-sensitized IL-22-deficient mice and found that they suffer from significantly higher airway hyperreactivity upon airway challenge. IL-22-deficiency led to increased eosinophil infiltration lymphocyte invasion and production of CCL17 (TARC), IL-5 and IL-13 in the lung. Mice treated with IL-22 before antigen challenge displayed reduced expression of CCL17 and IL-13 and significant amelioration of airway constriction and inflammation. We conclude that innate IL-22 limits airway inflammation, tissue damage and clinical decline in allergic lung disease.
PMCID: PMC3138740  PMID: 21789181
8.  Helicobacter pylori infection prevents allergic asthma in mouse models through the induction of regulatory T cells  
The Journal of Clinical Investigation  2011;121(8):3088-3093.
Atopic asthma is a chronic disease of the airways that has taken on epidemic proportions in the industrialized world. The increase in asthma rates has been linked epidemiologically to the rapid disappearance of Helicobacter pylori, a bacterial pathogen that persistently colonizes the human stomach, from Western societies. In this study, we have utilized mouse models of allergic airway disease induced by ovalbumin or house dust mite allergen to experimentally examine a possible inverse correlation between H. pylori and asthma. H. pylori infection efficiently protected mice from airway hyperresponsiveness, tissue inflammation, and goblet cell metaplasia, which are hallmarks of asthma, and prevented allergen-induced pulmonary and bronchoalveolar infiltration with eosinophils, Th2 cells, and Th17 cells. Protection against asthma was most robust in mice infected neonatally and was abrogated by antibiotic eradication of H. pylori. Asthma protection was further associated with impaired maturation of lung-infiltrating dendritic cells and the accumulation of highly suppressive Tregs in the lungs. Systemic Treg depletion abolished asthma protection; conversely, the adoptive transfer of purified Treg populations was sufficient to transfer protection from infected donor mice to uninfected recipients. Our results thus provide experimental evidence for a beneficial effect of H. pylori colonization on the development of allergen-induced asthma.
PMCID: PMC3148731  PMID: 21737881
9.  Mast Cells in Allergic Asthma and Beyond 
Yonsei Medical Journal  2010;51(6):797-807.
Mast cells have been regarded for a long time as effector cells in IgE mediated type I reactions and in host defence against parasites. However, they are resident in all environmental exposed tissues and express a wide variety of receptors, suggesting that these cells can also function as sentinels in innate immune responses. Indeed, studies have demonstrated an important role of mast cells during the induction of life-saving antibacterial responses. Furthermore, recent findings have shown that mast cells promote and modulate the development of adaptive immune responses, making them an important hinge of innate and acquired immunity. In addition, mast cells and several mast cell-produced mediators have been shown to be important during the development of allergic airway diseases. In the present review, we will summarize findings on the role of mast cells during the development of adaptive immune responses and highlight their function, especially during the development of allergic asthma.
PMCID: PMC2995967  PMID: 20879044
Asthma; mast cells; mediators; allergy
10.  AHR-Enhancing γδ T Cells Develop in Normal Untreated Mice and Fail to Produce IL-4/13, Unlike TH2 Cells and NKT Cells1 
Allergic airway hyperresponsiveness (AHR) in OVA-sensitized and challenged mice, mediated by allergen-specific Th2 cells and Th2-like iNKT cells, develops under the influence of enhancing and inhibitory γδ T cells. The AHR-enhancing cells belong to the Vγ1+ γδ T cell subset, cells that are capable of increasing IL-5 and IL-13 levels in the airways in a manner like Th2 cells. They also synergize with iNKT cells in mediating AHR. However, unlike Th2 cells, the AHR-enhancers arise in untreated mice, and we show here that they exhibit their functional bias already as thymocytes, at an HSAhi maturational stage. In further contrast to Th2 cells and also unlike iNKT cells, they could not be stimulated to produce IL-4 and IL-13, consistent with their synergistic dependence on iNKT cells in mediating AHR. Mice deficient in IFN-γ, TNFRp75 or IL-4 did not produce these AHR-enhancing γδ T cells, but in the absence of IFN-γ, their spontaneous development was restored by adoptive transfer of IFN-γ competent dendritic cells from untreated donors. Intra-peritoneal injection of OVA/alum restored development of the AHR-enhancers in all of the mutant strains, indicating that the enhancers still can be induced when they fail to develop spontaneously, and that they themselves need not express TNFRp75, IFN-γ or IL-4 in order to exert their function. We conclude that both the development and the cytokine potential of the AHR-enhancing γδ T cells differs critically from that of Th2 cells and NKT cells, despite similar influences of these cell populations on AHR.
PMCID: PMC2688721  PMID: 19201853
T cells; Allergy; Lung; Spleen and Lymph Nodes; Transgenic/Knockout Mice
11.  Evidence that CD8+ Dendritic Cells Enable the Development of γδ T Cells that Modulate Airway Hyperresponsiveness2 
Airway hyperresponsiveness (AHR), a hallmark of asthma and several other diseases, can be modulated by γδ T cells. In mice sensitized and challenged with ovalbumin, AHR depends on allergen-specific αβ T cells, but Vγδ1+ γδ T cells spontaneously enhance AHR, whereas Vγ4+ γδ T cells after being induced by airway challenge suppress AHR. The activity of these γδ T cell modulators is allergen-nonspecific, and how they develop is unclear. We now show that CD8 is essential for the development of both the AHR-suppressor and enhancer γδ T cells although neither type needs to express CD8 itself. Both cell types encounter CD8-expressing non-T cells in the spleen, and their functional development in an otherwise CD8-negative environment can be restored with transferred spleen cell preparations containing CD8+ DC, but not CD8+ T cells or CD8− DC. Our findings suggest that CD8+ DC in the lymphoid tissues enable an early step in the development of γδ T cells, through direct cell-contact. DC-expressed CD8 might take part in this interaction.
PMCID: PMC2493442  PMID: 18566396
Rodent; dendritic cells; T cells; cell differentiation; spleen and lymph nodes
12.  Arhgef1 Is Required by T Cells for the Development of Airway Hyperreactivity and Inflammation 
Rationale: Arhgef1 is an intracellular protein, expressed by hematopoietic cells, that regulates signaling by both G protein–coupled receptors and RhoA, and, consequently, is required for appropriate migration and adhesion of diverse leukocyte populations.
Objectives: To evaluate a possible contribution for Arhgef1 in the development of airway inflammation and airway hyperreactivity.
Methods: Arhgef1-deficient (Arhgef1−/−) and wild-type (WT) mice were sensitized and airway challenged, followed by measurement of airway responsiveness to inhaled methacholine. Inflammation was assessed by several parameters that included flow cytometric analysis and histology. Arhgef1-deficient recipients were reconstituted with WT T lymphocytes before sensitization and challenge, and again measured for airway responsiveness and inflammation. Cytokine production in response to specific antigen was measured in cultures of isolated leukocytes from lung and spleen and compared with the levels generated in lung and spleen explant cultures.
Measurements and Main Results: Arhgef1−/− mice display significantly reduced airway hyperreactivity, Th2 cytokine production, and lung inflammation, despite intact systemic immunity. After airway challenge of Arhgef1−/− mice, antigen-specific T cells were present in mutant lungs, but were found to interact with CD11c+ cells at a significantly reduced frequency. Adoptive transfer of WT T cells into Arhgef1−/− mice restored airway hyperreactivity and inflammation.
Conclusions: These data demonstrate that T cells depend on Arhgef1 to promote lung inflammation. Moreover, a deficiency in Arhgef1 results in reduced T cell–CD11c+ antigen-presenting cell interaction, and likely underscores the inability of Arhgef1−/− mice to mount an adaptive immune response to airway challenge.
PMCID: PMC2049063  PMID: 17463415
airway hyperreactivity; cytokines; lung inflammation; T cells
13.  Mast cells and the development of allergic airway disease 
Murine models have highlighted the importance of T-cells and TH2 cytokines in development of allergen-induced airway disease. In contrast, the role of mast cells for the development of allergic airway disease has been controversial. Recent studies in murine models demonstrate a significant contribution of mast cells during the development of airway hyperresponsiveness and airway inflammation. Furthermore these models have allowed identifying certain mast cell-produced mediators (e.g. histamine and leukotriene B4) to be involved in the recruitment of effector T-cells into the lung. Additionally, mast cell-produced TNF can directly activate TH2 cells and contribute to the development of allergic airway disease. These new findings demonstrate a complex role of mast cells and their mediators, not only as effector cells, but also during sensitization and development of allergic airway disease. Therefore mast cells and certain mast cell-produced mediators might be an interesting target for the prevention and treatment of allergic asthma.
PMCID: PMC2259396  PMID: 18315833
14.  Invasive and noninvasive methods for studying pulmonary function in mice 
Respiratory Research  2007;8(1):63.
The widespread use of genetically altered mouse models of experimental asthma has stimulated the development of lung function techniques in vivo to characterize the functional results of genetic manipulations. Here, we describe various classical and recent methods of measuring airway responsiveness in vivo including both invasive methodologies in anesthetized, intubated mice (repetitive/non-repetitive assessment of pulmonary resistance (RL) and dynamic compliance (Cdyn); measurement of low-frequency forced oscillations (LFOT)) and noninvasive technologies in conscious animals (head-out body plethysmography; barometric whole-body plethysmography). Outlined are the technical principles, validation and applications as well as the strengths and weaknesses of each methodology. Reviewed is the current set of invasive and noninvasive methods of measuring murine pulmonary function, with particular emphasis on practical considerations that should be considered when applying them for phenotyping in the laboratory mouse.
PMCID: PMC2039738  PMID: 17868442
15.  Soluble Triggering Receptor Expressed on Myeloid Cells 1 Is Released in Patients with Stable Chronic Obstructive Pulmonary Disease 
Chronic obstructive pulmonary disease (COPD) is increasingly recognized as a systemic disease that is associated with increased serum levels of markers of systemic inflammation. The triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently identified activating receptor on neutrophils, monocytes, and macrophage subsets. TREM-1 expression is upregulated by microbial products such as the toll-like receptor ligand lipoteichoic acid of Gram-positive or lipopolysaccharides of Gram-negative bacteria. In the present study, sera from 12 COPD patients (GOLD stages I–IV, FEV1 51 ± 6%) and 10 healthy individuals were retrospectively analyzed for soluble TREM-1 (sTREM-1) using a newly developed ELISA. In healthy subjects, sTREM-1 levels were low (median 0.25 ng/mL, range 0–5.9 ng/mL). In contrast, levels of sTREM-1 in sera of COPD patients were significantly increased (median 11.68 ng/mL, range 6.2–41.9 ng/mL, P<.05). Furthermore, serum levels of sTREM-1 showed a significant negative correlation with lung function impairment. In summary, serum concentrations of sTREM-1 are increased in patients with COPD. Prospective studies are warranted to evaluate the relevance of sTREM-1 as a potential marker of the disease in patients with COPD.
PMCID: PMC2246041  PMID: 18317529
16.  Importance of Myeloid Dendritic Cells in Persistent Airway Disease after Repeated Allergen Exposure 
Rationale: There is conflicting information about the development and resolution of airway inflammation and airway hyperresponsiveness (AHR) after repeated airway exposure to allergen in sensitized mice.
Methods: Sensitized BALB/c and C57BL/6 mice were exposed to repeated allergen challenge on 3, 7, or 11 occasions. Airway function in response to inhaled methacholine was monitored; bronchoalveolar lavage fluid inflammatory cells were counted; and goblet cell metaplasia, peribronchial fibrosis, and smooth muscle hypertrophy were quantitated on tissue sections. Bone marrow–derived dendritic cells were generated after differentiation of bone marrow cells in the presence of growth factors.
Results: Sensitization to ovalbumin (OVA) in alum, followed by three airway exposures to OVA, induced lung eosinophilia, goblet cell metaplasia, mild peribronchial fibrosis, and peribronchial smooth muscle hypertrophy; increased levels of interleukin (IL)-4, IL-5, IL-13, granulocyte-macrophage colony–stimulating factor, transforming growth factor-β1, eotaxin-1, RANTES (regulated on activation, normal T-cell expressed and secreted), and OVA-specific IgG1 and IgE; and resulted in AHR. After seven airway challenges, development of AHR was markedly decreased as was the production of IL-4, IL-5, and IL-13. Levels of IL-10 in both strains and the level of IL-12 in BALB/c mice increased. After 11 challenges, airway eosinophilia and peribronchial fibrosis further declined and the cytokine and chemokine profiles continued to change. At this time point, the number of myeloid dendritic cells and expression of CD80 and CD86 in lungs were decreased compared with three challenges. After 11 challenges, intratracheal instillation of bone marrow–derived dendritic cells restored AHR and airway eosinophilia.
Conclusions: These data suggest that repeated allergen exposure leads to progressive decreases in AHR and allergic inflammation, through decreases in myeloid dendritic cell numbers.
PMCID: PMC2662981  PMID: 16192450
airway hyperresponsiveness; chronic asthma; cytokine; dendritic cells; eosinophil
17.  Airway Hyperresponsiveness in the Absence of CD4+ T Cells after Primary but Not Secondary Challenge 
CD4+ T cells have been shown to play a role in the development of airway hyperresponsivness (AHR) and airway eosinophilia in mice using ablation as well as adoptive transfer experiments. However, as other T cell subsets (CD8, NKT) may play a role in these models, we examined the responses of sensitized CD4-deficient mice after either primary or secondary airway allergen challenge. After sensitization, CD4-deficiency in mice was not associated with airway eosinophilia, allergen-specific IgE, or elevated levels of interleukin (IL)-4 or IL-13. Increases in lung CD8 T cells and IL-5 were observed and shown to be essential for AHR as demonstrated after CD8 T cell depletion or anti–IL-5 treatment. In contrast to the response of sensitized CD4-deficient mice to primary allergen challenge, they failed to develop AHR after secondary allergen challenge. Although the importance of this CD4+ T cell–independent pathway in normal mice is unclear at this time, these studies identify the diversity of the cellular pathway, which may contribute to the development of AHR after primary allergen exposure of sensitized mice.
PMCID: PMC2715306  PMID: 15845865
airway hyperresponsiveness; CD4 T cells; inflammation; secondary challenge

Results 1-17 (17)