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1.  Aryl hydrocarbon receptor control of a disease tolerance defense pathway 
Nature  2014;511(7508):184-190.
Summary
Disease tolerance is the ability of the host to reduce the impact of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (“endotoxin tolerance”). We found that a first exposure to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase 2, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR complex-associated Src kinase activity promoted IDO1 phosphorylation and signaling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in gram-negative and gram-positive infections, pointing to a role for AhR in contributing to host fitness.
doi:10.1038/nature13323
PMCID: PMC4098076  PMID: 24930766
2.  Abrogated transforming growth factor beta receptor II (TGFβRII) signalling in dendritic cells promotes immune reactivity of T cells resulting in enhanced atherosclerosis 
European Heart Journal  2012;34(48):3717-3727.
Aims
The importance of transforming growth factor beta (TGFβ) as an immune regulatory cytokine in atherosclerosis has been established. However, the role of TGFβ signalling in dendritic cells (DCs) and in DC-mediated T cell proliferation and differentiation in atherosclerosis is unknown.
Methods and results
Here, we investigated the effect of disrupted TGFβ signalling in DCs on atherosclerosis by using mice carrying a transgene resulting in functional inactivation of TGFβ receptor II (TGFβRII) signalling in CD11c+ cells (Apoe−/−CD11cDNR). Apoe−/−CD11cDNR mice exhibited an over two-fold increase in the plaque area compared with Apoe−/− mice. Plaques of Apoe−/−CD11cDNR mice showed an increase in CD45+ leucocyte content, and specifically in CD3+, CD4+ and CD8+ cells, whereas macrophage content was not affected. In lymphoid organs, Apoe−/−CD11cDNR mice had equal amounts of CD11c+ cells, and CD11c+CD8+ and CD11c+CD8− subsets, but showed a subtle shift in the CD11c+CD8− population towards the more inflammatory CD11c+CD8−CD4− DC subset. In addition, the number of plasmacytoid-DCs decreased. Maturation markers such as MHCII, CD86 and CD40 on CD11chi cells did not change, but the CD11cDNR DCs produced more TNFα and IL-12. CD11c+ cells from CD11cDNR mice strongly induced T-cell proliferation and activation, resulting in increased amounts of effector T cells producing high amounts of Th1 (IFN-γ), Th2 (IL-4, IL-10), Th17 (IL-17), and Treg (IL-10) cytokines.
Conclusion
Here, we show that loss of TGFβRII signalling in CD11c+ cells induces subtle changes in DC subsets, which provoke uncontrolled T cell activation and maturation. This results in increased atherosclerosis and an inflammatory plaque phenotype during hypercholesterolaemia.
doi:10.1093/eurheartj/ehs106
PMCID: PMC3869966  PMID: 22613345
Atherosclerosis; Inflammation; TGFβ; Dendritic cell
4.  Targeting macrophage Histone deacetylase 3 stabilizes atherosclerotic lesions 
EMBO Molecular Medicine  2014;6(9):1124-1132.
Macrophages are key immune cells found in atherosclerotic plaques and critically shape atherosclerotic disease development. Targeting the functional repertoire of macrophages may hold novel approaches for future atherosclerosis management. Here, we describe a previously unrecognized role of the epigenomic enzyme Histone deacetylase 3 (Hdac3) in regulating the atherosclerotic phenotype of macrophages. Using conditional knockout mice, we found that myeloid Hdac3 deficiency promotes collagen deposition in atherosclerotic lesions and thus induces a stable plaque phenotype. Also, macrophages presented a switch to anti-inflammatory wound healing characteristics and showed improved lipid handling. The pro-fibrotic phenotype was directly linked to epigenetic regulation of the Tgfb1 locus upon Hdac3 deletion, driving smooth muscle cells to increased collagen production. Moreover, in humans, HDAC3 was the sole Hdac upregulated in ruptured atherosclerotic lesions, Hdac3 associated with inflammatory macrophages, and HDAC3 expression inversely correlated with pro-fibrotic TGFB1 expression. Collectively, we show that targeting the macrophage epigenome can improve atherosclerosis outcome and we identify Hdac3 as a potential novel therapeutic target in cardiovascular disease.
doi:10.15252/emmm.201404170
PMCID: PMC4197860  PMID: 25007801
atherosclerosis; epigenetics; fibrosis; lipids; macrophages
5.  Development of β-Lactoglobulin-Specific Chimeric Human IgEκ Monoclonal Antibodies for In Vitro Safety Assessment of Whey Hydrolysates 
PLoS ONE  2014;9(8):e106025.
Background
Cow’s milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow’s milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays.
Objective
An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays.
Methods
Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains.
Results
After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5–10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow’s milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow’s milk allergic children.
Conclusion
Usage of our ‘unlimited’ source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow’s milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.
doi:10.1371/journal.pone.0106025
PMCID: PMC4143325  PMID: 25153680
6.  Immune Modulating Effects of NKT Cells in a Physiologically Low Dose Leishmania major Infection Model after αGalCer Analog PBS57 Stimulation 
Leishmaniasis is a parasitic infection affecting ∼12 million people worldwide, mostly in developing countries. Treatment options are limited and no effective vaccines exist to date. Natural Killer T (NKT) cells are a conserved innate-like lymphocyte population with immunomodulating effects in various settings. A number of reports state a role of NKT cells in different models of Leishmania infection. Here, we investigated the effect of NKT cells in a physiologically relevant, intradermal low dose infection model. After inoculation of 103 infectious-stage L. major, comparable numbers of skin-immigrating NKT cells in both susceptible BALB/c mice and resistant C57BL/6 mice were noted. Compared to their wild type counterparts, NKT cell-deficient mice on a C57BL/6 background were better able to contain infection with L. major and showed decreased IL-4 production in cytokine analysis performed 5 and 8 weeks after infection. Low doses of the NKT cell stimulating αGalCer analog PBS57 applied at the time of infection led to disease exacerbation in C57BL/6 wild-type, but not NKT-deficient mice. The effect was dependent both on the timing and amount of PBS57 administered. The effect of NKT cell stimulation by PBS57 proved to be IL-4 dependent, as it was neutralized in IL-4-deficient C57BL/6 or anti-IL-4 antibody-treated wild-type mice. In contrast to C57BL/6 mice, administration of PBS57 in susceptible BALB/c mice resulted in an improved course of disease. Our results reveal a strain- and cytokine-dependent regulatory role of NKT cells in the development of immunity to low dose L. major infections. These effects, probably masked in previous studies using higher parasite inocula, should be considered in future therapy and immunization approaches.
Author Summary
Cutaneous leishmaniasis is a disease affecting about 12 million people worldwide. It is transmitted by a sand fly and primarily affects people in developing countries. To date there are no effective vaccines. Many of the treatments available have serious side effects and resistance mechanisms are becoming an increasingly prevalent problem. Natural killer T (NKT) cells are a unique T cell population recognizing glycolipids. Their role in immune processes, especially in infectious diseases, is incompletely understood. In the current study, we investigated the role of NKT cells in Leishmania infections in detail. We found that NKT cells can significantly alter the development of immunity, however in different directions depending on the host's genetic background. Their natural effect on infection can be increased when applying the stimulating antigen alpha-Galactosyl-Ceramide (αGalCer) or its analogs (in our study PBS57). Our results show that the effect of these cells in resistant mice (which are generally reminiscent of the situation in humans) is largely mediated by cytokine secretion, in particular IL-4, a Th2 cytokine. We conclude that NKT cells influence the course of Leishmania infection and that therapeutically modulating their function could be beneficial both to treat existing infections, as well as potentially develop desperately needed, effective vaccines.
doi:10.1371/journal.pntd.0002917
PMCID: PMC4072590  PMID: 24967701
7.  The CD200-CD200 Receptor Inhibitory Axis Controls Arteriogenesis and Local T Lymphocyte Influx 
PLoS ONE  2014;9(6):e98820.
The role of the CD200 ligand-CD200 receptor (CD200-CD200R) inhibitory axis is highly important in controlling myeloid cell function. Since the activation of myeloid cells is crucial in arteriogenesis, we hypothesized that disruption of the CD200-CD200R axis promotes arteriogenesis in a murine hindlimb ischemia model. Female Cd200−/− and wildtype (C57Bl/6J) mice underwent unilateral femoral artery ligation. Perfusion recovery was monitored over 7 days using Laser-Doppler analysis and was increased in Cd200−/− mice at day 3 and 7 after femoral artery ligation, compared to wildtype. Histology was performed on hindlimb muscles at baseline, day 3 and 7 to assess vessel geometry and number and inflammatory cell influx. Vessel geometry in non-ischemic muscles was larger, and vessel numbers in ischemic muscles were increased in Cd200−/− mice compared to wildtype. Furthermore, T lymphocyte influx was increased in Cd200−/− compared to wildtype. CD200R agonist treatment was performed in male C57Bl/6J mice to validate the role of the CD200-CD200R axis in arteriogenesis. CD200R agonist treatment after unilateral femoral artery ligation resulted in a significant decrease in vessel geometry, perfusion recovery and T lymphocyte influx at day 7 compared to isotype treatment. In this study, we show a causal role for the CD200-CD200R inhibitory axis in arteriogenesis in a murine hindlimb ischemia model. Lack of CD200R signaling is accompanied by increased T lymphocyte recruitment to the collateral vasculature and results in enlargement of preexisting collateral arteries.
doi:10.1371/journal.pone.0098820
PMCID: PMC4045841  PMID: 24897500
8.  The role of co-inhibitory signals in spontaneous tolerance of weakly mismatched transplants 
Immunobiology  2011;216(8):918-924.
The immune system of female H-2b (C57BL/6) mice is a strong responder against the male minor-H antigen. However rejection or acceptance of such weakly mismatched grafts depends on the type of tissue transplanted. The mechanism responsible for such spontaneous graft acceptance, and its relationship to the natural mechanisms of tolerance of self antigens is unknown. Co-inhibitory molecules negatively regulate immune responses, and are important for self tolerance. We examined whether co-inhibitory molecules play a critical role in “spontaneous” allograft tolerance. Naïve or donor sensitized diabetic female C57BL/6 (B6) wild type (WT), PD-1−/−, and BTLA−/− mice were transplanted with freshly isolated syngeneic male islet grafts. The role of co-inhibitors during priming of anti-donor responses and graft challenge was also assessed using monoclonal antibodies targeting co-inhibitory receptors. Among the co-inhibitor (CTLA-4, PD-1) specific antibodies tested, only anti-PD-1 showed some potential to prevent spontaneous acceptance of male islet grafts. All BTLA−/− and almost all PD-1−/− recipients maintained the ability to spontaneously accept male islet grafts. While spontaneous graft acceptance in naïve recipients was only weakly PD-1 dependent, tolerance induced by the accepted islets was found to be highly PD-1 dependent. Furthermore, spontaneous graft acceptance in pre-sensitized recipients showed an absolute requirement for recipient PD-1 but not BTLA. Thus, the PD-1 pathway, involved in self tolerance, plays a critical role in spontaneous tolerance induced by weakly mismatched grafts in naïve recipients and spontaneous graft acceptance in pre-sensitized recipients.
doi:10.1016/j.imbio.2011.01.007
PMCID: PMC4030676  PMID: 21281982
Co-inhibitory molecules; HY antigen; islet transplantation; PD-1; Tolerance
9.  Bifidobacterium breve and Lactobacillus rhamnosus treatment is as effective as budesonide at reducing inflammation in a murine model for chronic asthma 
Respiratory Research  2014;15(1):46.
Background
Asthma is estimated to affect as many as 300 million people worldwide and its incidence and prevalence are rapidly increasing throughout the world, especially in children and within developing countries. Recently, there has been a growing interest in the use of potentially beneficial bacteria for allergic diseases. This study is aimed at exploring the therapeutic effects of long-term treatment with two different beneficial bacterial strains (Bifidobacterium breve M-16 V and Lactobacillus rhamnosus NutRes1) and a glucocorticoid (budesonide), as a reference treatment, on inflammatory response in a murine model for chronic allergic asthma.
Methods
To mimic the chronic disease in asthmatic patients, we used the murine ovalbumin-induced asthma model combined with prolonged allergen exposure. Airway function; pulmonary airway inflammation; airway remodelling, mRNA expression of pattern recognition receptors, Th-specific cytokines and transcription factors in lung tissue; mast cell degranulation; in vitro T cell activation; and expression of Foxp3 in blood Th cells were examined.
Results
Lactobacillus rhamnosus reduced lung resistance to a similar extent as budesonide treatment in chronically asthmatic mice. Pulmonary airway inflammation, mast cell degranulation, T cell activation and airway remodelling were suppressed by all treatments. Beneficial bacteria and budesonide differentially modulated the expression of toll-like receptors (TLRs), nod-like receptors (NLRs), cytokines and T cell transcription factors. Bifidobacterium breve induced regulatory T cell responses in the airways by increasing Il10 and Foxp3 transcription in lung tissue as well as systemic by augmenting the mean fluorescence intensity of Foxp3 in blood CD4+ T cells.
Conclusion
These findings show that Bifidobacterium breve M-16 V and Lactobacillus rhamnosus NutRes1 have strong anti-inflammatory properties that are comparable to budesonide and therefore may be beneficial in the treatment of chronic asthma.
doi:10.1186/1465-9921-15-46
PMCID: PMC4029990  PMID: 24735374
Allergic asthma; Beneficial bacteria; Glucocorticoids; Regulatory T cell
10.  Adrenergic β2 Receptor Activation Stimulates Anti-Inflammatory Properties of Dendritic Cells In Vitro 
PLoS ONE  2014;9(1):e85086.
Vagal nerve efferent activation has been shown to ameliorate the course of many inflammatory disease states. This neuro-modulatory effect has been suggested to rest on acetylcholine receptor (AChR) activation on tissue macrophages or dendritic cells (DCs). In more recent studies, vagal anti-inflammatory activity was shown involve adrenergic, splenic, pathways. Here we provide evidence that the adrenergic, rather than cholinergic, receptor activation on bone marrow derived DCs results in enhanced endocytosis uptake, enhanced IL-10 production but a decreased IL-6, IL-12p70 and IL-23 production. In antigen specific T cell stimulation assays, adrenergic β2 receptor activation on bone marrow DCs led to an enhanced potential to induce Foxp3 positive suppressive Treg cells. These effects were independent of IL10-R activation, TGFβ release, or retinoic acid (RA) secretion. Hence, adrenergic receptor β2 activation modulates DC function resulting in skewing towards anti-inflammatory T cell phenotypes.
doi:10.1371/journal.pone.0085086
PMCID: PMC3898911  PMID: 24465481
11.  Alterations in Regulatory T Cells Induced by Specific Oligosaccharides Improve Vaccine Responsiveness in Mice 
PLoS ONE  2013;8(9):e75148.
Prophylactic vaccinations are generally performed to protect naïve individuals with or without suppressed immune responsiveness. In a mouse model for Influenza vaccinations the specific alterations of CD4+CD25+Foxp3+ regulatory T-cells (Tregs) in the immune modulation induced by orally supplied oligosaccharides containing scGOS/lcFOS/pAOS was assessed. This dietary intervention increased vaccine specific DTH responses. In addition, a significant increased percentage of T-bet+ (Th1) activated CD69+CD4+ T cells (p<0.001) and reduced percentage of Gata-3+ (Th2) activated CD69+CD4+T cells (p<0.001) was detected in the mesenteric lymph nodes (MLN) of mice receiving scGOS/lcFOS/pAOS compared to control mice. Although no difference in the number or percentage of Tregs (CD4+Foxp3+) could be determined after scGOS/lcFOS/pAOS intervention, the percentage of CXCR3 + /T-bet+ (Th1-Tregs) was significantly reduced (p<0.05) in mice receiving scGOS/lcFOS/pAOS as compared to mice receiving placebo diets. Moreover, although no absolute difference in suppressive capacity could be detected, an alteration in cytokine profile suggests a regulatory T cell shift towards a reducing Th1 suppression profile, supporting an improved vaccination response.
In conclusion
These data are indicative for improved vaccine responsiveness due to reduced Th1 suppressive capacity in the Treg population of mice fed the oligosaccharide specific diet, showing compartmentalization within the Treg population. The modulation of Tregs to control immune responses provides an additional arm of intervention using alternative strategies possibly leading to the development of improved vaccines.
doi:10.1371/journal.pone.0075148
PMCID: PMC3779252  PMID: 24073243
12.  Trefoil factor 2 negatively regulates Type 1 immunity against Toxoplasma gondii 
Interleukin 12 (IL-12)-mediated Type 1 inflammation confers host-protection against the parasitic protozoan Toxoplasma gondii. However, production of interferon gamma (IFN-γ), another Type 1 inflammatory cytokine, also drives lethality from excessive injury to the intestinal epithelium. As mechanisms that restore epithelial barrier function following infection remain poorly understood, this study investigated the role of Trefoil factor 2 (TFF2), a well-established regulator of mucosal tissue repair. Paradoxically, TFF2 antagonized IL-12 release from DC’s and macrophages, which protected TFF2 deficient mice (TFF2−/−) from T. gondii pathogenesis. Dysregulated intestinal homeostasis in naïve TFF2−/− mice correlated with increased IL-12/23p40 levels and enhanced T cell recruitment at baseline. Infected TFF2−/− mice displayed low rates of parasite replication and reduced gut immunopathology, whereas WT mice experienced disseminated infection and lethal ileitis. p38 MAPK activation and IL-12p70 production was more robust from TFF2−/− CD8+ DC compared to WT CD8+ DC and treatment of WT DC with rTFF2 suppressed TLR-induced IL-12/23p40 production. Neutralization of IFN-γ and IL-12 in TFF2−/− animals abrogated resistance shown by enhanced parasite replication and infection-induced morbidity. Hence, TFF2 regulated intestinal barrier function and Type 1 cytokine release from myeloid phagocytes, which dictated the outcome of oral T. gondii infection in mice.
doi:10.4049/jimmunol.1103374
PMCID: PMC3436937  PMID: 22896633
macrophage; epithelial cells; injury; inflammation
13.  IgA EGFR antibodies mediate tumour killing in vivo 
EMBO Molecular Medicine  2013;5(8):1213-1226.
Currently all approved anti-cancer therapeutic monoclonal antibodies (mAbs) are of the IgG isotype, which rely on Fcgamma receptors (FcγRs) to recruit cellular effector functions. In vitro studies showed that targeting of FcαRI (CD89) by bispecific antibodies (bsAbs) or recombinant IgA resulted in more effective elimination of tumour cells by myeloid effector cells than targeting of FcγR. Here we studied the in vivo anti-tumour activity of IgA EGFR antibodies generated using the variable sequences of the chimeric EGFR antibody cetuximab. Using FcαRI transgenic mice, we demonstrated significant in vivo anti-tumour activity of IgA2 EGFR against A431 cells in peritoneal and lung xenograft models, as well as against B16F10-EGFR cells in a lung metastasis model in immunocompetent mice. IgA2 EGFR was more effective than cetuximab in a short-term syngeneic peritoneal model using EGFR-transfected Ba/F3 target cells. The in vivo cytotoxic activity of IgA2 EGFR was mediated by macrophages and was significantly decreased in the absence of FcαRI. These results support the potential of targeting FcαRI for effective antibody therapy of cancer.
The study reveals that IgA antibodies directed against EGFR and engaging Fcalpha receptor (FcαRI) on effector cells, have in vivo anti-cancer activity. These data support the development of novel immunotherapeutic strategies based on targeting FcαRI.
doi:10.1002/emmm.201201929
PMCID: PMC3944462  PMID: 23918228
antibody therapy; EGFR; Fcalpha receptor I; IgA; tumour immunology
14.  Passive Immunization with Hypochlorite-oxLDL Specific Antibodies Reduces Plaque Volume in LDL Receptor-Deficient Mice 
PLoS ONE  2013;8(7):e68039.
Aims
New strategies to overcome complications of cardiovascular diseases are needed. Since it has been demonstrated that atherosclerosis is an inflammatory disease, modulation of the immune system may be a promising approach. Previously, it was suggested that antibodies may confer protective effects on the development of atherosclerosis. In this study, we hypothesised that passive immunization with anti-oxLDL IgM antibodies specific for hypochlorite (HOCl) may be athero-protective in mice.
Methods and Results
Monoclonal mouse IgM antibodies were produced and the antibody with specificity for hypochlorite-oxLDL (HOCl-oxLDL) (Moab A7S8) was selected. VH sequence determination revealed that Moab A7S8 is a natural IgM antibody. Atherosclerosis in LDLr−/− mice was induced by a perivascular collar placement around the right carotid artery in combination with feeding a high-fat diet. Subsequently, the mice were treated every six days with 500 µg Moab A7S8, non-relevant IgM or with PBS and the carotid arteries and aortic roots were studied for atherosclerosis. Passive immunization with this Moab A7S8 resulted in a significant reduced plaque volume formation in LDLr−/− mice when compared with PBS treatment (P = 0.002 and P = 0.035). Cholesterol levels decreased by 20% when mice were treated with Moab A7S8 compared to PBS. Furthermore, anti-oxLDL specific IgM and IgG antibody production increased significantly in the Moab A7S8 treated mice in comparison with PBS treated mice.
Conclusion
Our data show that passive immunization with a natural IgM antibody, directed to HOCl-oxLDL, can reduce atherosclerotic plaque development. We postulate that specific antibody therapy may be developed for use in human cardiovascular diseases.
doi:10.1371/journal.pone.0068039
PMCID: PMC3713002  PMID: 23874490
15.  Lipocalin 2 deactivates macrophages and worsens pneumococcal pneumonia outcomes 
The Journal of Clinical Investigation  2013;123(8):3363-3372.
Macrophages play a key role in responding to pathogens and initiate an inflammatory response to combat microbe multiplication. Deactivation of macrophages facilitates resolution of the inflammatory response. Deactivated macrophages are characterized by an immunosuppressive phenotype, but the lack of unique markers that can reliably identify these cells explains the poorly defined biological role of this macrophage subset. We identified lipocalin 2 (LCN2) as both a marker of deactivated macrophages and a macrophage deactivator. We show that LCN2 attenuated the early inflammatory response and impaired bacterial clearance, leading to impaired survival of mice suffering from pneumococcal pneumonia. LCN2 induced IL-10 formation by macrophages, skewing macrophage polarization in a STAT3-dependent manner. Pulmonary LCN2 levels were tremendously elevated during bacterial pneumonia in humans, and high LCN2 levels were indicative of a detrimental outcome from pneumonia with Gram-positive bacteria. Our data emphasize the importance of macrophage deactivation for the outcome of pneumococcal infections and highlight the role of LCN2 and IL-10 as determinants of macrophage performance in the respiratory tract.
doi:10.1172/JCI67911
PMCID: PMC3726165  PMID: 23863624
16.  NKT Cell-TCR Expression Activates Conventional T Cells in Vivo, but Is Largely Dispensable for Mature NKT Cell Biology 
PLoS Biology  2013;11(6):e1001589.
Natural killer T (NKT) cell development depends on recognition of self-glycolipids via their semi-invariant Vα14i-TCR. However, to what extent TCR-mediated signals determine identity and function of mature NKT cells remains incompletely understood. To address this issue, we developed a mouse strain allowing conditional Vα14i-TCR expression from within the endogenous Tcrα locus. We demonstrate that naïve T cells are activated upon replacement of their endogenous TCR repertoire with Vα14i-restricted TCRs, but they do not differentiate into NKT cells. On the other hand, induced TCR ablation on mature NKT cells did not affect their lineage identity, homeostasis, or innate rapid cytokine secretion abilities. We therefore propose that peripheral NKT cells become unresponsive to and thus are independent of their autoreactive TCR.
Author Summary
Immune system natural killer T (NKT) cells help to protect against certain strains of bacteria and viruses, and suppress the development of autoimmune diseases and cancer. However, NKT cells are also central mediators of allergic responses. The recognition of one's own glycolipid antigens (self-glycolipids) in the thymus via the unique Vα14i T cell receptor, Vα14i-TCR, triggers the NKT cell developmental program, which differs considerably from that of conventional T cells. We generated a mouse model to investigate whether the Vα14i-TCR on mature NKT cells constantly recognizes self-glycolipids and to assess whether this TCR is required for survival and continued NKT cell identity. Switching the peptide-recognizing TCR of a mature conventional T cell to a glycolipid-recognizing Vα14i-TCR led to activation of the T cells, indicating that this TCR is also autoreactive on peripheral T cells or can signal autonomously. But TCR ablation did not affect the half-life, characteristic gene expression or innate functions of mature NKT cells. Therefore, the inherently autoreactive Vα14i-TCR is dispensable for the functions of mature peripheral NKT cells after instructing thymic NKT cell development. Thus the Vα14i-TCR serves a similar function to pattern-recognition receptors, in mediating immune recognition of foreign invasion or diseased cells.
doi:10.1371/journal.pbio.1001589
PMCID: PMC3708704  PMID: 23853545
17.  Immune Adjuvant Efficacy of CpG Oligonucleotide in Cancer Treatment Is Founded Specifically upon TLR9 Function in Plasmacytoid Dendritic Cells 
Cancer research  2011;71(20):6428-6437.
The differences in function, location, and migratory pattern of conventional dendritic cells (cDC) and plasmacytoid DCs (pDC) not only point to specialized roles in immune responses but also signify additive and interdependent relationships required to clear pathogens. We studied the in vivo requirement of cross-talk between cDCs and pDCs for eliciting antitumor immunity against in situ released tumor antigens in the absence or presence of the Toll-like receptor (TLR) 9 agonist CpG. Previous data indicated that CpG boosted tumor-specific T-cell responses after in vivo tumor destruction and increased survival after tumor rechallenges. The present study shows that cDCs are indispensable for cross-presentation of ablation-released tumor antigens and for the induction of long-term antitumor immunity. Depletion of pDCs or applying this model in type I IFN receptor–deficient mice abrogated CpG-mediated responses. CD8α+ cDCs and the recently identified merocytic cDCs were dependent on pDCs for CpG-induced upregulation of CD80. Moreover, DC transfer studies revealed that merocytic cDCs and CD8α+ cDCs were most susceptible to pDC help and subsequently promoted tumor-free survival in a therapeutic setting. By transferring wild-type pDCs into TLR9-deficient mice, we finally showed that TLR9 expression in pDCs is sufficient to benefit from CpG as an adjuvant. These studies indicate that the efficacy of CpG in cancer immunotherapy is dependent on cross-talk between pDCs and specific subsets of cDCs.
doi:10.1158/0008-5472.CAN-11-2154
PMCID: PMC3653311  PMID: 21788345
18.  Depletion of Regulatory T Cells in a Mouse Experimental Glioma Model through Anti-CD25 Treatment Results in the Infiltration of Non-Immunosuppressive Myeloid Cells in the Brain 
The recruitment and activation of regulatory T cells (Tregs) in the micro-environment of malignant brain tumors has detrimental effects on antitumoral immune responses. Hence, local elimination of Tregs within the tumor micro-environment represents a highly valuable tool from both a fundamental and clinical perspective. In the syngeneic experimental GL261 murine glioma model, Tregs were prophylactically eliminated through treatment with PC61, an anti-CD25 mAb. This resulted in specific elimination of CD4+CD25hiFoxp3+ Treg within brain-infiltrating lymphocytes and complete protection against subsequent orthotopic GL261 tumor challenge. Interestingly, PC61-treated mice also showed a pronounced infiltration of CD11b+ myeloid cells in the brain. Phenotypically, these cells could not be considered as Gr-1+ myeloid-derived suppressor cells (MDSC) but were identified as F4/80+ macrophages and granulocytes.
doi:10.1155/2013/952469
PMCID: PMC3655451  PMID: 23710206
19.  The Mucosal Adjuvant Cholera Toxin B Instructs Non-Mucosal Dendritic Cells to Promote IgA Production Via Retinoic Acid and TGF-β 
PLoS ONE  2013;8(3):e59822.
It is currently unknown how mucosal adjuvants cause induction of secretory immunoglobulin A (IgA), and how T cell-dependent (TD) or -independent (TI) pathways might be involved. Mucosal dendritic cells (DCs) are the primary antigen presenting cells driving TI IgA synthesis, by producing a proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), Retinoic Acid (RA), TGF-β or nitric oxide (NO). We hypothesized that the mucosal adjuvant Cholera Toxin subunit B (CTB) could imprint non-mucosal DCs to induce IgA synthesis, and studied the mechanism of its induction. In vitro, CTB-treated bone marrow derived DCs primed for IgA production by B cells without the help of T cells, yet required co-signaling by different Toll-like receptor (TLR) ligands acting via the MyD88 pathway. CTB-DC induced IgA production was blocked in vitro or in vivo when RA receptor antagonist, TGF-β signaling inhibitor or neutralizing anti-TGF-β was added, demonstrating the involvement of RA and TGF-β in promoting IgA responses. There was no major involvement for BAFF, APRIL or NO. This study highlights that synergism between CTB and MyD88-dependent TLR signals selectively imprints a TI IgA-inducing capacity in non-mucosal DCs, explaining how CTB acts as an IgA promoting adjuvant.
doi:10.1371/journal.pone.0059822
PMCID: PMC3603891  PMID: 23527272
20.  Distinctive Cytokines as Biomarkers Predicting Fatal Outcome of Severe Staphylococcus aureus Bacteremia in Mice 
PLoS ONE  2013;8(3):e59107.
Invasive Staphylococcus aureus infections are frequently associated with bacteraemia. To support clinical decisions on antibiotic therapy, there is an urgent need for reliable markers as predictors of infection outcome. In the present study in mice, bacteraemia was established by intravenous inoculation of a clinical S. aureus isolate at the LD50 inoculum. As potential biomarkers for fatal outcome, blood culture (qualitative and quantitative), serum levels of C-reactive protein (CRP), as well as 31 selected cytokines and chemokines were assessed during the first three days of infection. A positive S. aureus blood culture, the quantitative blood culture, CRP levels, and levels of eight cytokines were indicative for the presence of S. aureus bacteraemia. However, only tumor necrosis factor (TNF) α, interleukin (IL) 1α, and keratinocyte chemoattractant (KC; a functional homologue of human IL-8) were each significantly elevated in eventually non-surviving infected mice versus eventually surviving infected mice. In severe S. aureus bacteraemia in mice, TNF-α, IL-1α, and KC are biomarkers predicting fatal outcome of infection. KC was a biomarker elevated irrespective the progression of infection, which is very interesting regarding clinical application in view of the heterogeneity of patients experiencing bacteraemia in this respect.
doi:10.1371/journal.pone.0059107
PMCID: PMC3592822  PMID: 23520553
21.  In Vivo Ablation of Plasmacytoid Dendritic Cells Inhibits Autoimmunity through Expansion of Myeloid-Derived Suppressor Cells 
Autoimmunity ensues upon breakdown of tolerance mechanism and priming of self-reactive T cells. Plasmacytoid dendritic cells (pDCs) constitute a unique cell subset that participates in the activation of autoreactive T cells but also has been shown to be critically involved in the induction of self-tolerance. However, their functional importance during the priming phase of an organ-specific autoimmune response remains unclear. In this study, we demonstrate that absence of pDCs during myelin antigenic challenge resulted in amelioration of experimental autoimmune encephalomyelitis and reduced disease severity. This was accompanied by significantly decreased frequency of myelin-specific T cells in the draining lymph nodes and inhibition of Th1 and Th17 immune responses. Unexpectedly, in vivo ablation of pDCs increased myelopoiesis in the bone marrow and specifically induced the generation of CD11bhiGr1+ myeloid-derived suppressor cells (MDSCs). Furthermore, we demonstrate that pDC depletion enhanced the mobilization of MDSCs in the spleen, and that sorted MDSCs could potently suppress CD4+ T cell responses in vitro. Importantly, pDC-depleted mice showed increased levels of MCP-1 in the draining lymph nodes, and in vivo administration of MCP-1 increased the frequency and absolute numbers of MDSCs in the periphery of treated mice. Together, our results reveal that absence of pDCs during the priming of an autoimmune response leads to increased mobilization of MDSCs in the periphery in an MCP-1–dependent manner and subsequent amelioration of autoimmunity.
doi:10.4049/jimmunol.1201897
PMCID: PMC3586977  PMID: 23382560
22.  Plasmacytoid dendritic cells protect against atherosclerosis by tuning T cell proliferation and activity 
Circulation research  2011;109(12):1387-1395.
Rationale
Unlike conventional dendritic cells (cDC), plasmacytoid DCs (pDC) are poor in antigen presentation and critical for type I interferon response. While proposed to be present in human atherosclerotic lesions, their role in atherosclerosis remains elusive.
Objective
To investigate the role of pDC in atherosclerosis.
Methods and Results
We show that pDC are scarcely present in human atherosclerotic lesions, and almost absent in mouse plaques. Surprisingly, pDC depletion by 120G8 mAb administration was seen to promote plaque T cell accumulation and exacerbate lesion development and progression in LDLr−/− mice. PDC depletion was accompanied by increased CD4+ T cell proliferation, IFN-γ expression by splenic T cells and plasma IFN-γ levels. Lymphoid tissue pDC from atherosclerotic mice showed increased indoleamine 2,3-dioxygenase (IDO) expression and IDO blockage abrogated the pDC suppressive effect on T cell proliferation.
Conclusion
Our data reveal a protective role for pDC in atherosclerosis, possibly by dampening T cell proliferation and activity in peripheral lymphoid tissue, rendering pDC an interesting target for future therapeutic interventions.
doi:10.1161/CIRCRESAHA.111.256529
PMCID: PMC3237962  PMID: 22021930
Plasmacytoid dendritic cells; atherosclerosis; immune tolerance; T cells
23.  Expression and Function of Transforming Growth Factor β in Melioidosis 
Infection and Immunity  2012;80(5):1853-1857.
Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast Asia and northern Australia. An important controller of the immune system is the pleiotropic cytokine transforming growth factor β (TGF-β), of which Smad2 and Smad3 are the major signal transducers. In this study, we aimed to characterize TGF-β expression and function in experimental melioidosis. TGF-β expression was determined in 33 patients with culture-proven infection with B. pseudomallei and 30 healthy controls. We found that plasma TGF-β concentrations were strongly elevated during melioidosis. In line with this finding, TGF-β expression in C57BL/6 mice intranasally inoculated with B. pseudomallei was enhanced as well. To assess the role of TGF-β, we inhibited TGF-β using a selective murine TGF-β antibody. Treatment of mice with anti-TGF-β antibody resulted in decreased lung Smad2 phosphorylation. TGF-β blockade appeared to be protective: mice treated with anti-TGF-β antibody and subsequently infected with B. pseudomallei showed diminished bacterial loads. Moreover, less distant organ injury was observed in anti-TGF-β treated mice as shown by reduced blood urea nitrogen (BUN) and aspartate transaminase (AST) values. However, anti-TGF-β treatment did not have an effect on survival. In conclusion, TGF-β is upregulated during B. pseudomallei infection and plays a limited but proinflammatory role during experimental melioidosis.
doi:10.1128/IAI.05534-11
PMCID: PMC3347439  PMID: 22331429
24.  Natural killer T cells in adipose tissue prevent insulin resistance 
The Journal of Clinical Investigation  2012;122(9):3343-3354.
Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under high-fat diet conditions. Here, we focused on the role of iNKT cells under normal conditions. We showed that iNKT cell–deficient mice on a low-fat diet, considered a normal diet for mice, displayed a distinctive insulin resistance phenotype without overt adipose tissue inflammation. Insulin resistance was characterized by adipocyte dysfunction, including adipocyte hypertrophy, increased leptin, and decreased adiponectin levels. The lack of liver abnormalities in CD1d-null mice together with the enrichment of CD1d-restricted iNKT cells in both mouse and human adipose tissue indicated a specific role for adipose tissue–resident iNKT cells in the development of insulin resistance. Strikingly, iNKT cell function was directly modulated by adipocytes, which acted as lipid antigen-presenting cells in a CD1d-mediated fashion. Based on these findings, we propose that, especially under low-fat diet conditions, adipose tissue–resident iNKT cells maintain healthy adipose tissue through direct interplay with adipocytes and prevent insulin resistance.
doi:10.1172/JCI62739
PMCID: PMC3428087  PMID: 22863618
25.  A non-redundant role for plasmacytoid dendritic cells in host defense against the human fungal pathogen Aspergillus fumigatus 
Cell host & microbe  2011;9(5):415-424.
Summary:
While plasmacytoid dendritic cells (pDCs), a natural type I interferon (IFN) producing cell type, are regarded as critical for innate immunity to viruses, their role in defense against fungal infections remains unknown. We examined the interactions of pDCs with hyphae of the invasive human fungal pathogen Aspergillus fumigatus. Human pDCs spread over hyphae and inhibited their growth. Antifungal activity was retained in pDC lysates, did not require direct fungal contact, and was partially reversed by zinc. Incubation with hyphae resulted in pDC cytotoxicity, partly due to fungal gliotoxin secretion. Following hyphal stimulation, pDCs released proinflammatory cytokines via a TLR9-independent mechanism. Pulmonary challenge of mice with A. fumigatus resulted in a substantial influx of pDCs into lungs and pDC-depleted mice were hypersusceptible to invasive aspergillosis. These data demonstrate the antifungal activity of pDCs against A. fumigatus and establish their non-redundant role in host defenses against invasive aspergillosis in vivo.
doi:10.1016/j.chom.2011.04.007
PMCID: PMC3100664  PMID: 21575912

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