ABSTRACT
Chronic schistosome infections protect against allergic airway inflammation (AAI) via the induction of IL-10-producing splenic regulatory B (Breg) cells. Previous experiments have demonstrated that schistosome-induced pulmonary B cells can also reduce AAI, but act independently of IL-10. We have now further characterized the phenotype and inhibitory activity of these protective pulmonary B cells. We excluded a role for regulatory T (Treg) cell induction as putative AAI-protective mechanisms. Schistosome-induced B cells showed increased CD86 expression and reduced cytokine expression in response to Toll-like receptor (TLR) ligands compared with control B cells. To investigate the consequences for T cell activation we cultured ovalbumin (OVA)-pulsed, schistosome-induced B cells with OVA-specific transgenic T cells and observed less Th2 cytokine expression and T cell proliferation compared with control conditions. This suppressive effect was preserved even under optimal T cell stimulation by anti-CD3/28. Blocking of the inhibitory cytokines IL-10 or TGF-ß only marginally restored Th2 cytokine induction. These data suggest that schistosome-induced pulmonary B cells are impaired in their capacity to produce cytokines to TLR ligands and to induce Th2 cytokine responses independent of their antigen-presenting function. These findings underline the presence of distinct B cell subsets with different stimulatory or inhibitory properties even if induced by the same type of helminth.
Subject(s)
B-Lymphocytes, Regulatory/immunology , Lung/cytology , Respiratory Hypersensitivity/immunology , Schistosomiasis/immunology , Th2 Cells/immunology , Adoptive Transfer , Animals , Bronchoalveolar Lavage Fluid/immunology , Cytokines/analysis , Female , Immunophenotyping , Interleukin-10/metabolism , Lung/immunology , Mice , Mice, Inbred C57BL , Respiratory Hypersensitivity/prevention & control , Schistosomiasis/complications , Specific Pathogen-Free OrganismsABSTRACT
Helminth infection is frequently associated with the expansion of regulatory T cells (Tregs) and suppression of immune responses to bystander antigens. We show that infection of mice with the chronic gastrointestinal helminth Heligmosomoides polygyrus drives rapid polyclonal expansion of Foxp3(+)Helios(+)CD4(+) thymic (t)Tregs in the lamina propria and mesenteric lymph nodes while Foxp3(+)Helios(-)CD4(+) peripheral (p)Treg expand more slowly. Notably, in partially resistant BALB/c mice parasite survival positively correlates with Foxp3(+)Helios(+)CD4(+) tTreg numbers. Boosting of Foxp3(+)Helios(+)CD4(+) tTreg populations by administration of recombinant interleukin-2 (rIL-2):anti-IL-2 (IL-2C) complex increased worm persistence by diminishing type-2 responsiveness in vivo, including suppression of alternatively activated macrophage and granulomatous responses at the sites of infection. IL-2C also increased innate lymphoid cell (ILC) numbers, indicating that Treg functions dominate over ILC effects in this setting. Surprisingly, complete removal of Tregs in transgenic Foxp3-DTR mice also resulted in increased worm burdens, with "immunological chaos" evident in high levels of the pro-inflammatory cytokines IL-6 and interferon-γ. In contrast, worm clearance could be induced by anti-CD25 antibody-mediated partial depletion of early Treg, alongside increased T helper type 2 responses and without incurring pathology. These findings highlight the overarching importance of the early Treg response to infection and the non-linear association between inflammation and the prevailing Treg frequency.
Subject(s)
Immunity, Mucosal/drug effects , Macrophages/immunology , Nematospiroides dubius/immunology , Strongylida Infections/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Antibodies, Neutralizing/pharmacology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Gene Expression Regulation , Granulocytes/drug effects , Granulocytes/immunology , Granulocytes/parasitology , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-2/pharmacology , Interleukin-2 Receptor alpha Subunit/antagonists & inhibitors , Interleukin-2 Receptor alpha Subunit/genetics , Interleukin-2 Receptor alpha Subunit/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Macrophages/drug effects , Macrophages/parasitology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Nematospiroides dubius/drug effects , Parasite Load , Signal Transduction , Strongylida Infections/drug therapy , Strongylida Infections/parasitology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/parasitology , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/parasitology , Th17 Cells/drug effects , Th17 Cells/immunology , Th17 Cells/parasitology , Transcription Factors/genetics , Transcription Factors/immunologyABSTRACT
Foxp3 (forkhead box P3 transcription factor)-expressing regulatory T cells (Tregs) are essential for immunological tolerance, best illustrated by uncontrolled effector T-cell responses and autoimmunity upon loss of Foxp3 expression. Tregs can adopt specific effector phenotypes upon activation, reflecting the diversity of functional demands in the different tissues of the body. Here, we report that Foxp3(+)CD4(+) T cells coexpressing retinoic acid-related orphan receptor-γt (RORγt), the master transcription factor for T helper type 17 (Th17) cells, represent a stable effector Treg lineage. Transcriptomic and epigenetic profiling revealed that Foxp3(+)RORγt(+) T cells display signatures of both Tregs and Th17 cells, although the degree of similarity was higher to Foxp3(+)RORγt(-) Tregs than to Foxp3(-)RORγt(+) T cells. Importantly, Foxp3(+)RORγt(+) T cells were significantly demethylated at Treg-specific epigenetic signature genes such as Foxp3, Ctla-4, Gitr, Eos, and Helios, suggesting that these cells have a stable regulatory rather than inflammatory function. Indeed, adoptive transfer of Foxp3(+)RORγt(+) T cells in the T-cell transfer colitis model confirmed their Treg function and lineage stability in vivo, and revealed an enhanced suppressive capacity as compared with Foxp3(+)RORγt(-) Tregs. Thus, our data suggest that RORγt expression in Tregs contributes to an optimal suppressive capacity during gut-specific immune responses, rendering Foxp3(+)RORγt(+) T cells as an important effector Treg subset in the intestinal system.
Subject(s)
Colitis/immunology , Forkhead Transcription Factors/immunology , Immunity, Mucosal/drug effects , Nuclear Receptor Subfamily 1, Group F, Member 3/immunology , T-Lymphocytes, Regulatory/immunology , Adoptive Transfer , Animals , CTLA-4 Antigen/genetics , CTLA-4 Antigen/immunology , Carrier Proteins/genetics , Carrier Proteins/immunology , Cell Lineage , Colitis/genetics , Colitis/pathology , Colon/immunology , Colon/pathology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Epigenesis, Genetic/immunology , Female , Forkhead Transcription Factors/genetics , Glucocorticoid-Induced TNFR-Related Protein/genetics , Glucocorticoid-Induced TNFR-Related Protein/immunology , Inflammation , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/immunology , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Signal Transduction , T-Lymphocytes, Regulatory/pathology , T-Lymphocytes, Regulatory/transplantation , Transcription Factors/genetics , Transcription Factors/immunologyABSTRACT
The Gram-positive bacterium Streptococcus pneumoniae causes life-threatening infections, especially among immunocompromised patients. The host's immune system senses S. pneumoniae via different families of pattern recognition receptors, in particular the Toll-like receptor (TLR) family that promotes immune cell activation. Yet, while single TLRs are dispensable for initiating inflammatory responses against S. pneumoniae, the central TLR adapter protein myeloid differentiation factor 88 (MyD88) is of vital importance, as MyD88-deficient mice succumb rapidly to infection. Since MyD88 is ubiquitously expressed in hematopoietic and non-hematopoietic cells, the extent to which MyD88 signaling is required in different cell types to control S. pneumoniae is unknown. Therefore, we used novel conditional knockin mice to investigate the necessity of MyD88 signaling in distinct lung-resident myeloid and epithelial cells for the initiation of a protective immune response against S. pneumoniae. Here, we show that MyD88 signaling in lysozyme M (LysM)- and CD11c-expressing myeloid cells, as well as in pulmonary epithelial cells, is critical to restore inflammatory cytokine and antimicrobial peptide production, leading to efficient neutrophil recruitment and enhanced bacterial clearance. Overall, we show a novel synergistic requirement of compartment-specific MyD88 signaling in S. pneumoniae immunity.
Subject(s)
Epithelial Cells/immunology , Lung/immunology , Myeloid Differentiation Factor 88/immunology , Neutrophils/immunology , Pneumonia, Pneumococcal/immunology , Animals , CD11c Antigen/genetics , CD11c Antigen/immunology , Cell Communication/immunology , Epithelial Cells/microbiology , Gene Expression Regulation , Gene Knock-In Techniques , Lung/microbiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muramidase/genetics , Muramidase/immunology , Myeloid Differentiation Factor 88/genetics , Neutrophil Infiltration , Neutrophils/microbiology , Pneumonia, Pneumococcal/genetics , Pneumonia, Pneumococcal/microbiology , Signal Transduction , Streptococcus pneumoniae/growth & development , Streptococcus pneumoniae/immunology , Streptococcus pneumoniae/pathogenicityABSTRACT
During the past two decades, a growing interest surrounding the interaction between microbe-associated molecular patterns (MAMPs) and pattern recognition receptors has occurred. This attention is now driven alongside bacterial-derived metabolites, which impact immune cell differentiation and function. Hence, this review introduces the term meta-MAMP as a means to classify the microbial derived-metabolites, which influence the immune response by affecting specific cellular processes. We discuss two prominent examples of meta-MAMPs: the first, rapamycin (isolated from Streptomyces), was discovered in the 1970s and since then has been thoroughly studied. The second, soraphen A (isolated from Myxobacteria), was discovered in the early 1990s but only recently identified as a promising immunomodulator. Both meta-MAMPs are similar in their remarkable capacity to modulate T cell fate by targeting key metabolic pathways triggered upon T cell activation. In this context, we highlight the progress made in the field of immunometabolism and the possibility of modulating metabolic pathways such as cellular fatty acid metabolism as a strategy for immunomodulation. We focus on the use of microbial metabolites as auspicious agents for T cell fate modulation.
Subject(s)
Lymphocyte Activation/immunology , Pathogen-Associated Molecular Pattern Molecules/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , HumansABSTRACT
Intestinal infection with the mouse pathogen Citrobacter rodentium induces a strong local Th17 response in the colon. Although this inflammatory immune response helps to clear the pathogen, it also induces inflammation-associated pathology in the gut and thus, has to be tightly controlled. In this project, we therefore studied the impact of Foxp3(+) regulatory T cells (Treg) on the infectious and inflammatory processes elicited by the bacterial pathogen C. rodentium. Surprisingly, we found that depletion of Treg by diphtheria toxin in the Foxp3(DTR) (DEREG) mouse model resulted in impaired bacterial clearance in the colon, exacerbated body weight loss, and increased systemic dissemination of bacteria. Consistent with the enhanced susceptibility to infection, we found that the colonic Th17-associated T-cell response was impaired in Treg-depleted mice, suggesting that the presence of Treg is crucial for the establishment of a functional Th17 response after the infection in the gut. As a consequence of the impaired Th17 response, we also observed less inflammation-associated pathology in the colons of Treg-depleted mice. Interestingly, anti-interleukin (IL)-2 treatment of infected Treg-depleted mice restored Th17 induction, indicating that Treg support the induction of a protective Th17 response during intestinal bacterial infection by consumption of local IL-2.
Subject(s)
Citrobacter rodentium/immunology , Colon/immunology , Enterobacteriaceae Infections/immunology , Immunity, Mucosal , T-Lymphocytes, Regulatory/immunology , Th17 Cells/immunology , Animals , Colon/microbiology , Colon/pathology , Enterobacteriaceae Infections/pathology , Mice , T-Lymphocytes, Regulatory/pathology , Th17 Cells/pathologyABSTRACT
A major concept in autoimmunity is that disruption of Foxp3(+) regulatory T cells (Tregs) predisposes to breach of tolerance. This is exemplified by the Foxp3-linked disorder termed IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked) which affects newborn children. There has been considerable clinical interest in the role of non-depleting anti-CD4 antibodies as a means of upregulating the function of Foxp3(+) Tregs in order to control detrimental inflammatory responses such as transplant rejection. However, according to the paradigm of a Treg-dependent mechanism of action, the effectiveness of anti-CD4 antibodies as a therapy for human autoimmune diseases is unclear considering that Treg function might be intrinsically impaired. Specifically, anti-CD4 therapy is expected to fail in patients suffering from the IPEX syndrome due to the lack of functional Foxp3(+) Tregs. Taking advantage of natural Foxp3 mutant scurfy (sf) mice closely resembling the IPEX syndrome, and genetically engineered mice depleted of Foxp3(+) Tregs, we report here that anti-CD4 treatment induces tolerance independent of Foxp3(+) Tregs. This so far undefined mechanism is dependent on the recessive non-infectious tolerization of autoreactive T cells. Treg-independent tolerance alone is powerful enough to suppress both the onset and severity of autoimmunity and reduces clinically relevant autoantibody levels and liver fibrosis. Mechanistically, tolerance induction requires the concomitant activation of autoreactive T cells and is associated with the down-regulation of the co-stimulatory TNF-receptor superfamily members OX40 and CD30 sustaining CD4(+) T cell survival. In the light of ongoing clinical trials, our results highlight an unexpected potency of anti-CD4 antibodies for the treatment of autoimmune diseases. Particularly, CD4 blockade might represent a novel therapeutic option for the human IPEX syndrome.
Subject(s)
Antilymphocyte Serum/pharmacology , Autoimmunity/drug effects , CD4 Antigens/immunology , Forkhead Transcription Factors/immunology , Animals , CD4 Antigens/genetics , Cell Survival , Diabetes Mellitus, Type 1/congenital , Diarrhea , Disease Models, Animal , Female , Forkhead Transcription Factors/deficiency , Forkhead Transcription Factors/genetics , Gene Expression Regulation/immunology , Genetic Diseases, X-Linked/drug therapy , Genetic Diseases, X-Linked/genetics , Genetic Diseases, X-Linked/immunology , Genetic Diseases, X-Linked/pathology , Humans , Immune System Diseases/congenital , Immune Tolerance/drug effects , Ki-1 Antigen/genetics , Ki-1 Antigen/immunology , Lymphocyte Activation , Male , Mice , Mice, Transgenic , Receptors, OX40/genetics , Receptors, OX40/immunology , Signal Transduction , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathologyABSTRACT
Foxp3(+) regulatory T cells (Tregs) have an essential role in immune and allograft tolerance. However, in both kidney and liver transplantation in humans, FOXP3(+) Tregs have been associated with clinical rejection. Therefore, the role and function of graft infiltrating Tregs have been of great interest. In the studies outlined, we demonstrated that Foxp3(+) Tregs were expanded in tolerant kidney allografts and in draining lymph nodes in the DBA/2 (H-2(d) ) to C57BL/6 (H-2(b) ) mouse spontaneous kidney allograft tolerance model. Kidney allograft tolerance was abrogated after deletion of Foxp3(+) Tregs in DEpletion of REGulatory T cells (DEREG) mice. Kidney allograft infiltrating Foxp3(+) Tregs (K-Tregs) expressed elevated levels of TGF-ß, IL-10, interferon gamma (IFN-γ), the transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) and chemokine receptor 3 (Cxcr3). These K-Tregs had the capacity to transfer dominant tolerance and demonstrate donor alloantigen-specific tolerance to skin allografts. This study demonstrated the crucial role, potency and specificity of graft infiltrating Foxp3(+) Tregs in the maintenance of spontaneously induced kidney allograft tolerance.
Subject(s)
Forkhead Transcription Factors/physiology , Graft Rejection/immunology , Graft Survival/immunology , Immune Tolerance/immunology , Kidney Transplantation , T-Lymphocytes, Regulatory/immunology , Tissue Donors , Transplantation Tolerance/immunology , Allografts , Animals , Cytokines/metabolism , Genes, Reporter , Inflammation Mediators , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred DBA , Skin Transplantation , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathologyABSTRACT
CD4(+) helper T cells orchestrate protective immunity against pathogens, yet can also induce undesired pathologies including allergies, transplant rejection and autoimmunity. Non-depleting CD4-specific antibodies such as clone YTS177.9 were found to promote long-lasting T cell tolerance in animal models. Thus, CD4 blockade could represent a promising therapeutic approach for human autoimmune diseases. However, the mechanisms underlying anti-CD4-induced tolerance are incompletely resolved. Particularly, multiple immune cells express CD4 including Foxp3(+) regulatory T cells (Tregs) and dendritic cells (DCs), both controlling the activation of CD4(+)Foxp3(-) helper T cells. Utilizing mixed leukocyte reactions (MLRs) reflecting physiological interactions between T cells and DCs, we report that anti-CD4 treatment inhibits CD4(+)Foxp3(-) T cell proliferation in an IL-2-independent fashion. Notably, YTS177.9 binding induces a rapid internalization of CD4 on both CD4(+)Foxp3(-) T cells and Foxp3(+) Tregs. However, no expansion or activation of immunosuppressive CD4(+)Foxp3(+) Tregs was observed following anti-CD4 treatment. Additionally, cytokine production, maturation and T cell priming capacity of DCs are not affected by anti-CD4 exposure. In line with these data, the selective ablation of Foxp3(+) Tregs from MLRs by the use of diphtheria toxin (DT)-treated bacterial artificial chromosome (BAC)-transgenic DEREG mice completely fails to abrogate the suppressive activity of multiple anti-CD4 antibodies. Instead, tolerization is associated with the defective expression of various co-stimulatory receptors including OX40 and CD30, suggesting altered signaling through the TCR complex. Consistent with our findings in mice, anti-CD4 treatment renders human CD4(+) T cells tolerant in the absence of Tregs. Thus, our results establish that anti-CD4 antibodies can directly tolerize pathogenic CD4(+)Foxp3(-) helper T cells. This has important implications for the treatment of human inflammatory diseases.
Subject(s)
Antilymphocyte Serum/immunology , CD4 Antigens/immunology , Forkhead Transcription Factors/immunology , Immune Tolerance/immunology , Lymphocyte Depletion , T-Lymphocytes, Regulatory/microbiology , Animals , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation , Cells, Cultured , Dendritic Cells/immunology , Humans , Interleukin-2/immunology , Ki-1 Antigen/biosynthesis , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, OX40/biosynthesisABSTRACT
BACKGROUND: Allergen-specific immunotherapy (SIT) has been used since 1911, yet its mechanism of action remains to be elucidated. There is evidence indicating that CD4(+)FOXP3(+) regulatory T cells (Treg cells) are induced during SIT in allergic patients. However, the contribution of these cells to SIT has not been evaluated in vivo. OBJECTIVE: To evaluate the in vivo contribution of (i) CD4(+) CD25(+) T cells during SIT and of (ii) SIT-generated inducible FOXP3(+) Treg cells during allergen exposure to SIT-mediated suppression of asthmatic manifestations. METHODS: We used a mouse model of SIT based on the classical OVA-driven experimental asthma. Treg cells were quantified by flow cytometry 24 and 96 h post SIT treatment. We depleted CD4(+) CD25(+) T cells prior to SIT, and CD4(+)FOXP3(+) T cells prior to allergen challenges to study their contribution to the suppression of allergic manifestations by SIT treatment. RESULTS: Our data show that depletion of CD4(+)CD25(+) T cells at the time of SIT treatment reverses the suppression of airway hyperresponsiveness (AHR), but not of airway eosinophilia and specific IgE levels in serum. Interestingly, the number of CD4(+)CD25(+)FOXP3(+) T cells is transiently increased after SIT in the spleen and blood, suggesting the generation of inducible and presumably allergen-specific Treg cells during treatment. Depletion of CD4(+)FOXP3(+) Treg cells after SIT treatment partially reverses the SIT-induced suppression of airway eosinophilia, but not of AHR and serum levels of specific IgE. CONCLUSION AND CLINICAL RELEVANCE: We conclude that SIT-mediated tolerance induction towards AHR requires CD4(+)CD25(+) T cells at the time of allergen injections. In addition, SIT generates CD4(+)CD25(+)FOXP3(+) T cells that contribute to the suppression of airway eosinophilia upon allergen challenges. Therefore, enhancing Treg cell number or their activity during and after SIT could be of clinical relevance to improve the therapeutic effects of SIT.
Subject(s)
Asthma/immunology , Asthma/therapy , Desensitization, Immunologic/methods , T-Lymphocytes, Regulatory/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Eosinophils/immunology , Female , Forkhead Transcription Factors/metabolism , Humans , Interleukin-2 Receptor alpha Subunit/metabolism , Mice , Mice, Inbred BALB C , Ovalbumin/administration & dosage , Ovalbumin/immunology , T-Lymphocytes, Regulatory/metabolism , Treatment OutcomeABSTRACT
The inflammatory response to lung infections must be tightly regulated, enabling pathogen elimination while maintaining crucial gas exchange. Using recently described "depletion of regulatory T cell" (DEREG) mice, we found that selective depletion of regulatory T cells (Tregs) during acute respiratory syncytial virus (RSV) infection enhanced viral clearance but increased weight loss, local cytokine and chemokine release, and T-cell activation and cellular influx into the lungs. Conversely, inflammation was decreased when Treg numbers and activity were boosted using interleukin-2 immune complexes. Unexpectedly, lung (but not draining lymph node) Tregs from RSV-infected mice expressed granzyme B (GzmB), and bone marrow chimeric mice with selective loss of GzmB in the Treg compartment displayed markedly enhanced cellular infiltration into the lung after infection. A crucial role for GzmB-expressing Tregs has not hitherto been described in the lung or during acute infections, but may explain the inability of children with perforin/GzmB defects to regulate immune responses to infection. The effects of RSV infection in mice with defective immune regulation closely parallel the observed effects of RSV in children with bronchiolitis, suggesting that the pathogenesis of bronchiolitis may involve an inability to regulate virus-induced inflammation.
Subject(s)
Bronchiolitis, Viral/immunology , Granzymes/metabolism , Pneumonia, Viral/immunology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Viruses/immunology , T-Lymphocytes, Regulatory/metabolism , Acute Disease , Animals , Antibodies/metabolism , Antigen-Antibody Complex/administration & dosage , Bronchiolitis, Viral/etiology , Bronchiolitis, Viral/prevention & control , Cell Movement/drug effects , Cells, Cultured , Child , Disease Models, Animal , Disease Progression , Granzymes/genetics , Granzymes/immunology , Humans , Interleukin-2/immunology , Interleukin-2/metabolism , Lung/immunology , Lung/pathology , Lung/virology , Lymphocyte Depletion , Mice , Respiratory Syncytial Virus Infections/complications , Respiratory Syncytial Virus Infections/therapy , Respiratory Syncytial Viruses/pathogenicity , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , T-Lymphocytes, Regulatory/virology , Viral Load/drug effects , Viral Load/immunologyABSTRACT
Recently, it was demonstrated that arteriogenesis is enhanced in mice deficient in regulatory T cells (CD4(+) CD25(+) FoxP3(+) T cell), which can suppress effector T cell responses. The present study investigates the effects of these regulatory T cells on arteriogenesis in more detail by either specific expanding or depleting regulatory T cells. Hind limb ischemia was induced by electro-coagulation of the femoral artery in mice. Regulatory T cells were either expanded by injecting mice with a complex of interleukin (IL)-2 with the IL-2 monoclonal antibody JES6-1, or depleted by anti-CD25 antibody or diphtheria toxin injections in DEREG mice (depletion of regulatory T cells). Blood flow restoration was monitored using laser Doppler perfusion imaging. Collateral arteries were visualized by immunohistochemistry. Regulatory T cell expansion led to a moderate though significant suppression of blood flow restoration after ischemia induction. Surprisingly, depletion of regulatory T cells resulted in minor increase on blood flow recovery. However, collateral and capillary densities in the post-ischemic skeletal muscle were significantly increased in DEREG mice depleted for regulatory T cells. The presence of regulatory T cells after ischemia induction when analysed in non-depleted DEREG mice could be demonstrated by green fluorescent protein staining only in lymph nodes in the ischemic area, and not in the ischemic muscle tissue. The current study demonstrates that, even under conditions of major changes in regulatory T cell content, the contribution of regulatory T cells to the regulation of the arteriogenic response is only moderate.
Subject(s)
Hindlimb/blood supply , Ischemia/physiopathology , Neovascularization, Pathologic/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacology , Diphtheria Toxin/administration & dosage , Diphtheria Toxin/pharmacology , Femoral Artery/pathology , Femoral Artery/physiopathology , Hindlimb/physiopathology , Interleukin-2/administration & dosage , Interleukin-2/immunology , Interleukin-2/pharmacology , Interleukin-2 Receptor alpha Subunit/immunology , Ischemia/blood , Ischemia/immunology , Lymph Nodes/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscle, Skeletal/blood supply , Muscle, Skeletal/physiopathologyABSTRACT
The prevalence of asthma has steadily increased during the last decade, probably as the result of changes in the environment, including reduced microbial exposure during infancy. Accordingly, experimental studies have shown that deliberate infections with live pathogens prevent the development of allergic airway diseases in mice. Bacterial extracts are currently used in children suffering from repeated upper respiratory tract infections. In the present study, we have investigated whether bacterial extracts, commercially available as Broncho-Vaxom (BV), could prevent allergic airway disease in mice. Oral treatment with BV suppressed airway inflammation through interleukin-10 (IL-10)-dependent and MyD88 (myeloid differentiation primary response gene (88))-dependent mechanisms and induced the conversion of FoxP3 (forkhead box P3)(-) T cells into FoxP3(+) regulatory T cells. Furthermore, CD4(+) T cells purified from the trachea of BV-treated mice conferred protection against airway inflammation when adoptively transferred into sensitized mice. Therefore, treatment with BV could possibly be a safe and efficient strategy to prevent the development of allergic diseases in children.
Subject(s)
Asthma , Bacteria , Respiratory System , T-Lymphocytes, Regulatory , Animals , Mice , Administration, Oral , Adoptive Transfer , Asthma/immunology , Asthma/prevention & control , Bacteria/cytology , Bacteria/immunology , CD4-Positive T-Lymphocytes/immunology , Cytokines/immunology , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Interleukin-10/metabolism , Mice, Inbred BALB C , Myeloid Differentiation Factor 88/metabolism , Respiratory System/immunology , T-Lymphocytes, Regulatory/immunologyABSTRACT
DC-SIGN, a type II membrane protein with a C-type lectin binding domain that is highly expressed on mucosal dendritic cells (DCs) and certain macrophages in vivo, binds to ICAM-3, ICAM-2, and human and simian immunodeficiency viruses (HIV and SIV). Virus captured by DC-SIGN can be presented to T cells, resulting in efficient virus infection, perhaps representing a mechanism by which virus can be ferried via normal DC trafficking from mucosal tissues to lymphoid organs in vivo. To develop reagents needed to characterize the expression and in vivo functions of DC-SIGN, we cloned, expressed, and analyzed rhesus macaque, pigtailed macaque, and murine DC-SIGN and made a panel of monoclonal antibodies (MAbs) to human DC-SIGN. Rhesus and pigtailed macaque DC-SIGN proteins were highly similar to human DC-SIGN and bound and transmitted HIV type 1 (HIV-1), HIV-2, and SIV to receptor-positive cells. In contrast, while competent to bind virus, murine DC-SIGN did not transmit virus to receptor-positive cells under the conditions tested. Thus, mere binding of virus to a C-type lectin does not necessarily mean that transmission will occur. The murine and macaque DC-SIGN molecules all bound ICAM-3. We mapped the determinants recognized by a panel of 16 MAbs to the repeat region, the lectin binding domain, and the extreme C terminus of DC-SIGN. One MAb was specific for DC-SIGN, failing to cross-react with DC-SIGNR. Most MAbs cross-reacted with rhesus and pigtailed macaque DC-SIGN, although none recognized murine DC-SIGN. Fifteen of the MAbs recognized DC-SIGN on DCs, with MAbs to the repeat region generally reacting most strongly. We conclude that rhesus and pigtailed macaque DC-SIGN proteins are structurally and functionally similar to human DC-SIGN and that the reagents that we have developed will make it possible to study the expression and function of this molecule in vivo.
Subject(s)
Cell Adhesion Molecules , Lectins, C-Type , Lectins/physiology , Receptors, Cell Surface/physiology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Epitope Mapping , Humans , Lectins/chemistry , Lectins/immunology , Lectins/metabolism , Macaca mulatta , Macaca nemestrina , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Rabbits , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/immunologyABSTRACT
Infections can influence concurrent and subsequent Th1 vs Th2 immune responses to Ags. Through pattern recognition of foreign unmethylated CpG dinucleotides, the vertebrate innate immune system can sense infectious danger and typically replies with a Th1-polarized adaptive immune response. We examined whether CpG-DNA exposure would influence subsequent responses to infection and soluble Ags. CpG-DNA injection led to local lymphadenopathy characterized by maintenance of cellular composition with some biasing toward elevated dendritic cell composition. Sustained local production of IL-12 and IFN-gamma from dendritic cells and T cells was shown. Prior injection by up to 2 wk with CpG-DNA protected BALB/c mice from Th2 driven lethal leishmaniasis. CpG-DNA injection by up to 5 wk before soluble Ag challenge resulted in the generation of Ag-specific CTL, Th1 recall responses to Ag, and Th1-polarized Ag-specific Abs. Thus, CpG-DNA instigated a local predisposition for intense CTL responses and Th1-polarized immune responses to subsequent infections or Ag challenge. The induction by the innate immune system of a locally contained hypersensitivity could represent a capacitating immune reaction yielding rapid conditioned responses to secondary infections.
Subject(s)
Antigens/immunology , CpG Islands/immunology , DNA/immunology , Lymphatic Diseases/immunology , Oligonucleotides/immunology , Th1 Cells/immunology , Adoptive Transfer , Animals , Antigens/administration & dosage , CD11 Antigens/biosynthesis , Cytotoxicity, Immunologic , DNA/administration & dosage , Female , Immunity, Cellular , Immunity, Innate , Immunization , Injections, Subcutaneous , Interferon-gamma/biosynthesis , Interleukin-12/biosynthesis , Kinetics , Leishmania major/immunology , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/prevention & control , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymph Nodes/pathology , Lymph Nodes/transplantation , Lymphatic Diseases/metabolism , Lymphatic Diseases/pathology , Lymphatic Diseases/prevention & control , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Oligonucleotides/administration & dosage , Ovalbumin/administration & dosage , Ovalbumin/immunology , T-Lymphocytes, Cytotoxic/immunology , Th1 Cells/metabolismSubject(s)
Antigen-Presenting Cells/immunology , CpG Islands/immunology , DNA, Bacterial/immunology , Adjuvants, Immunologic , Animals , DNA, Bacterial/therapeutic use , Dendritic Cells/immunology , Humans , Macrophage Activation/immunology , Shock, Septic/immunology , Shock, Septic/physiopathology , Th2 Cells/immunologyABSTRACT
Bacterial DNA and synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the immune system in a sequence-dependent manner. Here we investigated the potential of CpG-ODNs to cause proliferation, cytokine production, and regulation of surface molecules in human B-chronic lymphocytic leukemia (CLL) cells. CpG-ODN induced proliferation in both B-CLL cells and normal B cells; however, only B-CLL cells increased proliferative responses when CpG-ODN was added to co-cultures of CD40-ligand transfected mouse fibroblasts (CD40LF) and B cells. Production of interleukin-6 and tumor necrosis factor alpha was detectable at borderline levels, using CpG-ODN as the only stimulus. In contrast, when CpG-ODN was added to co-cultures of B cells and CD40LF, a strong increase in cytokine production occurred in B-CLL cells as well as in normal B cells. The surface molecules CD40, CD58, CD80, CD86, CD54, and MHC class I molecules were up-regulated in B-CLL cells, whereas CD95 expression was not influenced by CpG-ODN stimulation. The same pattern of surface molecule regulation was observed in normal B cells, but up-regulation of CD40 was significantly stronger in B-CLL cells. Costimulation with CpG-ODN and CD40LF resulted in further up-regulation of CD58, CD80, CD86, and MHC class I molecules. In contrast, CD95 expression induced by CD40-ligation was inhibited by CpG-ODN. CpG-ODN activated B-CLL cells acquired a strong stimulatory capacity toward T cells in allogeneic mixed lymphocyte reaction. This effect was completely inhibited by a combination of anti-CD80 and anti-CD86 monoclonal antibody. Taken together, these findings suggest the possible use of CpG-ODN for immunotherapeutic strategies in patients with B-CLL.
Subject(s)
Adjuvants, Immunologic/pharmacology , B-Lymphocyte Subsets/drug effects , Cytokines/biosynthesis , Gene Expression Regulation, Leukemic/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Neoplastic Stem Cells/drug effects , Oligodeoxyribonucleotides/pharmacology , Animals , Antigens, CD/biosynthesis , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/genetics , B-Lymphocyte Subsets/cytology , B-Lymphocyte Subsets/metabolism , B7-1 Antigen/immunology , B7-2 Antigen , CD40 Antigens/immunology , CD40 Ligand , Cell Division/drug effects , Coculture Techniques , Cytokines/genetics , Fibroblasts , Genes, MHC Class I , Histocompatibility Antigens Class I/biosynthesis , Humans , Immunophenotyping , Interleukin-6/biosynthesis , Interleukin-6/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Lymphocyte Cooperation , Lymphocyte Culture Test, Mixed , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice , Neoplastic Stem Cells/cytology , Neoplastic Stem Cells/metabolism , Transfection , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Receptors for conserved molecular patterns associated with microbial pathogens induce synthesis of co-stimulatory molecules and cytokines in immature dendritic cells (DC), as do antigen-reactive CD4 T helper cells via CD40 signaling. Once activated, antigen-presenting DC may activate CD8 T cell responses in a T helper cell-independent fashion. Using immunostimulatory CpG-oligonucleotides (ODN) mimicking bacterial CpG-DNA, we tested whether CpG-DNA bypasses the need for T helper cells in CTL responses towards proteins by directly activating antigen-presenting DC to transit into professional APC. We describe that immature DC in situ constitutively process soluble proteins and generate CD8 T cell determinants yet CD8 T cell responses remain abortive. Induction of primary antigen-specific CD8 cytotoxic T lymphocyte (CTL)-mediated responses becomes initiated in wild-type as well as T helper cell-deficient mice, provided soluble protein and CpG-ODN are draining into the same lymph node. Specifically we show that CpG-ODN trigger antigen-presenting immature DC within the draining lymph node to acutely up-regulate co-stimulatory molecules and produce IL-12. These results provide new insights for generating in vivo efficient CTL responses to soluble proteins which may influence vaccination strategies.
Subject(s)
DNA, Bacterial/pharmacology , Dendritic Cells/drug effects , Dinucleoside Phosphates/pharmacology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/physiology , Animals , CD40 Antigens/physiology , CD40 Ligand/physiology , Dendritic Cells/physiology , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Ovalbumin/immunologyABSTRACT
Bacterial DNA and the synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the adaptive immune system (lymphocytes) and the innate immune system (APCs) in a sequence-dependent manner. Here, we addressed whether CpG-ODNs affect hemopoiesis. Challenging mice with immunostimulatory CpG-ODN sequences led to transient splenomegaly, with a maximum increase of spleen weight at day 6. The induction of splenomegaly by CpG-ODNs was sequence-specific, dose-dependent, and associated with an increase in splenic cell count, in numbers of granulocyte-macrophage CFUs (GM-CFUs), and early erythroid progenitors (burst-forming units-erythroid). The transfer of spleen cells from CpG-ODN-pretreated animals into lethally irradiated syngeneic mice yielded an increase of spleen CFUs. Furthermore, the challenge of sublethally irradiated mice with CpG-ODNs caused radioprotective effects, in that recovery of GM-CFUs and cytotoxic T cell function was enhanced. The increase in GM-CFU and CTL function correlated with an enhanced resistance to Listeria infection in irradiated mice. We conclude from these data that CpG-ODNs trigger extramedullary hemopoiesis, and that this finding could be of therapeutic relevance in myelosuppression.