Showing papers by "Richard A. Flavell published in 2006"

Transforming growth factor-beta regulation of immune responses.

Abstract: Transforming growth factor-beta (TGF-beta) is a potent regulatory cytokine with diverse effects on hemopoietic cells. The pivotal function of TGF-beta in the immune system is to maintain tolerance via the regulation of lymphocyte proliferation, differentiation, and survival. In addition, TGF-beta controls the initiation and resolution of inflammatory responses through the regulation of chemotaxis, activation, and survival of lymphocytes, natural killer cells, dendritic cells, macrophages, mast cells, and granulocytes. The regulatory activity of TGF-beta is modulated by the cell differentiation state and by the presence of inflammatory cytokines and costimulatory molecules. Collectively, TGF-beta inhibits the development of immunopathology to self or nonharmful antigens without compromising immune responses to pathogens. This review highlights the findings that have advanced our understanding of TGF-beta in the immune system and in disease.

Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus

Abstract: The innate immune system recognizes viral dsRNA through two distinct pathways; the Toll-like receptor 3 (TLR3) pathway detects dsRNA phagocytosed in endosomes; the helicases retinoic acid-induced protein I (RIG-I) and melanoma differentiation-associated gene-5 (mda-5) detect cytoplasmic dsRNA generated during viral replication. Both RIG-I and mda-5 can bind polyriboinosinic:polyribocytidylic acid (polyI:C), the synthetic analog of viral dsRNA, and mediate type I IFN responses to polyI:C and multiple RNA viruses in vitro. We generated mda-5-deficient mice and showed that mda-5 is the dominant receptor mediating type I IFN secretion in response to polyI:C in vitro and in vivo. Moreover, mda-5−/− mice exhibited a selectively impaired antiviral response to encephalomyocarditis picornavirus, indicating functional specialization of mda-5 in vivo.

Caspases 3 and 7: key mediators of mitochondrial events of apoptosis.

Abstract: The current model of apoptosis holds that upstream signals lead to activation of downstream effector caspases. We generated mice deficient in the two effectors, caspase 3 and caspase 7, which died immediately after birth with defects in cardiac development. Fibroblasts lacking both enzymes were highly resistant to both mitochondrial and death receptor-mediated apoptosis, displayed preservation of mitochondrial membrane potential, and had defective nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, the early apoptotic events of Bax translocation and cytochrome c release were also delayed. We conclude that caspases 3 and 7 are critical mediators of mitochondrial events of apoptosis.

Natural regulatory T cells control the development of atherosclerosis in mice.

Abstract: Atherosclerosis is an immunoinflammatory disease elicited by accumulation of lipids in the artery wall and leads to myocardial infarction and stroke. Here, we show that naturally arising CD4(+)CD25(+) regulatory T cells, which actively maintain immunological tolerance to self and nonself antigens, are powerful inhibitors of atherosclerosis in several mouse models. These results provide new insights into the immunopathogenesis of atherosclerosis and could lead to new therapeutic approaches that involve immune modulation using regulatory T cells.

Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses.

Abstract: Engagement of Toll-like receptors (TLRs) on macrophages leads to activation of the mitogen-activated protein kinases (MAPKs), which contribute to innate immune responses. MAPK activity is regulated negatively by MAPK phosphatases (MKPs). MKP-1, the founding member of this family of dual-specificity phosphatases, has been implicated in regulating lipopolysaccharide (LPS) responses, but its role in TLR-mediated immune responses in vivo has not been defined. Here, we show that mice deficient in MKP-1 were highly susceptible to endotoxic shock in vivo, associated with enhanced production of proinflammatory cytokines TNF-α and IL-6 and an anti-inflammatory cytokine, IL-10. We further examined the regulation and function of MKP-1 in macrophages, a major cell type involved in endotoxic shock. MKP-1 was transiently induced by TLR stimulation through pathways mediated by both myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing IFN-β (TRIF). MKP-1 deficiency led to sustained activation of p38 MAPK and c-Jun N-terminal kinase (JNK) in LPS-treated macrophages. In response to TLR signals, MKP-1-deficient macrophages produced 5- to 10-fold higher IL-10, which could be blocked by a p38 MAPK inhibitor. Thus, p38 MAPK plays a critical role in mediating IL-10 synthesis in TLR signaling. TNF-α was found to be more abundant in MKP-1-deficient macrophages within 2 hours of TLR stimulation, but its production was rapidly down-regulated by IL-10. Our studies demonstrate that MKP-1 attenuates the activities of p38 MAPK and JNK to regulate both pro- and anti-inflammatory cytokines in TLR signaling. These results highlight the complex mechanisms by which the MAPKs regulate innate immunity.

Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity

Abstract: The live attenuated yellow fever vaccine 17D (YF-17D) is one of the most effective vaccines available, with a 65-yr history of use in >400 million people globally. Despite this efficacy, there is presently no information about the immunological mechanisms by which YF-17D acts. Here, we present data that suggest that YF-17D activates multiple Toll-like receptors (TLRs) on dendritic cells (DCs) to elicit a broad spectrum of innate and adaptive immune responses. Specifically, YF-17D activates multiple DC subsets via TLRs 2, 7, 8, and 9 to elicit the proinflammatory cytokines interleukin (IL)-12p40, IL-6, and interferon-α. Interestingly, the resulting adaptive immune responses are characterized by a mixed T helper cell (Th)1/Th2 cytokine profile and antigen-specific CD8+ T cells. Furthermore, distinct TLRs appear to differentially control the Th1/Th2 balance; thus, whilst MyD88-deficient mice show a profound impairment of Th1 cytokines, TLR2-deficient mice show greatly enhanced Th1 and Tc1 responses to YF-17D. Together, these data enhance our understanding of the molecular mechanism of action of YF-17D, and highlight the potential of vaccination strategies that use combinations of different TLR ligands to stimulate polyvalent immune responses.

Detrimental contribution of the Toll-like receptor (TLR)3 to influenza A virus-induced acute pneumonia.

Abstract: Influenza A virus (IAV) is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. Recently, we demonstrated, using an in vitro approach, that the pattern recognition Toll-like receptor (TLR)3 plays a key role in the immune response of lung epithelial cells to IAV. In view of these data and the fact that the functional role of TLR3 in vivo is still debated, we designed an investigation to better understand the role of TLR3 in the mechanisms of IAV pathogenesis and host immune response using an experimental murine model. The time-course of several dynamic parameters, including animal survival, respiratory suffering, viral clearance, leukocyte recruitment into the airspaces and secretion of critical inflammatory mediators, was compared in infected wild-type and TLR3−/− mice. First, we found that the pulmonary expression of TLR3 is constitutive and markedly upregulated following influenza infection in control mice. Notably, when compared to wild-type mice, infected TLR3−/− animals displayed significantly reduced inflammatory mediators, including RANTES (regulated upon activation, normal T cell expressed and secreted), interleukin-6, and interleukin-12p40/p70 as well as a lower number of CD8+ T lymphocytes in the bronchoalveolar airspace. More important, despite a higher viral production in the lungs, mice deficient in TLR3 had an unexpected survival advantage. Hence, to our knowledge, our findings show for the first time that TLR3-IAV interaction critically contributes to the debilitating effects of a detrimental host inflammatory response.

The Birc1e cytosolic pattern-recognition receptor contributes to the detection and control of Legionella pneumophila infection.

Abstract: Baculovirus inhibitor of apoptosis repeat-containing 1 (Birc1) proteins have homology to several germline-encoded receptors of the innate immune system. However, their function in immune surveillance is not clear. Here we describe a Birc1e-dependent signaling pathway that restricted replication of the intracellular pathogen Legionella pneumophila in mouse macrophages. Translocation of bacterial products into host-cell cytosol was essential for Birc1e-mediated control of bacterial replication. Caspase-1 was required for Birc1e-dependent antibacterial responses ex vivo in macrophages and in a mouse model of Legionnaires' disease. The interleukin 1β converting enzyme–protease-activating factor was necessary for L. pneumophila growth restriction, but interleukin 1β was not required. These results establish Birc1e as a nucleotide-binding oligomerization–leucine-rich repeat protein involved in the detection and control of intracellular L. pneumophila.

Signals mediated by transforming growth factor-beta initiate autoimmune encephalomyelitis, but chronic inflammation is needed to sustain disease.

Abstract: It is unclear whether TGF-β, a critical differentiation factor for T cells producing interleukin 17 (TH-17 cells), is required for the initiation of experimental autoimmune encephalomyelitis (EAE) in vivo. Here we show that mice whose T cells cannot respond to TGF-β signaling lack TH-17 cells and do not develop EAE despite the presence of T helper cell type 1 infiltrates in the spinal cord. Local but not systemic antibody blockade of TGF-β prevented TH-17 cell differentiation and the onset of EAE. The pathogen stimulus zymosan, like mycobacterium, induced TH-17 cells and initiated EAE, but the disease was transient and correlated with reduced production of interleukin 23. These data show that TGF-β is essential for the initiation of EAE and suggest that disease progression may require ongoing chronic inflammation and production of interleukin 23.

Role of the caspase-1 inflammasome in Salmonella typhimurium pathogenesis

Abstract: Caspase-1 is activated by a variety of stimuli after the assembly of the "inflammasome," an activating platform made up of a complex of the NOD-LRR family of proteins. Caspase-1 is required for the secretion of proinflammatory cytokines, such as interleukin (IL)-1beta and IL-18, and is involved in the control of many bacterial infections. Paradoxically, however, its absence has been reported to confer resistance to oral infection by Salmonella typhimurium. We show here that absence of caspase-1 or components of the inflammasome does not result in resistance to oral infection by S. typhimurium, but rather, leads to increased susceptibility to infection.

Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule.

Abstract: The mechanisms that govern homeostasis of complex systems have been elusive but can be illuminated by mutations that disrupt system behavior. Mutations in the gene encoding the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and hyperkalemia. We show that physiology in mice transgenic for genomic segments harboring wild-type (TgWnk4WT) or PHAII mutant (TgWnk4PHAII) Wnk4 is changed in opposite directions: TgWnk4PHAII mice have higher blood pressure, hyperkalemia, hypercalciuria and marked hyperplasia of the distal convoluted tubule (DCT), whereas the opposite is true in TgWnk4WT mice. Genetic deficiency for the Na-Cl cotransporter of the DCT (NCC) reverses phenotypes seen in TgWnk4PHAII mice, demonstrating that the effects of the PHAII mutation are due to altered NCC activity. These findings establish that Wnk4 is a molecular switch that regulates the balance between NaCl reabsorption and K+ secretion by altering the mass and function of the DCT through its effect on NCC.

Involvement of Toll-like receptor 5 in the recognition of flagellated bacteria

Abstract: Toll-like receptors (TLRs) are key components of the immune system that detect microbial infection and trigger antimicrobial host defense responses TLR5 is a sensor for monomeric flagellin, which is a component of bacterial flagella known to be a virulence factor In this study we generated TLR5-deficient mice and investigated the role of TLR5 signaling in the detection of flagellin and antibacterial immune responses to Salmonella typhimurium and Pseudomonas aeruginosa We found that TLR5 is essential for the recognition of bacterial flagellin both in vivo and ex vivo TLR5 contribution to antibacterial host response to ip infection with S typhimurium or intranasal administration of P aeruginosa may be masked by TLR4 or other sensing mechanisms By using radiation bone marrow chimera, we showed that upon ip injection of flagellin immune responses are mediated by lymphoid cells, whereas resident cells are required for the initiation of response upon intranasal flagellin administration These results suggest that flagellin recognition in different organs is mediated by distinct TLR5-expressing cells and provide insights into the cooperation of the TLR5 and TLR4 signaling pathways used by the innate immune system in the recognition of bacterial pathogens

The kinase TAK1 integrates antigen and cytokine receptor signaling for T cell development, survival and function

Abstract: The kinase TAK1 is critical for innate and B cell immunity. The function of TAK1 in T cells is unclear, however. We show here that T cell-specific deletion of the gene encoding TAK1 resulted in reduced development of thymocytes, especially of regulatory T cells expressing the transcription factor Foxp3. In mature thymocytes, TAK1 was required for interleukin 7-mediated survival and T cell receptor-dependent activation of transcription factor NF-kappaB and the kinase Jnk. In effector T cells, TAK1 was dispensable for T cell receptor-dependent NF-kappaB activation and cytokine production, but was important for proliferation and activation of the kinase p38 in response to interleukins 2, 7 and 15. Thus, TAK1 is essential for the integration of T cell receptor and cytokine signals to regulate the development, survival and function of T cells.

Anti-Mitochondrial Antibodies and Primary Biliary Cirrhosis in TGF-β Receptor II Dominant-Negative Mice

Abstract: Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver, characterized by lymphocytic infiltrates in portal tracts, selective destruction of biliary epithelial cells, and anti-mitochondrial Abs (AMAs). The elucidation of early events in the induction of tissue inflammation and autoimmunity in PBC has been hampered by the cryptic onset of the disease, the practical limitations in accessing the target tissue, and the lack of a suitable animal model. We demonstrate in this study that a mouse transgenic for directed expression of a dominant-negative form of TGF-β receptor type II (dnTGFβRII), under the direction of the CD4 promoter, mimics several key phenotypic features of human PBC, including spontaneous production of AMAs directed to the same mitochondrial autoantigens, namely PDC-E2, BCOADC-E2, and OGDC-E2. The murine AMAs also inhibit PDC-E2 activity. Moreover, there is lymphocytic liver infiltration with periportal inflammation analogous to the histological profile in human PBC. Additionally, the serum cytokine profile of affected mice mimics data in human PBC. The concomitant presence of these immunopathological features in the transgenic mice suggests that the TGF-βRII pathway is implicated in the pathogenesis of PBC. Finally, these data point away from initiation of autoimmunity by mechanisms such as molecular mimicry and more toward activation of an intrinsically self-reactive T cell repertoire in which necessary regulatory T cell influences are lacking.

Sepsis-induced suppression of lung innate immunity is mediated by IRAK-M

Abstract: Sepsis results in a state of relative immunosuppression, rendering critically ill patients susceptible to secondary infections and increased mortality. Monocytes isolated from septic patients and experimental animals display a “deactivated” phenotype, characterized by impaired inflammatory and antimicrobial responses, including hyporesponsiveness to LPS. We investigated the role of the LPS/TLR4 axis and its inhibitor, IL-1 receptor–associated kinase–M (IRAK-M), in modulating the immunosuppression of sepsis using a murine model of peritonitis-induced sepsis followed by secondary challenge by intratracheal Pseudomonas aeruginosa. Septic mice demonstrated impaired alveolar macrophage function and increased mortality when challenged with intratracheal Pseudomonas as compared with nonseptic controls. TLR2 and TLR4 expression was unchanged in the lung following sepsis, whereas levels of IRAK-M were upregulated. Macrophages from IRAK-M–deficient septic mice produced higher levels of proinflammatory cytokines ex vivo and greater costimulatory molecule expression in vivo as compared with those of their WT counterparts. Following sepsis and secondary intrapulmonary bacterial challenge, IRAK-M–/– animals had higher survival rates and improved bacterial clearance from lung and blood compared with WT mice. In addition, increased pulmonary chemokine and inflammatory cytokine production was observed in IRAK-M–/– animals, leading to enhanced neutrophil recruitment to airspaces. Collectively, these findings indicate that IRAK-M mediates critical aspects of innate immunity that result in an immunocompromised state during sepsis.

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

Abstract: Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-κB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Deletion of TLR3 Alters the Pulmonary Immune Environment and Mucus Production during Respiratory Syncytial Virus Infection

Abstract: The detection of a viral infection by pattern recognition receptors (PAMPs) is an integral part of antiviral immunity In these studies we have investigated the role of TLR3, which recognizes dsRNA, in Respiratory Syncytial virus (RSV) infection using B6 background mice with a TLR3 deletion Although we observed no changes in viral growth, we did find that TLR3-/- mice demonstrated significant increases in mucus production in the airways of RSV-infected mice The qualitative assessment was observed by examining differentially stained lungs, followed by immunohistochemical staining for gob5, a mucus-associated protein The histopathologic observations were verified using quantitative gene expression analyses examining gob5 gene expression Changes in pulmonary mucus production were accompanied by an increase in pulmonary IL-13 as well as IL-5 expression and eosinophils in the airways of TLR3-/- mice Examining leukocytes in the airway indicated an accumulation of eosinophils in TLR3-/- mice, but not wild-type mice, after RSV infection Isolated lung draining lymph node cells from TLR3-/- mice produced significant increases in Th2-type cytokines, IL-5, and IL-13, compared with wild-type TLR3+/+ mice only after RSV infection To demonstrate a causative link, we depleted TLR3-/- mice of IL-13 during RSV infection and found that mucus and gob5 expression in the lungs was attenuated Together, these studies highlight that although TLR3 may not be required for viral clearance, it is necessary to maintain the proper immune environment in the lung to avoid developing pathologic symptoms of disease

Microglia recognize double-stranded RNA via TLR3.

Abstract: Microglia are CNS resident innate immune cells of myeloid origin that become activated and produce innate proinflammatory molecules upon encountering bacteria or viruses. TLRs are a phylogenetically conserved diverse family of sensors for pathogen-associated molecular patterns that drive innate immune responses. We have recently shown that mice deficient in TLR3 (TLR3(-/-) mice) are resistant to lethal encephalitis and have reduced microglial activation after infection with West Nile virus, a retrovirus that produces dsRNA. We wished to determine whether microglia recognize dsRNA through the TLR3 pathway. In vitro, murine wild-type primary cultured microglia responded to synthetic dsRNA polyinosinic-polycytidylic acid (poly(I:C)) by increasing TLR3 and IFN-beta mRNA and by morphologic activation. Furthermore, wild-type microglia dose dependently secreted TNF-alpha and IL-6 after poly(I:C) challenge, whereas TLR3(-/-) microglia produced diminished cytokines. Activation of MAPK occurred in a time-dependent fashion following poly(I:C) treatment of wild-type microglia, but happened with delayed kinetics in TLR3(-/-) microglia. As an in vivo model of encephalitis, wild-type or TLR3(-/-) mice were injected intracerebroventricularly with poly(I:C) or LPS, and microglial activation was assessed by cell surface marker or phospho-MAPK immunofluorescence. After intracerebroventricular injection of poly(I:C), microgliosis was clearly evident in wild-type mice but was nearly absent in TLR3(-/-) animals. When taken together, our results demonstrate that microglia recognize dsRNA through TLR3 and associated signaling molecules and suggest that these cells are key sensors of dsRNA-producing viruses that may invade the CNS.

The roles for cytokines in the generation and maintenance of regulatory T cells.

Abstract: As an essential mechanism for self-tolerance, immune suppression has attracted much attention since the discovery of suppressor T cells, now called regulatory T cells (Tregs), in the 1990s. Different types of Tregs have been described based on distinct expression patterns of surface markers and cytokines. Cytokines are not only essential for function but also important for the generation of Tregs. Interleukin-2 (IL-2), transforming growth factor-beta, IL-10, and other immunoregulatory molecules have been shown to control the generation of Tregs. The presence of other types of cells, in particular antigen-presenting cells (APCs), is critical for the generation of Tregs. Cytokines can serve as either initiators or intermediates for the interactions between APCs and Tregs. This review discusses our current knowledge of how cytokines regulate the generation and maintenance of Tregs.

Cutting Edge: Deficiency in the E3 Ubiquitin Ligase Cbl-b Results in a Multifunctional Defect in T Cell TGF-β Sensitivity In Vitro and In Vivo

Abstract: Mice deficient in the E3 ubiquitin ligase Cbl-b have CD28-independent T cells and develop autoimmunity. We previously reported that Cbl-b-/- CD4+CD25- T effector cells are resistant in vitro to the antiproliferative effects of CD4+CD25+ regulatory T cells and TGF-beta. We have now asked whether the resistance noted in Cbl-b-/- T cells is restricted solely to TGF-beta's antiproliferative effects, whether the TGF-beta resistance has in vivo relevance, and whether a defect can be identified in the TGF-beta signaling pathway. We now demonstrate the following: 1) in vitro, Cbl-b deficiency prevents the TGF-beta-mediated induction of Foxp3+ functional regulatory T cells; 2) in vivo, Cbl-b-/- mice show a significantly enhanced response to a tumor that is strictly TGF-beta regulated; and 3) Cbl-b-/- T effector cells have defective TGF-beta-mediated Smad2 phosphorylation. These studies are the first to document that the E3 ubiquitin ligase Cbl-b plays an integral role in T cell TGF-beta signaling, and that its absence results in multifunctional TGF-beta-related defects that have important disease-related implications.

TLR3 Deletion Limits Mortality and Disease Severity due to Phlebovirus Infection

Abstract: TLR3 was the first member of the TLR family of pattern recognition receptors found to detect a conserved viral molecular pattern, dsRNA, yet supporting evidence for a major role in host defense against viral pathogens is limited. Punta Toro virus (PTV) has been shown to produce severe infection in mice, modeling disease caused by the related highly pathogenic Rift Valley fever phlebovirus in humans and domesticated ungulates. Using TLR3-deficient mice, we investigated the involvement of TLR3 in host defense against PTV infection. Compared with wild-type, TLR3(-/-) mice demonstrate increased resistance to lethal infection and have reduced liver disease associated with hepatotropic PTV infection. Infectious challenge produced comparable peak liver and serum viral loads; however, TLR3(-/-) mice were able to clear systemic virus at a slightly faster rate. Cytokine profiling suggests that TLR3 plays an important role in PTV pathogenesis through the overproduction of inflammatory mediators, which may be central to the observed differences in survival and disease severity. Compared with TLR3-deficient mice, IL-6, MCP-1, IFN-gamma, and RANTES were all present at higher levels in wild-type animals. Most dramatic was the exaggerated levels of IL-6 found systemically and in liver tissue of infected wild-type mice; however, IL-6-deficient animals were found to be more susceptible to lethal PTV infection. Taken together, we conclude that the TLR3-mediated response to PTV infection is detrimental to disease outcome and propose that IL-6, although critical to establishing antiviral defense, contributes to pathogenesis when released in excess, necessitating its controlled production as is seen with TLR3(-/-) mice.