Toll is a cell surface receptor with well described roles in the developmental patterning of invertebrates and innate immunity in adult Drosophila. Mammalian toll-like receptors represent a family of Toll orthologs that function in innate immunity by recognizing molecular motifs unique to pathogens or injured tissue. One member in this family of pattern recognition receptors, toll-like receptor 3 (TLR3), recognizes viral double-stranded RNA and host mRNA. We examined the expression and function of TLRs in the nervous system and found that TLR3 is expressed in the mouse central and peripheral nervous systems and is concentrated in the growth cones of neurons. Activation of TLR3 by the synthetic ligand polyinosine:polycytidylic acid (poly I:C) or by mRNA rapidly causes growth cone collapse and irreversibly inhibits neurite extension independent of nuclear factor κB. Mice lacking functional TLR3 were resistant to the neurodegenerative effects of poly I:C. Neonatal mice injected with poly I:C were found to have fewer axons exiting dorsal root ganglia and displayed related sensorimotor deficits. No effect of poly I:C was observed in mice lacking functional TLR3. Together, these findings provide evidence that an innate immune pattern recognition receptor functions autonomously in neurons to regulate axonal growth and advances a novel hypothesis that this class of receptors may contribute to injury and limited CNS regeneration.
Toll-like receptor-3; axon; polyinosine:polycytidylic acid; poly I:C; RNA; CNS; danger theory
Borrelia burgdorferi, the Lyme disease agent, causes joint inflammation in an experimental murine model. Inflammation occurs, in part, due to the ability of B. burgdorferi to induce the production of proinflammatory cytokines and a strong CD4+ T helper type 1 response. The mechanisms by which spirochetes induce these responses are not completely known, although transcription factors, such as NF-κB in phagocytic cells, initiate the proinflammatory cytokine burst. We show here that the mitogen-activated protein (MAP) kinase of 38 kDa (p38 MAP kinase) is involved in the proinflammatory cytokine production elicited by B. burgdorferi Ags in phagocytic cells and the development of murine Lyme arthritis. B. burgdorferi Ags activated p38 MAP kinase in vitro, and the use of a specific inhibitor repressed the spirochete-induced production of TNF-α. The infection of mice that are deficient for a specific upstream activator of the kinase, MAP kinase kinase 3, resulted in diminished proinflammatory cytokine production and the development of arthritis, without compromising the ability of CD4+ T cells to respond to borrelial Ags or the production of specific Abs. Overall, these data indicated that the p38 MAP kinase pathway plays an important role in B. burgdorferi-elicited inflammation and point to potential new therapeutic approaches to the treatment of inflammation induced by the spirochete.
Glucocorticoids are among the most commonly used anti-inflammatory agents. Despite the enormous efforts in elucidating the glucocorticoid-mediated anti-inflammatory actions, how glucocorticoids tightly control overactive inflammatory response is not fully understood. Here we show that glucocorticoids suppress bacteria-induced inflammation by enhancing IRAK-M, a central negative regulator of Toll-like receptor signalling. The ability of glucocorticoids to suppress pulmonary inflammation induced by non-typeable Haemophilus influenzae is significantly attenuated in IRAK-M-deficient mice. Glucocorticoids improve the survival rate after a lethal non-typeable Haemophilus influenzae infection in wild-type mice, but not in IRAK-M-deficient mice. Moreover, we show that glucocorticoids and non-typeable Haemophilus influenzae synergistically upregulate IRAK-M expression via mutually and synergistically enhancing p65 and glucocorticoid receptor binding to the IRAK-M promoter. Together, our studies unveil a mechanism by which glucocorticoids tightly control the inflammatory response and host defense via the induction of IRAK-M and may lead to further development of anti-inflammatory therapeutic strategies.
Glucocorticoids strongly suppress inflammation. Here the authors show that this suppression is mediated by induction of the negative inflammatory regulator IRAK-M, and demonstrate its important role in host defense against the pneumonia-causative bacterium, non-typeable Haemophilus influenzae.
The cytokine IL-9, derived primarily from T-helper (Th)-9 lymphocytes, promotes expansion of the Th2 subset and is implicated in the mechanisms of allergic asthma. We hypothesize that IL-9 also plays a role in human allergic contact dermatitis (ACD). To investigate this hypothesis, skin biopsy specimens of positive patch test sites from non-atopic patients were assayed using qPCR and immunohistochemistry. Along with Th2 associated cytokines, IFN-γ, IL-4, and IL-17A, expression of IL-9, and PU.1, a Th9-associated transcription factor, were elevated when compared to paired normal skin. Immunohistochemistry on ACD skin biopsies identified PU.1+CD3+, and PU.1+CD4+ cells, consistent with Th9 lymphocytes, in the inflammatory infiltrate. PBMC from nickel-allergic patients, but not non-allergic controls, show significant IL-9 production in response to nickel. Blocking studies with monoclonal antibodies to HLA-DR (but not HLA-A, B, C) or chloroquine significantly reduced this nickel-specific IL-9 production. Additionally, blockade of IL-9 or IL-4 enhanced allergen-specific IFN-γ production. A contact hypersensitivity model using IL-9−/− mice, shows enhanced Th1 lymphocyte immune responses, when compared to WT mice, consistent with our human in vitro data. This study demonstrates that IL-9, through its direct effects on Th1 and ability to promote IL-4 secretion, has a regulatory role for Th1 lymphocytes in ACD.
Regulatory T cells (Tregs), which express CD4 and FOXP3, are critical for modulating the immune response and promoting immune tolerance. Consequently, methods to expand Tregs for therapeutic use are of great interest. While transfer of Tregs after massive ex vivo expansion can be achieved, in vivo expansion of Tregs would be more practical. Here, we demonstrate that targeting the CD45 tyrosine phosphatase with a tolerogenic anti-CD45RB mAb acutely increases Treg numbers in WT mice, even in absence of exogenous antigen. Treg expansion occurred through substantial augmentation of homeostatic proliferation in the preexisting Treg population. Moreover, anti-CD45RB specifically increased Treg proliferation in response to cognate antigen. Compared with conventional T cells, Tregs differentially regulate their conjugation with DCs. Therefore, we determined whether CD45 ligation could alter interactions between Tregs and DCs. Live imaging showed that CD45 ligation specifically reduced Treg motility in an integrin-dependent manner, resulting in enhanced interactions between Tregs and DCs in vivo. Increased conjugate formation, in turn, augmented nuclear translocation of nuclear factor of activated T cells (NFAT) and Treg proliferation. Together, these results demonstrate that Treg peripheral homeostasis can be specifically modulated in vivo to promote Treg expansion and tolerance by increasing conjugation between Tregs and DCs.
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.
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.
Atherosclerosis; Inflammation; TGFβ; Dendritic cell
As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an anti-virulence agent against diverse bacteria including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest new therapeutic strategies to counter pathogens.
Interchromosomal associations can regulate gene expression but little is known about the molecular basis of such associations. In response to antigen stimulation, naïve T cells can differentiate into Th1, Th2 and Th17 cells expressing IFN-γ, IL-4 and IL-17, respectively. We previously reported that in naïve T cells, the IFN-γ locus is associated with the Th2 cytokine locus. Here we show that the Th2 locus additionally associates with the IL-17 locus. This association requires a DNase I hypersensitive region (RHS6) at the Th2 locus. RHS6 and the IL-17 promoter both bear Oct-1 binding sites. Deletion of either of these sites or Oct-1 gene impairs the association. Oct-1 and CTCF bind their cognate sites cooperatively and CTCF-deficiency similarly impairs the association. Finally, defects in the association lead to enhanced IL-17 induction. Collectively, our data indicate Th17 lineage differentiation is restrained by the Th2 locus via interchromosomal associations organized by Oct-1 and CTCF.
Salmonella lacking the TCA enzyme aconitase trigger NLRP3 inflammasome activation in infected macrophages, leading to elevated inflammatory responses and reduced virulence.
Microbial infection triggers assembly of inflammasome complexes that promote caspase-1–dependent antimicrobial responses. Inflammasome assembly is mediated by members of the nucleotide binding domain leucine-rich repeat (NLR) protein family that respond to cytosolic bacterial products or disruption of cellular processes. Flagellin injected into host cells by invading Salmonella induces inflammasome activation through NLRC4, whereas NLRP3 is required for inflammasome activation in response to multiple stimuli, including microbial infection, tissue damage, and metabolic dysregulation, through mechanisms that remain poorly understood. During systemic infection, Salmonella avoids NLRC4 inflammasome activation by down-regulating flagellin expression. Macrophages exhibit delayed NLRP3 inflammasome activation after Salmonella infection, suggesting that Salmonella may evade or prevent the rapid activation of the NLRP3 inflammasome. We therefore screened a Salmonella Typhimurium transposon library to identify bacterial factors that limit NLRP3 inflammasome activation. Surprisingly, absence of the Salmonella TCA enzyme aconitase induced rapid NLRP3 inflammasome activation. This inflammasome activation correlated with elevated levels of bacterial citrate, and required mitochondrial reactive oxygen species and bacterial citrate synthase. Importantly, Salmonella lacking aconitase displayed NLRP3- and caspase-1/11–dependent attenuation of virulence, and induced elevated serum IL-18 in wild-type mice. Together, our data link Salmonella genes controlling oxidative metabolism to inflammasome activation and suggest that NLRP3 inflammasome evasion promotes systemic Salmonella virulence.
The endocytosis regulator dynamin 2 is required for the regulation of S1PR1 internalization and continued S1PR1 signaling in low S1P environments.
Sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) is critical for lymphocyte egress from lymphoid organs. Lymphocytes encounter low S1P concentrations near exit sites before transmigration, yet S1PR1 signaling is rapidly terminated after exposure to S1P. How lymphocytes maintain S1PR1 signaling in a low S1P environment near egress sites is unknown. Here we identify dynamin 2, an essential component of endocytosis, as a novel regulator of T cell egress. Mice with T cell–specific dynamin 2 deficiency had profound lymphopenia and impaired egress from lymphoid organs. Dynamin 2 deficiency caused impaired egress through regulation of S1PR1 signaling, and transgenic S1PR1 overexpression rescued egress in dynamin 2 knockout mice. In low S1P concentrations, dynamin 2 was essential for S1PR1 internalization, which enabled continuous S1PR1 signaling and promoted egress from both thymus and lymph nodes. In contrast, dynamin 2–deficient cells were only capable of a pulse of S1PR1 signaling, which was insufficient for egress. Our results suggest a possible mechanism by which T lymphocytes positioned at exit portals sense low S1P concentrations, promoting their egress into circulatory fluids.
Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models are unable to support development of human innate immune cells, including myeloid cells and NK cells. Here we describe a mouse strain, called MI(S)TRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked in to their respective mouse loci. The human cytokines support the development and function of monocytes/macrophages and natural killer cells derived from human fetal liver or adult CD34+ progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MI(S)TRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.
Mucus production by goblet cells of the large intestine serves as a crucial anti microbial protective mechanism at the interface between the eukaryotic and prokaryotic cells of the mammalian intestinal ecosystem. However, the regulatory pathways involved in goblet cell-induced mucus secretion remain largely unknown. Here we demonstrate that the NLRP6 inflammasome, a recently described regulator of colonic microbiota composition and bio-geographical distribution, is a critical orchestrator of goblet cell mucin granule exocytosis. NLRP6 deficiency leads to defective autophagy in goblet cells and abrogated mucin secretion into the large intestinal lumen. Consequently, NLRP6 inflammasome-deficient mice are unable to clear enteric pathogens from the mucosal surface, rendering them highly susceptible to persistent infection. This study identifies the first innate immune regulatory pathway governing goblet cell mucus secretion, linking non-hematopoietic inflammasome signaling to autophagy and highlighting the goblet cell as a critical innate immune player in the control of intestinal host-microbial mutualism.
NLRP6; inflammasome; goblet cell; mucin; autophagy; Citrobacter infection
The development and optimization of immune therapies in patients has been hampered by the lack of preclinical models in which their effects on human immune cells can be studied. As a result, observations that have been made in preclinical studies have suggested mechanisms of drug action in murine models that may not be confirmed in clinical studies. We have utilized a humanized mouse reconstituted with human hematopoetic stem cells to circumvent these limitations. We have studied the effects of teplizumab in this model, a Fc receptor non-binding humanized monoclonal anti-CD3 antibody that has been used to treat patients with Type 1 diabetes mellitus. A novel mechanism of action was identified where human gut tropic CCR6+ T cells leave the circulation and secondary lymph organs and migrate to the small intestine. They become producers of IL-10 which can be detected in the peripheral circulation. Blockade of migration of T cells to the small intestine by natalizumab abolishes the treatment effects of teplizumab. Direct translation of these findings was possible in patients with Type 1 diabetes treated with teplizumab since we found there is increased expression of IL-10 by CD4+CD25highCCR6+FoxP3 cells when they emerge into the peripheral circulation. These findings demonstrate that humanized mice may be used to identify novel immunologic mechanisms that occur in patients treated with immune modulators.
To directly study complex human hemato-lymphoid system physiology and respective system-associated diseases in vivo, human-to-mouse xenotransplantation models for human blood and blood-forming cells and organs have been developed over the past three decades. We here review the fundamental requirements and the remarkable progress made over the past few years in improving these systems, the current major achievements reached by use of these models, and the future challenges to more closely model and study human health and disease and to achieve predictive preclinical testing of both prevention measures and potential new therapies.
humanized mice; stem cells; hematopoiesis; infectious disease; cancer
RNA is known to be an abundant and important structural component of the nuclear matrix, including long noncoding RNAs (lncRNA). Yet the molecular identities, functional roles, and localization dynamics of lncRNAs that influence nuclear architecture remain poorly understood. Here, we describe one lncRNA, Firre, that interacts with the nuclear matrix factor hnRNPU, through a 156 bp repeating sequence and Firre localizes across a ~5 Mb domain on the X-chromosome. We further observed Firre localization across at least five distinct trans-chromosomal loci, which reside in spatial proximity to the Firre genomic locus on the X-chromosome. Both genetic deletion of the Firre locus or knockdown of hnRNPU resulted in loss of co-localization of these trans-chromosomal interacting loci. Thus, our data suggest a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes.
YopM is a leucine-rich repeat (LRR)-containing effector in several Yersinia species, including Yersinia pestis and Y. pseudotuberculosis. Different Yersinia strains encode distinct YopM isoforms with variable numbers of LRRs but conserved C-terminal tails. A 15-LRR isoform in Y. pseudotuberculosis YPIII was recently shown to bind and inhibit caspase-1 via a YLTD motif in LRR 10, and attenuation of YopM− YPIII was reversed in mice lacking caspase-1, indicating that caspase-1 inhibition is a major virulence function of YopMYPIII. To determine if other YopM proteins inhibit caspase-1, we utilized Y. pseudotuberculosis strains natively expressing a 21-LRR isoform lacking the YLTD motif (YopM32777) or ectopically expressing a Y. pestis 15-LRR version with a functional (YopMKIM) or inactivated (YopMKIM D271A) YLTD motif. Results of mouse and macrophage infections with these strains showed that YopM32777, YopMKIM, and YopMKIM D271A inhibit caspase-1 activation, indicating that the YLTD motif is dispensable for this activity. Analysis of YopMKIM deletion variants revealed that LRRs 6 to 15 and the C-terminal tail are required to inhibit caspase-1 activation. YopM32777, YopMKIM, and YopMKIM deletion variants were purified, and binding partners in macrophage lysates were identified. Caspase-1 bound to YopMKIM but not YopM32777. Additionally, YopMKIM bound IQGAP1 and the use of Iqgap1−/− macrophages revealed that this scaffolding protein is important for caspase-1 activation upon infection with YopM−
Y. pseudotuberculosis. Thus, while multiple YopM isoforms inhibit caspase-1 activation, their variable LRR domains bind different host proteins to perform this function and the LRRs of YopMKIM target IQGAP1, a novel regulator of caspase-1, in macrophages.
Activation of caspase-1, mediated by macromolecular complexes termed inflammasomes, is important for innate immune defense against pathogens. Pathogens can, in turn, subvert caspase-1-dependent responses through the action of effector proteins. For example, the Yersinia effector YopM inhibits caspase-1 activation by arresting inflammasome formation. This caspase-1 inhibitory activity has been studied in a specific YopM isoform, and in this case, the protein was shown to act as a pseudosubstrate to bind and inhibit caspase-1. Different Yersinia strains encode distinct YopM isoforms, many of which lack the pseudosubstrate motif. We studied additional isoforms and found that these YopM proteins inhibit caspase-1 activation independently of a pseudosubstrate motif. We also identified IQGAP1 as a novel binding partner of the Yersinia pestis YopMKIM isoform and demonstrated that IQGAP1 is important for caspase-1 activation in macrophages infected with Yersinia. Thus, this study reveals new insights into inflammasome regulation during Yersinia infection.
Inflammasome pathways are important in chronic diseases, but it is not known how the signalling is sustained after initiation. Inflammasome activation is dependent on stimuli such as LPS and ATP that provide two distinct signals resulting in rapid production of IL-1β, with lack of response to repeat stimulation. Here we report that adenosine is a key regulator of inflammasome activity, increasing the duration of the inflammatory response via the A2A receptor. Adenosine does not replace signals provided by stimuli such as LPS or ATP, but sustains inflammasome activity via a cAMP/PKA/CREB/HIF-1α pathway. In the setting of lack of IL-1β responses after previous exposure to LPS, adenosine can supersede this tolerogenic state and drive IL-1β production. These data reveal that inflammasome activity is sustained, after initial activation, by A2A receptor-mediated signalling.
Following immune reconstitution, hematopoietic stem cell transplant patients often display reduced immune function and are especially susceptible to lung infections. In a mouse model of syngeneic bone marrow transplantation (BMT), we previously reported that PGE2 is overproduced in lungs of BMT mice, significantly impairing host defense against Pseudomonas aeruginosa. This impairment in host defense post-BMT is also marked by diminished alveolar macrophage (AM) phagocytosis, bacterial killing, and production of TNF-α and cysteinyl leukotrienes. However, a mechanism by which overproduction of PGE2 suppresses pulmonary host defense post-BMT is unknown. As IL-1R–associated kinase (IRAK)-M is a known inhibitor of MyD88-dependent IL-1R/TLR signaling and macrophage function, we sought to determine whether IRAK-M is involved in PGE2-induced immunosuppression post-BMT. We found that IRAK-M expression is elevated 3.5-fold in BMT AMs relative to control AMs, and this is related to AM overproduction of PGE2. Furthermore, genetic ablation of IRAK-M in the bone marrow of BMT mice restores host defense against P. aeruginosa. Despite AM overproduction of PGE2 and elevated E prostanoid 2 receptor expression, AM phagocytosis, killing, and production of cysteinyl leukotrienes and TNF-α are restored in the absence of IRAK-M post-BMT. Also, treatment with PGE2 does not inhibit AM phagocytosis in the absence of IRAK-M. These data suggest that the absence of IRAK-M in the hematopoietic compartment post-BMT enhances pulmonary host defense and mitigates AM sensitivity to the inhibitory effects of PGE2. Therefore, strategies to limit IRAK-M elevation post-BMT may be efficacious in reducing patient susceptibility to infection.
Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, since a flagellin-deficient mutant of B. melitensis was found to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunologic standoff between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.
Oxalate nephropathy with renal failure is caused by multiple disorders causing hyperoxaluria due to either overproduction of oxalate (primary hyperoxaluria) or excessive absorption of dietary oxalate (enteric hyperoxaluria). To study the etiology of renal failure in crystal-induced kidney disease, we created a model of progressive oxalate nephropathy by feeding mice a diet high in soluble oxalate (high oxalate in the absence of dietary calcium). Renal histology was characterized by intratubular calcium-oxalate crystal deposition with an inflammatory response in the surrounding interstitium. Oxalate nephropathy was not found in mice fed a high oxalate diet that also contained calcium. NALP3, also known as cryopyrin, has been implicated in crystal-associated diseases such as gout and silicosis. Mice fed the diet high in soluble oxalate demonstrated increased NALP3 expression in the kidney. Nalp3-null mice were completely protected from the progressive renal failure and death that occurred in wild-type mice fed the diet high in soluble oxalate. NALP3-deficiency did not affect oxalate homeostasis, thereby excluding differences in intestinal oxalate handling to explain the observed phenotype. Thus, progressive renal failure in oxalate nephropathy results primarily from NALP3-mediated inflammation.
Oxalate; crystal nephropathy; NALP3; inflammasome
Nitrogen dioxide (NO2) is an environmental pollutant and endogenously generated oxidant associated with the development, severity, and exacerbation of asthma. NO2 exposure is capable of allergically sensitizing mice to the innocuous inhaled antigen ovalbumin (OVA), promoting neutrophil and eosinophil recruitment, and a mixed Th2/Th17 response upon antigen challenge that is reminiscent of severe asthma. However, the identity of IL-17A–producing cells and the mechanisms governing their ontogeny in NO2-promoted allergic airway disease remain unstudied. We measured the kinetics of lung inflammation after antigen challenge in NO2-promoted allergic airway disease, including inflammatory cells in bronchoalveolar lavage and antigen-specific IL-17A production from the lung. We determined that IL-17A+ cells were predominately CD4+T cell receptor (TCR)β+ Th17 cells, and that a functional IL-1 receptor was required for Th17, but not Th2, cytokine production after in vitro antigen restimulation of lung cells. The absence of natural killer T cells, γδ T cells, or the inflammasome scaffold nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain (Nlrp)3 did not affect the development of NO2-promoted allergic inflammation or IL-17A production. Similarly, neutrophil depletion or the neutralization of IL-1α during sensitization exerted no effect on these parameters. However, the absence of caspase-1 significantly reduced IL-17A production from lung cells without affecting Th2 cytokines or lung inflammation. Finally, the intranasal administration of IL-1β and the inhalation of antigen promoted allergic sensitization that was reflected by neutrophilic airway inflammation and IL-17A production from CD4+TCRβ+ Th17 cells subsequent to antigen challenge. These data implicate a role for caspase-1 and IL-1β in the IL-1 receptor–dependent Th17 response manifest in NO2-promoted allergic airway disease.
asthma; Th17; IL-1R; IL-17; nitrogen dioxide
Type 2 inflammatory cytokines including interleukin-4 (IL-4), -5, -9, and -13 drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial and the importance of IL-9 vs. IL-4 producing CD4+ effector T cells in Type 2 immunity is incompletely defined. Herein, we generated IL-9 deficient and IL-9 fluorescent reporter mice that demonstrated an essential role for this cytokine in the early Type 2 immunity against Nippostrongylus brasiliensis. Whereas Th9 cells and Type 2 Innate Lymphoid Cells (ILC2) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells caused rapid worm expulsion, marked basophilia and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and non-redundant role for Th9 cells and IL-9 in host protective Type 2 immunity against parasitic worm infection.
B cells are exposed to high levels of CD40 ligand (CD40L, CD154) in chronic inflammatory diseases. In addition, B cells expressing both CD40 and CD40L have been identified in human diseases such as autoimmune diseases and lymphoma. However, how such constitutively CD40-activated B cells under inflammation may impact on T cell response remains unknown. Using a mouse model in which B cells express a CD40 ligand transgene (CD40LTg) and receive autocrine CD40/CD40L signaling, we show that CD40LTg B cells stimulated memory-like CD4 and CD8 T cells to express IL-10. This IL-10 expression by CD8 T cells was dependent on IFN-I and Programmed cell death protein 1, and was critical for CD8 T cells to counter-regulate their over activation. Furthermore, adoptive transfer of naïve CD8 T cells in RAG-1−/− mice normally induces colitis in association with IL-17 and IFNγ cytokine production. Using this model, we show that adoptive co-transfer of CD40LTg B cells, but not wild type B cells, significantly reduced IL-17 response and regulated colitis in association with IL-10 induction in CD8 T cells. Thus, B cells expressing CD40L can be a therapeutic goal to regulate inflammatory CD8 T cell response by IL-10 induction. 194
The growth arrest and DNA damage-inducible 45β (GADD45β) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45β in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45β as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45β mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45β protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45β−/− mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45β in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45β stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45β plays an essential role during chondrocyte terminal differentiation.