The tumour microenvironment thwarts conventional immunotherapy through multiple immunologic mechanisms, such as the secretion of the transforming growth factor-β (TGF-β), which stunts local tumour immune responses. Therefore, high doses of interleukin-2 (IL-2), a conventional cytokine for metastatic melanoma, induces only limited responses. To overcome the immunoinhibitory nature of the tumour microenvironment, we developed nanoscale liposomal polymeric gels (nanolipogels; nLGs) of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers that can deliver small hydrophobic molecular inhibitors and water-soluble protein cytokines in a sustained fashion to the tumour microenvironment. nLGs releasing TGF-β inhibitor and IL-2 significantly delayed tumour growth, increased survival of tumour-bearing mice, and increased the activity of natural killer cells and of intratumoral-activated CD8+ T-cell infiltration. We demonstrate that the efficacy of nLGs in tumour immunotherapy results from a crucial mechanism involving activation of both innate and adaptive immune responses.

There are no mucosal adjuvant formulations licensed for human use, despite protection against many mucosally-transmitted infections probably requiring immunity at the site of pathogen entry1. Polyethyleneimines (PEI) are organic polycations used as nucleic acid transfection reagents in vitro, and gene and DNA vaccine delivery vehicles in vivo2, 3. Here we show that PEI has unexpected and unusually potent mucosal adjuvant activity in conjunction with viral subunit glycoprotein antigens. Single intranasal administration of influenza HA or HSV-2 gD with PEI elicited robust protection from otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen that were taken up by antigen presenting cells in vitro and in vivo, promoted DC trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host dsDNA that triggered Irf-3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use.

Microbial penetration of the intestinal epithelial barrier triggers inflammatory responses that include induction of the bactericidal C-type lectin RegIIIγ. Systemic administration of flagellin, a bacterial protein that stimulates Toll-like receptor 5 (TLR5), induces epithelial expression of RegIIIγ and protects mice from intestinal colonization with antibiotic-resistant bacteria. Flagellin-induced RegIIIγ expression is IL-22-dependent, but how TLR signaling leads to IL-22 expression is incompletely defined. Using conditional depletion of lamina propria dendritic cell (LPDC) subsets, we demonstrated that CD103+ CD11b+ LPDCs, but not monocyte-derived CD103− CD11b+ LPDCs, expressed high amounts of IL-23 following bacterial flagellin administration and drove IL-22-dependent RegIIIγ production. Maximal expression of IL-23 subunits IL-23p19 and IL-12p40 occurred within 60 minutes of exposure to flagellin. IL-23 subsequently induced a burst of IL-22 followed by sustained RegIIIγ expression. Thus, CD103+ CD11b+ LPDCs, in addition to promoting long-term tolerance to ingested antigens, also rapidly produce IL-23 in response to detection of flagellin in the lamina propria.

Increasing evidence suggests that innate immunity plays an important role in alcohol-induced liver injury and most studies have focused on positive regulation of innate immunity. The main objective of this study was to investigate the negative regulator of innate immunity, IL-1/Toll-like receptor (TLR) signaling pathways and interleukin receptor-associated kinase-M (IRAK-M) in alcoholic liver injury. We established an alcohol-induced liver injury model using wild type and IRAK-M deficient B6 mice and investigated the possible mechanisms. We found that in the absence of IRAK-M, liver damage by alcohol was worse with higher alanine transaminase (ALT), more immune cell infiltration and increased numbers of IFNγ producing cells. We also found enhanced phagocytic activity in CD68+ cells. Moreover, our results revealed altered gut bacteria after alcohol consumption and this was more striking in the absence of IRAK-M. Our study provides evidence that IRAK-M plays an important role in alcohol-induced liver injury and IRAK-M negatively regulates the innate and possibly the adaptive immune response in the liver reacting to acute insult by alcohol. In the absence of IRAK-M, the hosts developed worse liver injury, enhanced gut permeability and altered gut microbiota.

We all have been taught that the immune system is educated in the thymus; however, where the immune system receives the second lesson in order to be tolerant against non-harmful pathogens, such as commensal bacteria, has never been addressed. Considering that commensal bacteria colonize the intestine and that regulatory T (Treg) cells are enriched in this organ, one could think that the intestine is the place where this second lesson would occur. This idea was now sustained by the work of Lathrop et al., which sheds new light on the complex mechanism of peripheral tolerance induction.

Bacterial pneumonia remains a significant cause of mortality in the United States. The innate immune response is the first line of defense against invading bacteria. Neutrophil recruitment to the lungs is the first step in a multistep sequence leading to bacterial clearance. Ligand interaction with pattern-recognizing receptors (PRRs) leads to chemokine production, which drives neutrophils to the site of infection. Although we demonstrated that RIP2 is important for host defense in the lungs against Escherichia coli, the individual roles of NOD1 and NOD2 in pulmonary defense have not been addressed. Here, we explored the role of NOD2 in neutrophil-mediated host defense against an extracellular pathogen, E. coli. We found enhanced bacterial burden and reduced neutrophil and cytokine/chemokine levels in the lungs of NOD2−/− mice following E. coli infection. Furthermore, we observed reduced activation of NF-κB and mitogen-activated protein kinases (MAPKs) in the lungs of NOD2−/− mice upon E. coli challenge. Moreover, NOD2−/− neutrophils show impaired intracellular bacterial killing. Using NOD2/RIP2−/− mice, we observed bacterial burden and neutrophil accumulation in the lungs similar to those seen with NOD2−/− mice. In addition, bone marrow-derived macrophages obtained from NOD2/RIP2−/− mice demonstrate a reduction in activation of NF-κB and MAPKs similar to that seen with NOD2−/− mice in response to E. coli. These findings unveil a previously unrecognized role of the NOD2-RIP2 axis for host defense against extracellular Gram-negative bacteria. This pathway may represent a novel target for the treatment of lung infection/inflammation.

Intracellular (clade B) ovalbumin (ov)-serpin protease inhibitors play an important role in tissue homeostasis by protecting cells from death in response to hypoosmotic stress, heat shock and other stimuli. Whether these serpins influence immunological tolerance and the risk for autoimmune diseases is not known. We found that a fraction of young autoimmune diabetes-prone non-obese diabetic (NOD) mice had elevated levels of autoantibodies against a member of clade B family known as serpinB13. High levels of anti-serpinB13 antibodies were accompanied by low levels of anti-insulin autoantibodies, reduced numbers of islet-associated T-cells and delayed onset of diabetes. Exposure to anti-serpinB13 monoclonal antibody (mAb) alone also decreased islet inflammation and co-administration of this reagent and a suboptimal dose of anti-CD3 mAb accelerated recovery from diabetes. In a fashion similar to that discovered in the NOD model, a deficiency in humoral activity against serpinB13 was associated with early onset of human type 1 diabetes. These findings suggest that in addition to limiting exposure to proteases within the cell, clade B serpins help to maintain homeostasis by inducing protective humoral immunity.

Commensal bacterial sensing by Toll-like receptors (TLRs) is critical for maintaining intestinal homeostasis, but can lead to colitis in the absence of IL-10. While TLRs are expressed in multiple cell types in the colon, the cell type(s) responsible for the development of colitis currently unknown. Here, we generated mice that are selectively deficient in MyD88 in various cellular compartments in an IL-10−/− setting. While epithelial expression of MyD88 was dispensable, MyD88 expression in the mononuclear phagocyte (MNP) compartment was required for colitis development. Specifically, phenotypically distinct populations of colonic MNPs expressed high levels IL-1β, IL-23 and IL-6 and promoted Th17 responses in the absence of IL-10. Thus, gut bacterial sensing through MyD88 in MNPs drives inflammatory bowel disease (IBD) when unopposed by IL-10.

Background

Conditional gene knockout (cKO) mediated by the Cre/LoxP system is indispensable for exploring gene functions in mice. However, a major limitation of this method is that gene KO is not reversible. A number of methods have been developed to overcome this, but each method has its own limitations.

Results

We describe a simple method we have named LOFT [LoxP-flippase (FLP) recognition target (FRT) Trap], which is capable of reversible cKO and free of the limitations associated with existing techniques. This method involves two alleles of a target gene: a standard floxed allele, and a multi-functional allele bearing an FRT-flanked gene-trap cassette, which inactivates the target gene while reporting its expression with green fluorescent protein (GFP); the trapped allele is thus a null and GFP reporter by default, but is convertible into a wild-type allele. The floxed and trapped alleles can typically be generated using a single construct bearing a gene-trap cassette doubly flanked by LoxP and FRT sites, and can be used independently to achieve conditional and constitutive gene KO, respectively. More importantly, in mice bearing both alleles and also expressing the Cre and FLP recombinases, sequential function of the two enzymes should lead to deletion of the target gene, followed by restoration of its expression, thus achieving reversible cKO. LOFT should be generally applicable to mouse genes, including the growing numbers of genes already floxed; in the latter case, only the trapped alleles need to be generated to confer reversibility to the pre-existing cKO models. LOFT has other applications, including the creation and reversal of hypomorphic mutations. In this study we proved the principle of LOFT in the context of T-cell development, at a hypomorphic allele of Baf57/Smarce1 encoding a subunit of the chromatin-remodeling Brg/Brahma-associated factor (BAF) complex. Interestingly, the FLP used in the current work caused efficient reversal in peripheral T cells but not thymocytes, which is advantageous for studying developmental epigenetic programming of T-cell functions, a fundamental issue in immunology.

Conclusions

LOFT combines well-established basic genetic methods into a simple and reliable method for reversible gene targeting, with the flexibility of achieving traditional constitutive and conditional KO.

The chronic autoimmune diseases include multiple complex genetic disorders. Recently, genome-wide association studies (GWAS) have identified a large number of major loci, with many associations shared between various autoimmune diseases. These associations highlight key roles for lymphocyte activation and prioritize specific cytokine pathways and mechanisms of host-microbe recognition. Despite success in identifying loci, comprehensive models of disease pathogenesis are currently lacking. Future efforts comparing association patterns between autoimmune diseases may be particularly illustrative. New genomic technologies applied to classic genetic studies involving twins, early onset cases, and phenotypic extremes may provide key insights into developmental and gene-environment interactions in autoimmunity.

NLRs (nucleotide-binding domain leucine-rich repeat containing receptors; NOD-like receptors) are a class of pattern recognition receptor (PRR) that respond to host perturbation from either infectious agents or cellular stress1,2. The function of most NLR family members has not been characterized and their role in instructing adaptive immune responses remains unclear 2,3. NLRP10 (also known as PYNOD, NALP10, PAN5 and NOD8) is the only NLR lacking the putative ligand binding leucine rich repeat domain, and has been postulated to be a negative regulator of other NLR members including NLRP34–6. We did not find evidence that NLRP10 functions through an inflammasome to regulate caspase-1 activity nor that it regulates other inflammasomes. Instead, Nlrp10−/− mice had a profound defect in helper T cell-driven immune responses to a diverse array of adjuvants including lipopolysaccharide (LPS), aluminium hydroxide (alum) and complete Freund’s adjuvant (CFA). Adaptive immunity was impaired in the absence of NLRP10 due to a dendritic cell (DC) intrinsic defect in emigration from inflamed tissues while upregulation of DC costimulatory molecules and chemotaxis to CCR7-dependent and independent ligands remained intact. The loss of antigen transport to the draining LN by this migratory DC subset resulted in an almost absolute loss in naïve CD4+ T cell priming, highlighting the critical link between diverse innate immune stimulation, NLRP10 activity and the immune function of mature DCs.

Recent studies have shown that IL-17 can contribute beneficially to pathogen defense, but also that excessive IL-17 levels are associated with chronic inflammation and autoimmune disorders. So far, the role of IL-17 in viral infections and type 1 diabetes is ambiguous. In this study, we used IL-17A eGFP bicistronic reporter mouse strains to analyze in situ production of IL-17A. Upon Klebsiella pneumonia bacterial infection, CD4+ and γδ T cells produce IL-17A. In contrast, CD4+ or CD8+ T cells do not produce IL-17A in response to acute or protracted viral infection with lymphocytic choriomeningitis virus (LCMV), or during autoimmune diabetes development in the CD8-driven LCMV-induced model of type 1 diabetes. We conclude that viral elimination and type 1 diabetes can occur in the absence of detectable IL-17A production, suggesting IL-17A is not essential in these settings.

The Th17 cytokine, IL-22, regulates host immune responses to extracellular pathogens. Whether IL-22 plays a role in viral infection, however, is poorly understood. We report here that Il22−/− mice were more resistant to lethal West Nile virus (WNV) encephalitis, but had similar viral loads in the periphery compared to wild type (WT) mice. Viral loads, leukocyte infiltrates, proinflammatory cytokines and apoptotic cells in the central nervous system (CNS) of Il22−/− mice were also strikingly reduced. Further examination showed that Cxcr2, a chemokine receptor that plays a non-redundant role in mediating neutrophil migration, was significantly reduced in Il22−/− compared to WT leukocytes. Expression of Cxcr2 ligands, cxcl1 and cxcl5, was lower in Il22−/− brains than wild type mice. Correspondingly, neutrophil migration from the blood into the brain was attenuated following lethal WNV infection of Il22−/− mice. Our results suggest that IL-22 signaling exacerbates lethal WNV encephalitis likely by promoting WNV neuroinvasion.

To investigate the respective contributions of TLR versus IL-1R mediated signals in MyD88 dependent control of Mycobacterium tuberculosis, we compared the outcome of M. tuberculosis infection in MyD88, TRIF/MyD88, IL-1R1, and IL-1β-deficient mice. All four strains displayed acute mortality with highly increased pulmonary bacterial burden suggesting a major role for IL-1β signaling in determining the MyD88 dependent phenotype. Unexpectedly, the infected MyD88 and TRIF/MyD88-deficient mice, rather than being defective in IL-1β expression, displayed increased cytokine levels relative to wild-type animals. Similarly, infected mice deficient in caspase-1 and ASC, which have critical functions in inflammasome-mediated IL-1β maturation, showed unimpaired IL-1β production and importantly, were considerably less susceptible to infection than IL-1β deficient mice. Together our findings reveal a major role for IL-1β in host resistance to M. tuberculosis and indicate that during this infection the cytokine can be generated by a mechanism that does not require TLR signaling or caspase-1. The Journal of Immunology, 2010, 184: 3326–3330.

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and the leading cause of chronic liver disease in the Western world. Twenty percent of NAFLD individuals develop chronic hepatic inflammation (non-alcoholic steatohepatitis, NASH) associated with cirrhosis, portal hypertension and hepatocellular carcinoma, yet causes of progression from NAFLD to NASH remain obscure. Here, we show that the NLRP6 and NLRP3 inflammasomes and the effector protein IL-18 negatively regulate NAFLD/NASH progression, as well as multiple aspects of metabolic syndrome via modulation of the gut microbiota. Different animal models reveal that inflammasome deficiency-associated changes in the configuration of the gut microbiota are associated with exacerbated hepatic steatosis and inflammation through influx of TLR4 and TLR9 agonists into the portal circulation, leading to enhanced hepatic TNF-α expression that drives NASH progression. Furthermore, co-housing of inflammasome-deficient animals to wild type mice results in exacerbation of hepatic steatosis, glucose intolerance, and obesity. Thus, altered interactions between the gut microbiota and the host, produced by defective NLRP3 and NLRP6 inflammasome sensing, may govern the rate of progression of multiple metabolic syndrome-associated abnormalities, highlighting the central role of the microbiota in the pathogenesis of heretofore seemingly unrelated systemic auto-inflammatory and metabolic disorders.

Various cell types in both lymphoid and non-lymphoid tissues produce the anti-inflammatory cytokine interleukin (IL)-10 during murine cytomegalovirus (MCMV) infection. The functions of IL-10 in the liver during acute infection and the cells that generate this cytokine at this site have not been extensively investigated. In this study, we demonstrate that the production of IL-10 in the liver is elevated in C57BL/6 mice during late acute MCMV infection. Using IL-10 green fluorescence protein (GFP) reporter knock-in mice, designated IL-10-internal ribosomal entry site (IRES)-GFP-enhanced reporter (tiger), NK cells are identified as major IL-10 expressing cells in the liver after infection, along with T cells and other leukocytes. In the absence of IL-10, mice exhibit marked elevations in proinflammatory cytokines and in the numbers of mononuclear cells and lymphocytes infiltrating the liver during this infection. IL-10-deficiency also enhances liver injury without improving viral clearance from this site. Collectively, the results indicate that IL-10-producing cells in the liver provide protection from collateral injury by modulating the inflammatory response associated with MCMV infection.

IL-10 is an anti-inflammatory cytokine that regulates the extent of host immunity to infection by exerting suppressive effects on different cell types. Herpes viruses induce IL-10 to modulate the virus-host balance towards their own benefit, resulting in prolonged virus persistence. To define the cellular and molecular players involved in IL-10 modulation of herpes virus-specific immunity, we studied mouse cytomegalovirus (MCMV) infection. Here we demonstrate that IL-10 specifically curtails the MCMV-specific CD4 T cell response by suppressing the bidirectional crosstalk between NK cells and myeloid dendritic cells (DCs). In absence of IL-10, NK cells licensed DCs to effectively prime MCMV-specific CD4 T cells and we defined the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 to regulate this bidirectional NK/DC interplay. Consequently, markedly enhanced priming of MCMV-specific CD4 T cells in Il10−/− mice led to faster control of lytic viral replication, but this came at the expense of TNF-α mediated immunopathology. Taken together, our data show that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive immune cell crosstalk, thereby impacting beneficially on the ensuing virus-host balance for both the virus and the host.

Author Summary

Cytomegalovirus (CMV) infections are very widespread in mammalian hosts. Despite the fact that CMVs are usually well controlled by the immune system, they cause persistent life-long infection and have evolved a number of strategies to effectively modulate or hide from host immunity. Since the establishment of an immunosuppressive environment favors virus persistence, IL-10 is one of the host targets that CMVs actively use to tune the virus-host balance toward their own benefit, resulting in prolonged virus persistence and hence increased chance for horizontal transmission. Here, we delineate the mechanisms of how IL-10 exerts its powerful immune-suppressing function in the context of murine cytomegalovirus (MCMV) infection. We found that IL-10 specifically restrains the priming of MCMV-specific CD4 T cell responses by suppressing dendritic cell (DC) - natural killer cell (NK) crosstalk during acute MCMV infection. Target molecules mediating this bi-directional crosstalk between DCs and NK cells were the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 and all of them were markedly suppressed by IL-10. A consequence resulting from this impeded DC-NK cross-talk by IL-10, leading to poor priming of MCMV-specific CD4 T cell responses was increased lytic CMV persistence and reduced development of host tissue damage. Our study indicates that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive crosstalk, thereby imparting on the ensuing virus-host balance for the benefit of both the virus and the host.

BACKGROUND & AIMS

Acute pancreatitis is characterized by early activation of intracellular proteases followed by acinar cell death and inflammation. Activation of damage-associated molecular pattern (DAMP) receptors and a cytosolic complex termed the inflammasome initiates forms of inflammation. In this study, we examined whether DAMP-receptors and the inflammasome provide the link between cell death and the initiation of inflammation in pancreatitis.

METHODS

Acute pancreatitis was induced by caerulein stimulation in wild-type mice and mice deficient in components of the inflammasome (ASC, NLRP3, caspase-1), Toll-like receptor 9 (TLR9), or the purinergic receptor P2X7. Resident and infiltrating immune cell populations and pro-IL-1β expression were characterized in control and caerulein-treated adult murine pancreas. TLR9 expression was quantified in pancreatic cell populations. Additionally, wild-type mice were pretreated with a TLR9 antagonist prior to induction of acute pancreatitis by caerulein or retrograde bile duct infusion of taurolithocholic acid 3-sulfate (TLCS).

RESULTS

Caspase-1, ASC, and NLPR3 were required for inflammation in acute pancreatitis. Genetic deletion of Tlr9 reduced pancreatic edema, inflammation, and pro-IL-1β expression in pancreatitis. TLR9 was expressed in resident immune cells of the pancreas, which are predominantly macrophages. Pretreatment with the TLR9 antagonist IRS954 reduced pancreatic edema, inflammatory infiltrate, and apoptosis. Pretreatment with IRS954 reduced pancreatic necrosis and lung inflammation in TLCS-induced acute pancreatitis.

CONCLUSIONS

Components of the inflammasome, specifically ASC, caspase-1, and NLRP3, are required for the development of inflammation in acute pancreatitis. TLR9 and P2X7 are important DAMP receptors upstream of inflammasome activation, and their antagonism could provide a new therapeutic strategy for treating acute pancreatitis.

Background and Aims

Chronic infection with the bacterial pathogen Helicobacter pylori causes gastric disorders ranging from chronic gastritis to gastric adenocarcinoma. Only a subset of infected individuals will develop overt disease; the large majority remains asymptomatic despite lifelong colonization. This study aims to elucidate the differential susceptibility to H. pylori that is found both across and within populations.

Methods

We have established a C57BL/6 mouse model of H. pylori infection with a strain that is capable of delivering the virulence factor CagA into host cells through the activity of a Cag-pathogenicity island-encoded type IV secretion system.

Results

Mice infected at 5–6 weeks of age with CagA+ H. pylori rapidly develop gastritis, gastric atrophy, epithelial hyperplasia and metaplasia in a type IV secretion system-dependent manner. In contrast, mice infected during the neonatal period with the same strain are protected from preneoplastic lesions. Their protection results from the development of H. pylori-specific peripheral immunological tolerance, which requires TGF-β signalling and is mediated by long-lived, inducible regulatory T-cells, and which controls the local CD4+ T-cell responses that trigger premalignant transformation. Tolerance to H. pylori develops in the neonatal period due to a biased Treg to T-effector cell ratio, and is favoured by prolonged low-dose exposure to antigen.

Conclusions

Using a novel CagA+ H. pylori infection model, we report here that the development of tolerance to H. pylori protects from gastric cancer precursor lesions. The age at initial infection may thus account for the differential susceptibility of infected individuals to H. pylori-associated disease manifestations.

Pneumonia is a common cause of morbidity and mortality and the most frequent source of sepsis. Bacteria that try to invade normally sterile body sites are recognized by innate immune cells through pattern recognition receptors, among which toll-like receptors (TLRs) feature prominently. Interleukin-1 receptor (IL-1R)–associated kinase (IRAK)-M is a proximal inhibitor of TLR signaling expressed by epithelial cells and macrophages in the lung. To determine the role of IRAK-M in host defense against bacterial pneumonia, IRAK-M-deficient (IRAK-M−/−) and normal wild-type (WT) mice were infected intranasally with Klebsiella pneumoniae. IRAK-M mRNA was upregulated in lungs of WT mice with Klebsiella pneumonia, and the absence of IRAK-M resulted in a strongly improved host defense as reflected by reduced bacterial growth in the lungs, diminished dissemination to distant body sites, less peripheral tissue injury and better survival rates. Although IRAK-M−/− alveolar macrophages displayed enhanced responsiveness toward intact K. pneumoniae and Klebsiella lipopolysaccharide (LPS) in vitro, IRAK-M−/− mice did not show increased cytokine or chemokine levels in their lungs after infection in vivo. The extent of lung inflammation was increased in IRAK-M−/− mice shortly after K. pneumoniae infection, as determined by semiquantitative scoring of specific components of the inflammatory response in lung tissue slides. These data indicate that IRAK-M impairs host defense during pneumonia caused by a common gram-negative respiratory pathogen.

Genome-wide association studies (GWAS) in immune-mediated diseases have identified over 150 associated genomic loci. Many of these loci play a role in T cell responses, and regulation of T cell differentiation plays a critical role in immune-mediated diseases; however, the relationship between implicated disease loci and T cell differentiation is incompletely understood. To further address this relationship, we examined differential gene expression in naïve human CD4+ T cells, as well as in in vitro differentiated Th1, memory Th17-negative and Th17-enriched CD4+ T cells subsets using microarray and RNASeq. We observed a marked enrichment for increased expression in memory CD4+ compared to naïve CD4+ T cells of genes contained among immune–mediated disease loci. Within memory T cells, expression of disease-associated genes was typically increased in Th17-enriched compared to Th17-negative cells. Utilizing RNASeq and promoter methylation studies, we identified a differential regulation pattern for genes solely expressed in Th17 cells (IL17A and CCL20) compared to genes expressed in both Th17 and Th1 cells (IL23R and IL12RB2), where high levels of promoter methylation are correlated to near zero RNASeq levels for IL17A and CCL20. These findings have implications for human Th17 celI plasticity and for the regulation of Th17-Th1 expression signatures. Importantly, utilizing RNASeq we found an abundant isoform of IL23R terminating before the transmembrane domain that was enriched in Th17 cells. In addition to molecular resolution, we find that RNASeq provides significantly improved power to define differential gene expression and identify alternative gene variants relative to microarray analysis. The comprehensive integration of differential gene expression between cell subsets with disease-association signals, and functional pathways provides insight into disease pathogenesis.

Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld form a complex via the interaction through ‘WW-PPXY’ motifs. The Itch-Cyld complex sequentially cleaved K63-linked ubiquitin chains and catalyzed K48-linked ubiquitination on the kinase Tak1 to terminate inflammatory tumor necrosis factor signaling. Reconstitution of wild-type Cyld but not mutant Cyld(Y485A), which cannot associate with Itch, blocked the sustained Tak1 activation and proinflammatory cytokine production by Cyld−/− bone marrow-derived macrophages. Itch or Cyld deficiency resulted in chronic production of tumor-promoting cytokines by the tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have uncovered an Itch-Cyld mediated regulatory mechanism in innate inflammatory cells.

Innate and adaptive immunity play important protective roles by combating herpes simplex virus 1 (HSV-1) infection. Transforming growth factor β (TGF-β) is a key negative cytokine regulator of both innate and adaptive immune responses. Yet, it is unknown whether TGF-β signaling in either immune compartment impacts HSV-1 replication and latency. We undertook genetic approaches to address these issues by infecting two different dominant negative TGF-β receptor type II transgenic mouse lines. These mice have specific TGF-β signaling blockades in either T cells or innate cells. Mice were ocularly infected with HSV-1 to evaluate the effects of restricted innate or adaptive TGF-β signaling during acute and latent infections. Limiting innate cell but not T cell TGF-β signaling reduced virus replication in the eyes of infected mice. On the other hand, blocking TGF-β signaling in either innate cells or T cells resulted in decreased latency in the trigeminal ganglia of infected mice. Furthermore, inhibiting TGF-β signaling in T cells reduced cell lysis and leukocyte infiltration in corneas and trigeminal ganglia during primary HSV-1 infection of mice. These findings strongly suggest that TGF-β signaling, which generally functions to dampen immune responses, results in increased HSV-1 latency.

Recognition of microbial patterns by host receptors is the first step in a multistep sequence leading to neutrophil-dependent host resistance. Although the role of membrane-bound sensors in bacterial recognition has been examined in detail, the importance of cytosolic sensors in the lungs is largely unexplored. In this context, there is a major lack of understanding related to the downstream signaling mediators, such as cells and/or molecules, during acute extracellular Gram-negative bacterial pneumonia. In order to determine the role of NOD-like receptors (NLRs), we used an experimental Escherichia coli infection model using mice deficient in the gene coding for the NLR adaptor, receptor-interacting protein 2 (RIP2). RIP2−/− mice with E. coli infection displayed higher bacterial burden and reduced neutrophil recruitment and tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), macrophage inflammatory protein 2 (MIP-2), and CXCL5/LIX expression, along with attenuated histopathological changes in the lungs. Decreased IL-17A levels were observed, along with lower numbers of IL-17A-producing T cells, in RIP2−/− mice after infection. RIP2−/− mice also show reduced IL-6 and IL-23 levels in the lungs, along with decreased activation of STAT3 after infection. Furthermore, activation of NF-κB and mitogen-activated protein kinases (MAPKs) and expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in the lungs of infected RIP2−/− mice were attenuated following infection. Although neutrophil mobilization to the blood was impaired in RIP2−/− mice following infection, the expression of CD62P, CD11a/18, CD11b, and CXCR2 on blood and lung neutrophils was not altered between infected wild-type (WT) and RIP2−/− mice. Thus, RIP2 contributes to neutrophil-dependent host defense against an extracellular Gram-negative pathogen via (i) IL-17A regulation and (ii) neutrophil mobilization to the blood.

We have created a high-resolution linkage map of Miscanthus sinensis, using genotyping-by-sequencing (GBS), identifying all 19 linkage groups for the first time. The result is technically significant since Miscanthus has a very large and highly heterozygous genome, but has no or limited genomics information to date. The composite linkage map containing markers from both parental linkage maps is composed of 3,745 SNP markers spanning 2,396 cM on 19 linkage groups with a 0.64 cM average resolution. Comparative genomics analyses of the M. sinensis composite linkage map to the genomes of sorghum, maize, rice, and Brachypodium distachyon indicate that sorghum has the closest syntenic relationship to Miscanthus compared to other species. The comparative results revealed that each pair of the 19 M. sinensis linkages aligned to one sorghum chromosome, except for LG8, which mapped to two sorghum chromosomes (4 and 7), presumably due to a chromosome fusion event after genome duplication. The data also revealed several other chromosome rearrangements relative to sorghum, including two telomere-centromere inversions of the sorghum syntenic chromosome 7 in LG8 of M. sinensis and two paracentric inversions of sorghum syntenic chromosome 4 in LG7 and LG8 of M. sinensis. The results clearly demonstrate, for the first time, that the diploid M. sinensis is tetraploid origin consisting of two sub-genomes. This complete and high resolution composite linkage map will not only serve as a useful resource for novel QTL discoveries, but also enable informed deployment of the wealth of existing genomics resources of other species to the improvement of Miscanthus as a high biomass energy crop. In addition, it has utility as a reference for genome sequence assembly for the forthcoming whole genome sequencing of the Miscanthus genus.