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1.  Tuning innate immune activation by surface texturing of polymer microparticles: the role of shape in inflammasome activation1 
Polymeric microparticles have been widely investigated as platforms for delivery of drugs, vaccines, and imaging contrast agents, and are increasingly used in a variety of clinical applications. Microparticles activate the inflammasome complex and induce the processing and secretion of IL-1β, a key innate immune cytokine. Recent work suggests that while receptors are clearly important for particle phagocytosis, other physical characteristics especially shape, play an important role in the way microparticles activate cells. We examined the role of particle surface texturing not only on uptake efficiency but also on the subsequent immune cell activation of the inflammasome. Using a method based on emulsion processing of amphiphilic block copolymers, we prepared microparticles with similar overall sizes and surface chemistries, but having either smooth or highly micro-textured surfaces. In vivo, textured (budding) particles induced more rapid neutrophil recruitment to the injection site. In vitro, budding particles were more readily phagocytosed than smooth particles and induced more lipid raft recruitment to the phagosome. Remarkably, budding particles also induced stronger IL-1β secretion than smooth particles, through activation of the NLRP3 inflammasome. These findings demonstrate a pronounced role of particle surface topography in immune cell activation suggesting that shape is a major determinant of inflammasome activation.
PMCID: PMC3646559  PMID: 23427254
2.  T-bet Knockout Prevents Helicobacter felis-Induced Gastric Cancer1 
Helicobacter infection is the primary risk factor for gastric cancer, with the cytokine environment within the gastric mucosa the strongest predictor of disease risk. Elevated TNF-α, IL-1β, and low IL-10 are associated with the highest risk. In this study, we used C57BL/6 mice to identify T-bet as a central regulator of the cytokine environment during Helicobacter felis infection. We infected male and female C57BL/6 and C57BL/6-T-bet knockout (KO) liter mates with H. felis and examined the bacterial colonization, immune response, and mucosal damage at varying time points. T-bet KO mice maintained infection for 15 mo at similar levels to wild-type mice. Infection and immune response did not differ between male and female mice. Despite sustained infection, T-bet KO mice respond with a blunted Th1 response associated with preservation of parietal and chief cells and protection from the development of gastric cancer. Unexpectedly, T-bet KO mice develop a gastric environment that would not be expected based on the phenotype of T-bet KO CD4 cells alone. T-bet KO mice respond to H. felis infection with a markedly blunted IL-1β and TNF-α and elevated IL-10 levels. Activity of this one master regulator modulates the expression of the key gastric mucosal cytokines associated with gastric cancer and may be a target for therapy to restore immune balance clinically in patients at risk for gastric cancer.
PMCID: PMC3966469  PMID: 19535625
3.  Proteasomal degradation of herpes simplex virus capsids in macrophages releases DNA to the cytosol for recognition by DNA sensors1 
The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9, and that non-immune tissue cells can sense herpesvirus DNA in the nucleus. However, it remains unknown how and where myeloid cells, like macrophages and conventional dendritic cells, detect infections with herpesviruses. Here we demonstrate that the HSV-1 capsid was ubiquitinated in the cytosol and degraded by the proteasome, hence releasing genomic DNA into the cytoplasm for detection by DNA sensors. In this context, the DNA sensor IFI16 is important for induction of IFN-β in human macrophages after infection with HSV-1 and CMV. Viral DNA localized to the same cytoplasmic regions as IFI16, with DNA sensing being independent of viral nuclear entry. Thus, proteasomal degradation of herpesvirus capsids releases DNA to the cytoplasm for recognition by DNA sensors.
PMCID: PMC3578088  PMID: 23345332
4.  Type 1 IFN Mediates Cross-Talk between Innate and Adaptive Immunity That Abrogates Transplantation Tolerance1,2 
TLR activation of innate immunity prevents the induction of transplantation tolerance and shortens skin allograft survival in mice treated with costimulation blockade. The mechanism by which TLR signaling mediates this effect has not been clear. We now report that administration of the TLR agonists LPS (TLR4) or polyinosinic:polycytidylic acid (TLR3) to mice treated with costimulation blockade prevents alloreactive CD8+ T cell deletion, primes alloreactive CTLs, and shortens allograft survival. The TLR4- and MyD88-dependent pathways are required for LPS to shorten allograft survival, whereas polyinosinic:polycytidylic acid mediates its effects through a TLR3-independent pathway. These effects are all mediated by signaling through the type 1 IFN (IFN-αβ) receptor. Administration of IFN-β recapitulates the detrimental effects of TLR agonists on transplantation tolerance. We conclude that the type 1 IFN generated as part of an innate immune response to TLR activation can in turn activate adaptive immune responses that abrogate transplantation tolerance. Blocking of type 1 IFN-dependent pathways in patients may improve allograft survival in the presence of exogenous TLR ligands.
PMCID: PMC3896082  PMID: 17982052
5.  Chronic Alcohol-Induced microRNA-155 Contributes to Neuroinflammation in a TLR4-Dependent Manner in Mice 
PLoS ONE  2013;8(8):e70945.
Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation.
To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation.
Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation.
Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155.
Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.
PMCID: PMC3739772  PMID: 23951048
6.  Role of Interferon Regulatory Factor 7 in T Cell Responses during Acute Lymphocytic Choriomeningitis Virus Infection 
Journal of Virology  2012;86(20):11254-11265.
Type I interferons (IFNs), predominantly IFN-α and -β, play critical roles in both innate and adaptive immune responses against viral infections. Interferon regulatory factor 7 (IRF7), a key innate immune molecule in the type I IFN signaling pathway, is essential for the type I IFN response to many viruses, including lymphocytic choriomeningitis virus (LCMV). Here, we show that although IRF7 knockout (KO) mice failed to control the replication of LCMV in the early stages of infection, they were capable of clearing LCMV infection. Despite the lack of type I IFN production, IRF7 KO mice generated normal CD4+ T cell responses, and the expansion of naïve CD8+ T cells into primary CD8+ T cells specific for LCMV GP33–41 was relatively normal. In contrast, the expansion of the LCMV NP396-specific CD8+ T cells was severely impaired in IRF7 KO mice. We demonstrated that this defective CD8+ T cell response is due neither to an impaired antigen-presenting system nor to any intrinsic role of IRF7 in CD8+ T cells. The lack of a type I IFN response in IRF7 KO mice did not affect the formation of memory CD8+ T cells. Thus, the present study provides new insight into the impact of the innate immune system on viral pathogenesis and demonstrates the critical contribution of innate immunity in controlling virus replication in the early stages of infection, which may shape the quality of CD8+ T cell responses.
PMCID: PMC3457134  PMID: 22875973
7.  C21orf91 Genotypes Correlate With Herpes Simplex Labialis (Cold Sore) Frequency: Description of a Cold Sore Susceptibility Gene 
The Journal of Infectious Diseases  2011;204(11):1654-1662.
(See the editorial commentary by Cunningham and Booth, on pages 1645–7.)
Background. Herpes simplex virus type 1 (HSV-1) infects >70% of the United States population. We identified a 3-megabase region on human chromosome 21 containing 6 candidate genes associated with herpes simplex labialis (HSL, “cold sores”).
Methods. We conducted single nucleotide polymorphism (SNP) scans of the chromosome 21 region to define which of 6 possible candidate genes were associated with cold sore frequency. We obtained the annual HSL frequency for 355 HSV-1 seropositive individuals and determined the individual genotypes by SNPlex for linkage analysis and parental transmission disequilibrium testing (ParenTDT).
Results. Two-point linkage analysis showed positive linkage between cold sore frequency and 2 SNPs within the C21orf91 region, 1 of which is nonsynonymous. ParenTDT analysis revealed a strong association between another C21orf91 SNP, predicted to lie in the 3′ untranslated region, and frequent HSL (P = .0047). C21orf 91 is a predicted open reading frame of unknown function that encodes a cytosolic protein.
Conclusions. We evaluated candidate genes in the cold sore susceptibility region using fine mapping with 45 SNP markers. 2 complementary techniques identified C21orf91 as a gene of interest for susceptibility to HSL. We propose that C21orf91 be designated the Cold Sore Susceptibility Gene-1 (CSSG1).
PMCID: PMC3203230  PMID: 22039568
8.  Antibodies to cell surface proteins redirect intracellular trafficking pathways 
Antibody-mediated intracellular delivery of therapeutic agents has been considered for treatment of a variety of diseases. These approaches involve targeting cell-surface receptor proteins expressed by tumors or viral proteins expressed on infected cells. We examined the intracellular trafficking of a viral cell-surface-expressed protein, rabies G, with or without binding a specific antibody, ARG1. We found that antibody binding shifts the native intracellular trafficking pathway of rabies G in an Fc-independent manner. Kinetic studies indicate that the ARG1/rabies G complex progressively co-localized with clathrin, early endosomes, late endosomes, and lysosomes after addition to cells. This pathway was different from that taken by rabies G without addition of antibody, which localized with recycling endosomes. Findings were recapitulated using a cellular receptor with a well-defined endogenous recycling pathway. We conclude that antibody binding to cell-surface proteins induces redirection of intracellular trafficking of unbound or ligand bound receptors to a specific degradation pathway. These findings have broad implications for future developments of antibody-based therapeutics.
PMCID: PMC3315679  PMID: 21819978
Endosomes; degradation; antibody; trafficking; internalization
9.  CD200R1 Supports HSV-1 Viral Replication and Licenses Pro-Inflammatory Signaling Functions of TLR2 
PLoS ONE  2012;7(10):e47740.
The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1−/− mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1−/− peritoneal macrophages demonstrated a 70–75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam2CSK4 and to HSV-1. CD200R1−/− macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1−/− mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1−/− mice and CD200R1−/− fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in “licensing” pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence.
PMCID: PMC3474780  PMID: 23082204
10.  Trefoil factor 2 rapidly induces interleukin 33 to promote type 2 immunity during allergic asthma and hookworm infection 
The repair protein trefoil factor 2 promotes Th2 responses to helminth infection and allergens in part by inducing IL-33.
The molecular mechanisms that drive mucosal T helper type 2 (TH2) responses against parasitic helminths and allergens remain unclear. In this study, we demonstrate in mice that TFF2 (trefoil factor 2), an epithelial cell–derived repair molecule, is needed for the control of lung injury caused by the hookworm parasite Nippostrongylus brasiliensis and for type 2 immunity after infection. TFF2 is also necessary for the rapid production of IL-33, a TH2-promoting cytokine, by lung epithelia, alveolar macrophages, and inflammatory dendritic cells in infected mice. TFF2 also increases the severity of allergic lung disease caused by house dust mite antigens or IL-13. Moreover, TFF2 messenger RNA expression is significantly increased in nasal mucosal brushings during asthma exacerbations in children. These experiments extend the biological functions of TFF2 from tissue repair to the initiation and maintenance of mucosal TH2 responses.
PMCID: PMC3302229  PMID: 22329990
11.  IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice 
The Journal of Clinical Investigation  2012;122(10):3476-3489.
Alcoholic liver disease (ALD) is characterized by steatosis and upregulation of proinflammatory cytokines, including IL-1β. IL-1β, type I IL-1 receptor (IL-1R1), and IL-1 receptor antagonist (IL-1Ra) are all important regulators of the IL-1 signaling complex, which plays a role in inflammation. Furthermore, IL-1β maturation is dependent on caspase-1 (Casp-1). Using IL-1Ra–treated mice as well as 3 mouse models deficient in regulators of IL-1β activation (Casp-1 and ASC) or signaling (IL-1R1), we found that IL-1β signaling is required for the development of alcohol-induced liver steatosis, inflammation, and injury. Increased IL-1β was due to upregulation of Casp-1 activity and inflammasome activation. The pathogenic role of IL-1 signaling in ALD was attributable to the activation of the inflammasome in BM-derived Kupffer cells. Importantly, in vivo intervention with a recombinant IL-1Ra blocked IL-1 signaling and markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. Furthermore, physiological doses of IL-1β induced steatosis, increased the inflammatory and prosteatotic chemokine MCP-1 in hepatocytes, and augmented TLR4-dependent upregulation of inflammatory signaling in macrophages. In conclusion, we demonstrated that Casp-1–dependent upregulation of IL-1β and signaling mediated by IL-1R1 are crucial in ALD pathogenesis. Our findings suggest a potential role of IL-1R1 inhibition in the treatment of ALD.
PMCID: PMC3461900  PMID: 22945633
12.  Role of Specific Innate Immune Responses in Herpes Simplex Virus Infection of the Central Nervous System 
Journal of Virology  2012;86(4):2273-2281.
Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.
PMCID: PMC3302371  PMID: 22171256
13.  Type I Interferons Protect from Toll-like Receptor 9-Associated Liver Injury and Regulate IL-1 Receptor Antagonist in Mice 
Gastroenterology  2010;140(2):697-708.e4.
Background & Aims
Liver inflammation and injury are mediated by the innate immune response, which is regulated by Toll-like receptors (TLR). Activation of TLR9 induces Type I interferons (IFNs) via the interferon regulatory factor (IRF)-7. We investigated the roles of Type I IFNs in TLR9-associated liver injury in mice.
Liver injury was induced in wild-type (WT), IRF7-deficient, and IFN-α/s receptor-1 (IFNAR1)-deficient mice by administration of ligands for TLR9 or TLR2. Findings from mice were verified in cultured hepatocytes and liver mononuclear cells, and in vivo experiments using recombinant Type-I IFN and interleukin-1 receptor antagonist (IL-1ra).
Type I IFNs were upregulated during TLR9-associated liver injury in WT mice. IRF7- and IFNAR1-deficient mice, which have disruptions in Type I IFN production or signaling, respectively, had greater amounts of liver damage and inflammation, decreased recruitment of dendritic cells, and increased production of TNF-α by liver mononuclear cells (LMNC). These findings indicate that Type I IFNs have anti-inflammatory activities in liver. The IL-1ra, which is produced by LMNC and hepatocytes, is an IFN-regulated antagonist of the pro-inflammatory cytokine IL-1β; IRF7- and IFNAR1-deficient mice had decreased levels of IL-1ra, compared with WT mice. IL-1ra protected cultured hepatocytes from IL-1β-mediated sensitization to cytotoxicity from TNF-α. In vivo exposure to Type I IFN, which induced IL-1ra, or administration of IL-1ra reduced TLR9-associated liver injury; the protective effect of Type-I IFNs therefore appears to be mediated by IFN-dependent induction of IL-1ra.
Type I IFNs have anti-inflammatory effects mediated by endogenous IL-1ra which regulates the extent of TLR9-induced liver damage. Type I interferon signaling is therefore required for protection from immune-mediated liver injury.
PMCID: PMC3031737  PMID: 20727895
liver disease; immunology; innate immunity; bacterial DNA
14.  Interferon regulatory factor 3 and Type I interferons are protective in alcoholic liver injury in mice via cross-talk of parenchymal and myeloid cells 
Hepatology (Baltimore, Md.)  2011;53(2):649-660.
Alcoholic liver disease (ALD) features increased hepatic exposure to bacterial lipopolysaccharide (LPS). Toll-like receptor-4 (TLR4) recognizes LPS and activates signaling pathways depending on MyD88 or TRIF adaptors. We previously showed that MyD88 is dispensable in ALD. TLR4 induces Type-I interferons (IFN) in MyD88-independent manner that involves interferon regulatory factor-3 (IRF3). We fed alcohol or control diets to wild-type (WT) and IRF3 knock-out (KO) mice, and to mice with selective IRF3 deficiency in liver parenchymal and bone marrow-derived cells. Whole-body IRF3-KO mice were protected from alcohol-induced liver injury, steatosis and inflammation. In contrast to WT or bone-marrow specific IRF3-KO mice, deficiency of IRF3 only in parenchymal cells aggravated alcohol-induced liver injury, associated with increased pro-inflammatory cytokines, lower anti-inflammatory cytokine IL-10 and lower Type-I IFNs compared to WT mice. Co-culture of WT primary murine hepatocytes with liver mononuclear cells (LMNC) resulted in higher LPS-induced IL-10 and IFN-β, and lower TNF-α levels compared to LMNC alone. Type-I IFN was important since co-cultures of hepatocytes with LMNC from Type-I IFN receptor KO mice showed attenuated IL-10 levels compared to control co-cultures from WT mice. We further identified that Type-I IFNs potentiated LPS-induced IL-10 and inhibited inflammatory cytokine production in both murine macrophages and human leukocytes, indicating preserved cross-species effects. These findings suggest that liver parenchymal cells are the dominant source of Type-I IFN in TLR4/IRF3-dependent manner. Further, parenchymal cell-derived Type-I IFNs increase anti-inflammatory and suppress pro-inflammatory cytokines production by LMNC in paracrine manner. In conclusion, our results indicate that IRF3 activation in parenchymal cells and resulting type I IFNs have protective effects in ALD via modulation of inflammatory functions in macrophages. These results suggest potential therapeutic targets in ALD.
PMCID: PMC3069538  PMID: 21274885
alcoholic liver disease; toll-like receptor 4; interferons, type I; tumor necrosis factor alpha; interleukin 10
15.  CD14 Deficiency Impacts Glucose Homeostasis in Mice through Altered Adrenal Tone 
PLoS ONE  2012;7(1):e29688.
The toll-like receptors comprise one of the most conserved components of the innate immune system, signaling the presence of molecules of microbial origin. It has been proposed that signaling through TLR4, which requires CD14 to recognize bacterial lipopolysaccharide (LPS), may generate low-grade inflammation and thereby affect insulin sensitivity and glucose metabolism. To examine the long-term influence of partial innate immune signaling disruption on glucose homeostasis, we analyzed knockout mice deficient in CD14 backcrossed into the diabetes-prone C57BL6 background at 6 or 12 months of age. CD14-ko mice, fed either normal or high-fat diets, displayed significant glucose intolerance compared to wild type controls. They also displayed elevated norepinephrine urinary excretion and increased adrenal medullary volume, as well as an enhanced norepinephrine secretory response to insulin-induced hypoglycemia. These results point out a previously unappreciated crosstalk between innate immune- and sympathoadrenal- systems, which exerts a major long-term effect on glucose homeostasis.
PMCID: PMC3258240  PMID: 22253759
16.  Discovery of a novel TLR2 signaling inhibitor with anti-viral activity 
Antiviral research  2010;87(3):295-306.
Blockade of Toll-like receptor (TLR)-mediated inflammatory responses represents a new approach in the development of anti-inflammation therapeutics. In the present study, we have screened for TLR2-mediated inflammation inhibitors from a small molecule compound library using a sensitive cell line stably expressing TLR2, CD14, and an NF-κB-driven-luciferase reporter gene. Lymphocytic choriomeningitis virus (LCMV) was used as a virus model. This arenavirus activates a TLR2/CD14-dependent NF-κB signaling pathway. We have identified 10 potential anti-inflammatory compounds out of 101306 compounds. We further evaluated 1 of these positive compounds, E567. We demonstrated that compound E567 efficiently inhibits both LCMV and Herpes simplex virus 1 (HSV-1) induced cytokine responses in both human and mouse cell cultures. We also demonstrated that E567 inhibits cytokine responses in the mouse. Remarkably, E567 is also capable of inhibiting LCMV replication in mice. This is a new model for developing drugs for use in treating viral illnesses.
PMCID: PMC2924919  PMID: 20603154
Toll-like receptor (TLR); Lymphocytic choriomeningitis virus (LCMV); compound screening
17.  Mutations in Bone Marrow-Derived Stromal Stem Cells Unmask Latent Malignancy 
Stem Cells and Development  2010;19(8):1153-1166.
Neoplastic epithelia may remain dormant and clinically unapparent in human patients for decades. Multiple risk factors including mutations in tumor cells or the stromal cells may affect the switch from dormancy to malignancy. Gene mutations, including p53 mutations, within the stroma of tumors are associated with a worse clinical prognosis; however, it is not known if these stromal mutations can promote tumors in genetically at-risk tissue. To address this question, ApcMin/+ and ApcMin/+ Rag2−/− mice, which have a predilection to mammary carcinoma (as well as wild-type (wt) mice), received mesenchymal stem cells (MSC) with mutant p53 (p53MSC) transferred via tail vein injection. In the wt mouse, p53MSC circulated in the periphery and homed to the marrow cavity where they could be recovered up to a year later without apparent effect on the health of the mouse. No mammary tumors were found. However, in mice carrying the ApcMin/+ mutation, p53MSC homed to mammary tissue and significantly increased the incidence of mammary carcinoma. Tumor necrosis factor (TNF)-α-dependent factors elaborated from mesenchymal cells converted quiescent epithelia into clinically apparent disease. The increased cancer phenotype was completely preventable with neutralization of TNF-α or by transfer of CD4+ regulatory T cells from immune competent donors, demonstrating that immune competency to regulate inflammation was sufficient to maintain neoplastic dormancy even in the presence of oncogenic epithelial and stromal mutations. The significant synergy between host immunity and mesenchymal cells identified here may restructure treatments to restore an anticancer microenvironment.
PMCID: PMC3135253  PMID: 20199238
18.  Assembly and Immunological Properties of Newcastle Disease Virus-Like Particles Containing the Respiratory Syncytial Virus F and G Proteins ▿  
Journal of Virology  2010;85(1):366-377.
Human respiratory syncytial virus (RSV) is a serious respiratory pathogen in infants and young children as well as elderly and immunocompromised populations. However, no RSV vaccines are available. We have explored the potential of virus-like particles (VLPs) as an RSV vaccine candidate. VLPs composed entirely of RSV proteins were produced at levels inadequate for their preparation as immunogens. However, VLPs composed of the Newcastle disease virus (NDV) nucleocapsid and membrane proteins and chimera proteins containing the ectodomains of RSV F and G proteins fused to the transmembrane and cytoplasmic domains of NDV F and HN proteins, respectively, were quantitatively prepared from avian cells. Immunization of mice with these VLPs, without adjuvant, stimulated robust, anti-RSV F and G protein antibody responses. IgG2a/IgG1 ratios were very high, suggesting predominantly TH1 responses. In contrast to infectious RSV immunization, neutralization antibody titers were robust and stable for 4 months. Immunization with a single dose of VLPs resulted in the complete protection of mice from RSV replication in lungs. Upon RSV intranasal challenge of VLP-immunized mice, no enhanced lung pathology was observed, in contrast to the pathology observed in mice immunized with formalin-inactivated RSV. These results suggest that these VLPs are effective RSV vaccines in mice, in contrast to other nonreplicating RSV vaccine candidates.
PMCID: PMC3014159  PMID: 20980510
19.  Pattern Recognition Receptors and the Innate Immune Response to Viral Infection 
Viruses  2011;3(6):920-940.
The innate immune response to viral pathogens is critical in order to mobilize protective immunity. Cells of the innate immune system detect viral infection largely through germline-encoded pattern recognition receptors (PRRs) present either on the cell surface or within distinct intracellular compartments. These include the Toll-like receptors (TLRs), the retinoic acid-inducble gene I-like receptors (RLRs), the nucleotide oligomerization domain-like receptors (NLRs, also called NACHT, LRR and PYD domain proteins) and cytosolic DNA sensors. While in certain cases viral proteins are the trigger of these receptors, the predominant viral activators are nucleic acids. The presence of viral sensing PRRs in multiple cellular compartments allows innate cells to recognize and quickly respond to a broad range of viruses, which replicate in different cellular compartments. Here, we review the role of PRRs and associated signaling pathways in detecting viral pathogens in order to evoke production of interferons and cytokines. By highlighting recent progress in these areas, we hope to convey a greater understanding of how viruses activate PRR signaling and how this interaction shapes the anti-viral immune response.
PMCID: PMC3186011  PMID: 21994762
pattern recognition receptor; toll like receptor; nod like receptor; AIM2 like receptor; RIG-I like receptor; cytosolic DNA sensor; inflammasome; interferon; virus
20.  Herpes Simplex Virus Immediate-Early ICP0 Protein Inhibits Toll-Like Receptor 2-Dependent Inflammatory Responses and NF-κB Signaling ▿  
Journal of Virology  2010;84(20):10802-10811.
The discovery of the Toll-like receptors (TLRs) and their importance in the regulation of host responses to infection raised attention to the complex interplay between viral gene products and the host innate immune responses in determining the outcome of virus infection. Robust inflammatory cytokine responses are observed in herpes simplex virus (HSV)-infected animals and cells. Our studies have demonstrated that Toll-like receptor 2 (TLR2) activation by HSV results in NF-κB activation with concomitant inflammatory cytokine production and that TLR2 activation plays a critical role in HSV-induced pathology and mortality. Here we demonstrate that the HSV-1 immediate-early ICP0 protein reduces the TLR2-mediated inflammatory response to HSV 1 (HSV-1) infection. Expression of ICP0 alone is sufficient to block TLR2-driven responses to both viral and nonviral ligands at or downstream of the MyD88 adaptor and upstream of p65. ICP0 alone can also reduce the levels of MyD88 and Mal (TIRAP). In HSV-infected cells, the E3 ligase function of ICP0 and cellular proteasomal activity are required for the inhibitory activity. Our results argue for a model in which ICP0 promotes the degradation of TLR adaptor molecules and inhibition of the inflammatory response, much as it inhibits the interferon response by sequestration and degradation of interferon regulatory factor 3 (IRF-3).
PMCID: PMC2950559  PMID: 20686034
21.  Induction and Inhibition of Type I Interferon Responses by Distinct Components of Lymphocytic Choriomeningitis Virus▿  
Journal of Virology  2010;84(18):9452-9462.
Type I interferons (IFNs) play a critical role in the host defense against viruses. Lymphocytic choriomeningitis virus (LCMV) infection induces robust type I IFN production in its natural host, the mouse. However, the mechanisms underlying the induction of type I IFNs in response to LCMV infection have not yet been clearly defined. In the present study, we demonstrate that IRF7 is required for both the early phase (day 1 postinfection) and the late phase (day 2 postinfection) of the type I IFN response to LCMV, and melanoma differentiation-associated gene 5 (MDA5)/mitochondrial antiviral signaling protein (MAVS) signaling is crucial for the late phase of the type I IFN response to LCMV. We further demonstrate that LCMV genomic RNA itself (without other LCMV components) is able to induce type I IFN responses in various cell types by activation of the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5. We also show that expression of the LCMV nucleoprotein (NP) inhibits the type I IFN response induced by LCMV RNA and other RIG-I/MDA5 ligands. These virus-host interactions may play important roles in the pathogeneses of LCMV and other human arenavirus diseases.
PMCID: PMC2937596  PMID: 20592086
22.  Interleukin-10 Prevents Diet-Induced Insulin Resistance by Attenuating Macrophage and Cytokine Response in Skeletal Muscle 
Diabetes  2009;58(11):2525-2535.
Insulin resistance is a major characteristic of type 2 diabetes and is causally associated with obesity. Inflammation plays an important role in obesity-associated insulin resistance, but the underlying mechanism remains unclear. Interleukin (IL)-10 is an anti-inflammatory cytokine with lower circulating levels in obese subjects, and acute treatment with IL-10 prevents lipid-induced insulin resistance. We examined the role of IL-10 in glucose homeostasis using transgenic mice with muscle-specific overexpression of IL-10 (MCK-IL10).
MCK-IL10 and wild-type mice were fed a high-fat diet (HFD) for 3 weeks, and insulin sensitivity was determined using hyperinsulinemic-euglycemic clamps in conscious mice. Biochemical and molecular analyses were performed in muscle to assess glucose metabolism, insulin signaling, and inflammatory responses.
MCK-IL10 mice developed with no obvious anomaly and showed increased whole-body insulin sensitivity. After 3 weeks of HFD, MCK-IL10 mice developed comparable obesity to wild-type littermates but remained insulin sensitive in skeletal muscle. This was mostly due to significant increases in glucose metabolism, insulin receptor substrate-1, and Akt activity in muscle. HFD increased macrophage-specific CD68 and F4/80 levels in wild-type muscle that was associated with marked increases in tumor necrosis factor-α, IL-6, and C-C motif chemokine receptor-2 levels. In contrast, MCK-IL10 mice were protected from diet-induced inflammatory response in muscle.
These results demonstrate that IL-10 increases insulin sensitivity and protects skeletal muscle from obesity-associated macrophage infiltration, increases in inflammatory cytokines, and their deleterious effects on insulin signaling and glucose metabolism. Our findings provide novel insights into the role of anti-inflammatory cytokine in the treatment of type 2 diabetes.
PMCID: PMC2768157  PMID: 19690064
23.  Human and mouse colon cancer utilizes CD95 signaling for local growth and metastatic spread to liver 
Gastroenterology  2009;137(3):934-944.e4.
Background and aim
Analysis of clinical colon cancer specimens show alterations in the CD95 (Fas Ag/Fas L) pathway as tumors progress from local to metastatic disease, suggesting this pathway may play a role in invasive behavior of colon cancer. However, direct causality between these alterations and clinical disease progression has not been shown.
Surgically resected metastatic colon cancer samples were evaluated for Fas Ag/L and apoptosis. Alterations in the Fas signaling pathway found in human samples was recreated through a series of staged transfection experiments in the MC38 mouse colon cancer cell line and the effects on growth tested in vitro, and in vivo.
Expression of FLICE like inhibitory protein (FLIP) confers apoptosis resistance, increasing the incidence of primary tumors through a survival advantage by avoiding apoptosis and inducing Fas mediated proliferation. Co-expression of Fas L enables colon cancer cells to metastasize to the liver from local tumors as well as from intravenous injection of cells. MC38-FasL/FLIP colon cancer cells induce apoptosis in hepatocytes via activation of type 2 Fas Ag signaling, thus creating a niche conducive to tumor growth and fueling their own growth via Fas proliferative signaling.
Alterations in the Fas Ag pathway which inhibit apoptosis and increase Fas mediate proliferation directly increases local colon cancer growth and enhances metastasis to the liver. Delineating points in the pathway responsible for growth and metastasis will offer targets which may be exploited for therapy.
PMCID: PMC2763556  PMID: 19524576
24.  UNC93B1 Mediates Host Resistance to Infection with Toxoplasma gondii 
PLoS Pathogens  2010;6(8):e1001071.
UNC93B1 associates with Toll-Like Receptor (TLR) 3, TLR7 and TLR9, mediating their translocation from the endoplasmic reticulum to the endolysosome, hence allowing proper activation by nucleic acid ligands. We found that the triple deficient ‘3d’ mice, which lack functional UNC93B1, are hyper-susceptible to infection with Toxoplasma gondii. We established that while mounting a normal systemic pro-inflammatory response, i.e. producing abundant MCP-1, IL-6, TNFα and IFNγ, the 3d mice were unable to control parasite replication. Nevertheless, infection of reciprocal bone marrow chimeras between wild-type and 3d mice with T. gondii demonstrated a primary role of hemopoietic cell lineages in the enhanced susceptibility of UNC93B1 mutant mice. The protective role mediated by UNC93B1 to T. gondii infection was associated with impaired IL-12 responses and delayed IFNγ by spleen cells. Notably, in macrophages infected with T. gondii, UNC93B1 accumulates on the parasitophorous vacuole. Furthermore, upon in vitro infection the rate of tachyzoite replication was enhanced in non-activated macrophages carrying mutant UNC93B1 as compared to wild type gene. Strikingly, the role of UNC93B1 on intracellular parasite growth appears to be independent of TLR function. Altogether, our results reveal a critical role for UNC93B1 on induction of IL-12/IFNγ production as well as autonomous control of Toxoplasma replication by macrophages.
Author Summary
One third of the human population in the world is chronically infected with Toxoplasma gondii. While the majority of infected individuals are asymptomatic, toxoplasmosis is a major cause of congenital disease, abortion, and a life-threatening opportunistic disease in immunocompromised individuals. Early activation of the innate immune system and cytokine production by myeloid cells is required for establishment of protective immunity to T. gondii infection. In mice, a mutation in the UNC93B1 gene abolishes signaling via the intracellular innate immune receptors, namely Toll-like receptors (TLR) 3, 7 and 9, thus, named triple-deficiency (3d) mice. Our results demonstrate that the hyper-susceptibility of 3d mice to T. gondii infection is associated with impaired IL-12 production, delayed IFNγ production, and uncontrolled parasite replication in macrophages. Overall, our study reveals a critical role for UNC93B1 in the immunological control of T. gondii infection.
PMCID: PMC2928809  PMID: 20865117
25.  Newcastle Disease Virus-Like Particles Containing Respiratory Syncytial Virus G Protein Induced Protection in BALB/c Mice, with No Evidence of Immunopathology▿  
Journal of Virology  2009;84(2):1110-1123.
Respiratory syncytial virus (RSV) is the leading cause of serious respiratory infections in children as well as a serious cause of disease in elderly and immunosuppressed populations. There are no licensed vaccines available to prevent RSV disease. We have developed a virus-like particle (VLP) vaccine candidate for protection from RSV. The VLP is composed of the NP and M proteins of Newcastle disease virus (NDV) and a chimeric protein containing the cytoplasmic and transmembrane domains of the NDV HN protein and the ectodomain of the human RSV G protein (H/G). Immunization of mice with 10 or 40 μg total VLP-H/G protein by intraperitoneal or intramuscular inoculation stimulated antibody responses to G protein which were as good as or better than those stimulated by comparable amounts of UV-inactivated RSV. Immunization of mice with two doses or even a single dose of these particles resulted in the complete protection of mice from RSV replication in the lungs. Immunization with these particles induced neutralizing antibodies with modest titers. Upon RSV challenge of VLP-H/G-immunized mice, no enhanced pathology in the lungs was observed, although lungs of mice immunized in parallel with formalin-inactivated RSV (FI-RSV) showed the significant pathology that has previously been documented after immunization with FI-RSV. Thus, the VLP-H/G candidate vaccine was immunogenic in BALB/c mice and prevented replication of RSV in murine lungs, with no evidence of immunopathology. These data support further development of virus-like particle vaccine candidates for protection against RSV.
PMCID: PMC2798376  PMID: 19889768

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