Poly IC Treatment Research Articles

Production, Isolation, and Detection of Poly IC in Extracellular Vesicles from U937 Cells.

Double-stranded RNA is produced by viruses during their replicative cycle. It is a potent immune modulator and indicator of viral infection within the body. Extracellular vesicles (EVs) are lipid-bound particles released from cells homeostatically. Recent studies have shown that a commercially available dsRNA, poly inosinic: poly cytidylic acid (poly IC), can be detected within EVs. This finding opens the door for studying EVs as (1) carriers for dsRNA and (2) indicators of viral infection. To study dsRNA-containing EVs, we must have reliable methods for producing, isolating, and detecting them. This chapter uses U937, a pro-monocytic, human myeloid leukemia cell line, as the EV producer following poly IC treatment, and an immunoblot using an anti-dsRNA antibody (J2) for detection. Two methods for isolating the EVs and two methods for isolating the RNA from these EVs are described. Together, these methods effectively produce, isolate, and detect long dsRNA from EVs.

Hypersensitive response to interferon-stimulated gene (ISG)-inducing double-stranded RNA in American bullfrog tadpole fibroblasts

American bullfrogs are thought to be carriers of ranaviruses and contribute to their global spread via trade. Bullfrog tadpoles succumb to ranaviral infection's more severe and deadly effects than bullfrog adults. Presently, little is known about bullfrog tadpoles' innate antiviral immunity, possible due to the lack of available bullfrog tadpole cell lines. In this study, we describe a novel bullfrog tadpole fibroblast cell line named BullTad-leg. Its general cellular attributes, gene expression and function of class-A scavenger receptors (SR-As), and responses to poly IC (a synthetic dsRNA mimicking viral dsRNAs and a potent inducer of the interferon (IFN)-mediated antiviral responses) are investigated. Its abundant expression of vimentin corroborated with the cells' fibroblast morphology. BullTad-leg cells expressed transcripts of four SR-A members: SR-AI, SCARA3, SCARA4, and SCARA5, but transcripts of MARCO, the fifth SR-A member, were not detected. BullTad-leg cells expressed functional SR-As and could bind AcLDL. BullTad-leg cells exhibited cytotoxicity in response to poly IC treatment via SR-As. Additionally, very low doses of poly IC were able to induce dose-dependent expressions of ISGs including Mx, PKR, ISG20, and IFI35. This research sheds new light on the innate immune response, particularly SR-A biology and dsRNA responsiveness, in bullfrog tadpoles.

Dendritic cell expansion and activation enhances the adaptive immune response against TNBC brain metastases

Abstract Despite initial control by chemotherapy, Triple Negative Breast Cancer (TNBC) quickly acquires resistance and metastasizes to distal organs, including the brain. Median survival following diagnosis of TNBC brain metastases is a meager 7.3 months—indicative of a substantial unmet clinical need, with only a small subpopulation responding to checkpoint immunotherapy. As suboptimal antigen presentation can contribute to the weak T lymphocyte response to intracranial tumors, we hypothesized that enhancing dendritic cell (DC) presence in tumors may support intracranial T cell responses to tumors. Using a murine model of intracranial metastasis, we assessed immune cell frequency and function and in vivocytokine staining for flow cytometry to test whether Flt3L can be used to promote DC presence within tumors, and whether the addition of polyIC further enhances subsequent T cell responses. Flt3L treatment effectively increases the number of DC within the brains and meninges of mice, however these newly expanded populations exhibit lower proportions of the costimulatory molecule CD86 than the untreated control group, indicative of an immature phenotype. 24 hours after polyIC treatment, a higher percentage of DCs in brain tumors express CD86 but do not produce IL-12. Despite the lack of intratumoral IL-12, the combination of Flt3L and polyIC treatments led to a higher presence of T cells within tumors and their production of IFN-γ in the TME than either treatment alone. In conclusion, simply increasing the presence of DC within intracranial tumors is insufficient to promote T cell immunity; rather, deliberate activation of de novo DC is required for this full effect. Supported by grants from NIH (2T32CA009109-46 and 1R21CA226607-01A1) and the Schiff Foundation

Abstract 156: Role of Platelets in Maintaining Hemostasis and Coagulation Activation in Viral-induced Acute Respiratory Distress Syndrome (ards) in Mice

Platelets are crucial players during viral infections, as bridge hemostasis, inflammation, and immune response. RNA viruses like IAV are associated with pathologic platelet activation and thrombosis. We are currently investigating how platelet depletion before or post initial IAV infection can affect disease outcome. WT mice were infected with IAV (0.02HAU, intranasally) for 7 days to induce viral ARDS. Mice were injected with platelet depletion antibody R300 or control IgG at day 0 (prior) and day 3 (after infection). Mice treated with R300/control IgG were subjected to dsRNA mimetic poly IC intranasally for 3 days to induce virus-like ARDS. Global lung function was recorded, plasma and BALF were collected 7 or 3 dpi or final poly IC treatment, respectively. BALF cellularity and blood cellular composition was analyzed. Markers of lung injury and cytokines were measured. We observed that IAV infection leads to local lung inflammation, coagulation activation and ARDS-like lung dysfunction. Mice treated with R300 showed severe thrombocytopenia in blood. We observed more impaired alveolar hemostasis and increased hemorrhages into the airspace when platelet depletion was initiated 3 dpi compared to mice where platelets were depleted prior to infection. In addition, in dsRNA-induced ARDS model, platelet depletion resulted in increased signs of lung dysfunction, increased lung injury and local inflammation compared to mice with normal platelet levels. In conclusion, depletion of platelets after IAV infection resulted in increased alveolar hemorrhage and was associated with more pronounced lung inflammation, injury, and lung dysfunction. Our study suggests that interfering with platelet before viral infection can be beneficial as it limits the role of platelets as potential multiplicator of an initial pathologic immune reaction subsequently reducing collateral damage and organ dysfunction/failure. However, at later stages of viral infection, platelet activation is beneficial to facilitate tissue hemostasis as their absence later can result in increased hemorrhagic risk and more pronounced lung dysfunction. We aim to address the mechanism by which platelet contribute later to pathologic immune processes in dsRNA-induced or viral ARDS

Hypoxia-inducible Factor-1α Suppresses the Innate Immune Response in Cultured Human Proximal Tubular Cells.

Retinoic acid-inducible gene (RIG)-I like receptors (RLRs) are expressed on renal proximal tubular epithelial cells (RPTECs) in viral nephropathy, indicating the presence of RLR-mediated innate immune responses in RPTECs. Hypoxia is also known to affect innate immunity. This study investigated the effects of hypoxia, and hypoxia-inducible factor (HIF) on innate immunity in RPTECs. Primary human RPTECs were cultured under normoxic or hypoxic conditions and treated with a synthetic analog of double-stranded RNA (polyIC). The expression levels of RIG-I and MDA5, as RLRs, and IFNβ, IL6, and TNFα, as inflammatory mediators were evaluated using quantitative reverse transcription-polymerase chain reaction, western blotting, and lactate dehydrogenase activity (LDH) assays. To further investigate the role of hypoxia, a small interfering RNA was used to knockdown HIF1α. Under normoxic conditions, polyIC increased RIG-I, MDA5, and IFNβ mRNA expression in RPTECs by, 9.4±0.4-, 10.8±0.5-, and 4.0±0.1-fold, respectively, compared to control, and by 5.4±0.1-, 7.4±0.1-, and 2.4±0.3-fold, respectively, under hypoxic conditions, the rate of increase was lower than that under normoxic conditions (p<0.01). Protein expression showed a similar trend. Under hypoxic conditions, polyIC treatment with HIF1α knockdown in RPTECs increased RIG-I, MDA5, and IFNβ mRNA expression by 3.1±0.5-, 2.9±0.4-, and 6.1±0.4-fold, respectively, and cytotoxicity, demonstrated by LDH assay, was increased compared to that without knockdown (all p<0.01). Hypoxia suppresses polyIC-induced RLRs mediated innate immune responses in RPTECs via HIF1α.

NKAP Regulates Senescence and Cell Death Pathways in Hematopoietic Progenitors.

NKAP is a multi-functional nuclear protein that has been shown to be essential for hematopoiesis. Deletion of NKAP in hematopoietic stem cells (HSCs) was previously found to result in rapid lethality and hematopoietic failure. NKAP deficient cells also exhibited diminished proliferation and increased expression of the cyclin dependent kinase inhibitors (CDKIs) p19 Ink4d and p21 Cip1. To determine how dysregulation of CDKI expression contributes to the effects of NKAP deficiency, NKAP was deleted in mice also deficient in p19 Ink4d or p21 Cip1 using poly-IC treatment to induce Mx1-cre. Hematopoietic failure and lethality were not prevented by deficiency in either CDKI when NKAP was deleted. Inducible deletion of NKAP in cultured hematopoietic progenitors ex vivo resulted in a senescent phenotype and altered expression of numerous cell cycle regulators including the CDKI p16 INK4a. Interestingly, while combined deficiency in p16 INK4a and p21 Cip1 did not reverse the effect of NKAP deficiency on hematopoiesis in vivo, it did shift the consequence of NKAP deficiency from senescence to apoptosis in ex vivo cultures. These results suggest that NKAP may limit cellular stress that can trigger cell cycle withdrawal or cell death, a role critical for the maintenance of a viable pool of hematopoietic progenitors.

998 EMT-like phenotype in normal keratinocytes driven by TLR3 activation

Toll-Like-Receptor 3 (TLR3), a pattern recognition receptor that recognizes dsRNA, activation results in NFkB or interferon-regulatory factor 3 (IRF3) mediated gene transcription of inflammatory cytokines (e.g. IL-6) and type I and III interferons (e.g. IFN-λ). TLR3 also contributes to induced-pluripotent stem cell (IPS) programming, wound healing and skin regeneration through Wnt, SHH, and epithelial-mesenchymal transition (EMT) pathways. Our hypothesis is that TLR3 signaling within normal keratinocytes contributes to non-melanoma skin cancer (NMSC) through activation of EMT. In vitro, normal neonatal human embryonic keratinocytes (NHEKs) treated with a TLR3 agonist, polyinosinic:polycytidylic acid (poly IC), exhibit sustained EMT morphology changes that correlate with elevated levels of the intermediate filament Vimentin (∼2 fold), and transcription factors, ZEB1 (∼3-fold), SNAI1 (∼2-fold), and TWIST1 (∼2-fold), all key EMT markers. Poly IC treatment significantly increased levels of IL-6 (∼8-fold) and IFN-λ1 (∼200-fold) in normal keratinocytes. However, treatment of NHEKs with recombinant IL-6 or IFN-λ1 proteins do not induce an EMT-like morphology or increase in EMT gene expression. UVB radiation, the major player in NMSC development, also activates TLR3 signaling in keratinocytes. In vitro, NHEKs exposed to UVB (10mJ/cm2) exhibit similar EMT-like morphology and gene expression changes as poly IC treated cells. Chemical inhibition of TLR3 (CU CPT 4a) prior to poly IC or UVB exposure prevents EMT gene expression. We performed RNA-sequencing to investigate common pathways activated by both Poly IC and UVB to uncover additional downstream players responsible for driving the TLR3 induced EMT-phenotype. Taken together, TLR3 activation and the resulting EMT-like phenotype is a result of multiple players, and not likely driven by a single downstream mediator of TLR3 signaling.

TRAF6 Is Essential for Maintaining Hematopoietic Stem Cell Homeostasis

Tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), an E3 ubiquitin ligase downstream of Toll-like receptors (TLR), is required for mediating signals in response to foreign pathogens and stress molecules, and is implicated in the pathogenesis of MDS and AML. Although TLRs are expressed on normal HSC and TRAF6 is implicated in malignant HSC function, the normal physiological role of TRAF6 in HSC homeostasis and during hematopoiesis remains unknown. We find that TRAF6 is expressed in human and mouse HSPC (LT-HSC, ST-HSC, and MPP) at comparable or elevated levels relative to mature myeloid and lymphoid cells. To understand the role of TRAF6 in HSPC homeostasis, we generated hematopoietic-specific and inducible TRAF6 deleted mice by crossing Traf6-floxed with Vav-Cre (Traf6-HscKO) or Mx1-Cre (Traf6-iKO after PolyIC treatment) mice, respectively. Traf6-HscKO mice are born smaller and become moribund shortly after birth. Examination of peripheral blood (PB) and bone marrow (BM) revealed a significant expansion of myeloid cells and reduction of lymphoid cells. Moreover, moribund mice developed splenomegaly and extramedullary hematopoiesis. To determine whether the observed phenotype could be driven by loss of TRAF6 in mature myeloid cells, we generated mice in which TRAF6 is only deleted in myeloid cells by crossing Traf6-floxed with LysM-Cre mice (Traf6-MyKO). Interestingly, Traf6-MyKO mice did not develop myeloid expansion in the PB, BM, or spleen, indicating that TRAF6 plays a role in normal HSPC function.To determine the cell-intrinsic role of TRAF6 in hematopoiesis, we transplanted BM cells from Traf6-HscKO mice into lethally-irradiated recipient mice. The recipient mice with Traf6-HscKO BM cells similarly displayed myeloid-biased hematopoiesis in PB, BM, and spleens. Strikingly, LT-HSCs from Traf6-HscKO mice were significantly reduced in the BM of recipient mice. To exclude a possible effect of myeloid cells on the reduction in LT-HSC, we examined BM HSPC from Traf6-MyKO mice. Consistent with a role of TRAF6 in normal HSC function, the LT-HSC proportions and numbers were not affected in Traf6-MyKO mice. We next examined the functional consequences of deleting TRAF6 in HSC by performing competitive BM transplantation assays. Although initial homing to the BM was comparable between WT and Traf6-HscKO cells, the donor-derived chimerism of Traf6-HscKO cells was significantly reduced for myeloid and lymphoid populations 1 month post transplantation, and declined to below 5% after 4 months as compared with control mice. In addition, donor-derived HSC, HPC, and total BM cell chimerism of Traf6-HscKO cells was dramatically reduced. To examine the effects of TRAF6 deletion on HSC function after BM engraftment has been achieved, competitive BMT were performed with BM cells from Traf6-iKO mice. Upon deletion of Traf6 (PolyIC treatment 2 months post transplantation), total PB and BM chimerism, and chimerism of Traf6-deleted LT-HSC and HPC dramatically declined. Collectively, these findings indicate that TRAF6 is essential for normal HSPC function and homeostasis.To understand the function of TRAF6 in HSPC, HSC-enriched Lin-Sca1+Kit+(LSK) BM cells were isolated and examined for gene expression changes by RNA-sequencing. Genes directly implicated in cell cycle control were among the most differentially expressed in Traf6-deficient HSPC. Particularly, the cyclin-dependent kinase inhibitors (CDKIs) p21, p27 and p57 were significantly down-regulated in Traf6-deficient LSK cells as compared to normal LSK cells. CDKIs are negative regulators of cell cycle progression and involved in maintaining HSC quiescence. Consistent with the observed reduction in CDKI genes, LT-HSC and HPC (LSK) from Traf6-HscKO mice were less quiescent (lower proportion of G0 cells) and more actively cycling (higher proportion of G1/S/G2/M cells).Despite the established requirement of TRAF6 in myeloid and lymphoid cells during infection, our study uncovers a critical role of TRAF6 during normal HSC function and homeostasis. Our findings suggest that TRAF6 is a novel hematopoietic-requisite factor for maintaining HSC quiescence and controlling myeloid-biased differentiation. These findings reinforce the importance of innate immune pathway gene dosage and signaling requirements in normal and malignant HSPC. DisclosuresNo relevant conflicts of interest to declare.

337 Expansion of Dendritic Cells Using FLT3 Ligand to Treat Glioblastoma

Abstract FLT3L induces expansion and recruitment of plasmacytoid dendritic cells (pDCs) into the brain. By increasing antigen presentation, FLT3L agonists may enhance an immune response against glioblastoma (GBM). Previous results suggest that the antitumor response generated by FLT3 is secondary to production of interferon gamma. We studied the effect of FLT3L treatment of mice with a syngeneic GBM model. Under ACUC approval, 32 mice underwent implantation of 130 000 GL261 cells in the left striatum using a stereotactic frame. The presence of tumor was confirmed by bioluminescence imaging at day 7. Mice were randomly assigned to 4 groups: Control, FLT3 (courtesy of Celldex) treatment, Poly IC treatment, and FLT3+Poly IC treatment. Survival was assessed using log-rank analysis and described using Kaplan-Meier curves. A survival experiment demonstrated that FLT3L treatment of mice resulted in 50% long-term survival of mice and improved survival compared with control groups (P = .001). Addition of Poly IC (TLR3 agonist), did not add survival benefit. The present work suggests that an increase in antigen presentation by enhancing and recruiting dendritic cells into the brain and brain tumor may be beneficial and a potential therapeutic strategy for patients with GBM. The mechanism of this phenomenon is currently under investigation, but includes increased secretion of interferon gamma. Additionally, increased antigen presentation may aid the immune system mount a significant antitumor immune response. Further experiments evaluating the proportion of T cells that have been exposed to antigen are currently underway.

Type I IFN dependent and independent mechanisms of protection in C. difficile infection

Abstract Clostridium difficile infection (CDI) is increasingly prevalent in individuals in health care settings and in the community. C. difficile is a gram positive, anaerobic bacteria that forms spores resistant to common disinfectants. Upon infection, C. difficile spores germinate, divide and produce damaging toxins resulting in intestinal inflammation. While inflammatory mediators are known to affect disease severity, the role of type I IFN signaling in CDI has not been characterized. We hypothesized that type I IFN signaling was important for innate protection in CDI. IFNAR-deficient mice succumbed to CDI more rapidly than wild type mice. Since type I IFNs can reduce inflammasome activity, we assessed IL-1β expression in multiple tissues, and discovered that IFNAR mice expressed more IL-1β protein, and less insulin-like growth factor (IGF) transcripts in response to CDI. To investigate a potential treatment, we treated mice with the type I IFN-inducing dsRNA mimetic, polyIC. PolyIC treatment delayed onset of symptoms in survival studies. Surprisingly, the protective effect of polyIC was not a result of IFNAR signaling; treatment increased IGF and decreased IL-1β in both wild type and IFNAR-deficient mice. Finally, treating wild type mice with recombinant type I IFN also reduced disease severity. These findings suggest direct type I IFN signaling protects from CDI and treatment with polyIC may protect mice via a pathway independent of IFNAR signaling. Considering that recombinant type I IFN is already used clinically and novel treatment options for CDI are needed, further investigation of innate immune pathways may present new treatment options for this emerging infection.

Regulation of TLR3 Activation by S100A9.

Recognition of viral dsRNA by endosomal TLR3 activates innate immune response during virus infection. Trafficking of TLR3 to the endolysosomal compartment arising from fusion of late endosome (LE) with lysosome is required for recognition and detection of pathogen associated molecular patterns, which results in activation of the TLR3-dependent signaling cascade. Existing knowledge about the mechanism(s) and cellular factor(s) governing TLR3 trafficking is limited. In the current study, we identified intracellular S100A9 protein as a critical regulator of TLR3 trafficking. S100A9 was required for maturation of TLR3 containing early endosome (EE) into LE, the compartment that fuses with lysosome to form the endolysosomal compartment. A drastic reduction in cytokine production was observed in S100A9-knockout (KO) primary macrophages following RNA virus infection and treatment of cells with polyinosinic-polycytidylic acid (polyIC; a dsRNA mimetic that acts as a TLR3 agonist). Mechanistic studies revealed colocalization and interaction of S100A9 with TLR3 following polyIC treatment. S100A9-TLR3 interaction was critical for maturation of TLR3 containing EE into LE because TLR3 could not be detected in the LE of polyIC-treated S100A9-KO macrophages. Subsequently, TLR3 failed to colocalize with its agonist (i.e., biotin-labeled polyIC) in S100A9-deficient macrophages. The in vivo physiological role of S100A9 was evident from loss of cytokine production in polyIC-treated S100A9-KO mice. Thus, we identified intracellular S100A9 as a regulator of TLR3 signaling and demonstrated that S100A9 functions during pre-TLR3 activation stages by facilitating maturation of TLR3 containing EE into LE.

Class-A scavenger receptor function and expression in the rainbow trout (Oncorhynchus mykiss) epithelial cell lines RTgutGC and RTgill-W1

Class A scavenger receptors (SR-As) are cell surface receptors that bind a range of ligands, including modified low-density lipoproteins (mLDLs) and nucleic acids. Due to their ability to bind extracellular dsRNA, SR-As play an important role in the viral dsRNA initiated immune pathway. Most research on SR-As has focused on mammalian models, and there has been limited research on SR-As in fish. Thus, the presence of functional class A scavenger receptors (SR-As) were investigated in the rainbow trout cell lines, RTgutGC and RTgill-W1. SR-A ligand binding was assessed using fluorescently labeled acetylated-low density lipoprotein (acLDL) and synthetic dsRNA, polyinosinic:polycytidylic acid (poly IC), in combination with a series of known SR-A competitive ligands: fucoidan, dextran sulfate (DxSO4) and polyinosinic acid (poly I). Both cell lines were able to bind acLDL, which was blocked by SR-A competitive ligands. In RTgutGC, acLDL and poly IC competed for binding to the same surface receptor; however, in RTgill-W1 they did not. Poly IC-fluorescein binding was blocked by SR-A competitive ligands in RTgutGC but not RTgill-W1, suggesting an SR-A dependent dsRNA uptake mechanism in RTgutGC and an SR-A-independent update mechanism in RTgill-W1. Both cell lines responded to extracellular dsRNA treatment with the up-regulation of interferons (IFNs) and interferon stimulated genes (ISGs) as measured by quantitative (q)RT-PCR; however, RTgutGC expressed significantly higher transcript levels for both IFNs and ISGs compared with RTgill-W1 following extracellular poly IC treatment. Expression of SR-As, specifically a SCARA4-like sequence, was identified at the transcript level in both cell lines. These results suggest that both RTgill-W1 and RTgutGC express functional SR-As that are able to bind the classic SR-A ligand, acLDL. Although they both express SCARA4, the full SR-A expression profile; however, is likely different between the cell lines, as dsRNA uptake appears to be SR-A dependent in RTgutGC but SR-A-independent in RTgill-W1. Also, dsRNA uptake via SR-As appears to mediate a more robust antiviral response compared with a SR-A independent method of uptake. This study is the first to identify functional SR-As in rainbow trout epithelial cells, and contributes not only to a better understanding of modified LDL transport but also innate immunity in these economically important animals.

Sensitivity of drug-resistant mutants of hepatitis B virus to poly-IC.

The long-term benefits of antiviral treatment are limited by the resistance of hepatitis B virus (HBV). However, the effect of interferon (IFN)α treatment on drug-resistant HBVs is so far unknown. We, therefore, investigated the effects of IFN-α inducer poly-IC on the replication of HBV mutants resistant to drugs such as lamivudine (LAM), adefovir dipivoxil (ADV) and entecavir (ETV) in mice. HBV DNA and HBV DNA intermediate (RI) were employed as markers of the virus replication and 2',5'-oligoadenylate synthase (OAS) mRNA as a marker of IFN-α/β induction. Poly-IC inhibited wtHBV replication and increased levels of OAS mRNA. Compared to the wt virus, the capacity of virus replication was reduced in most LAMr and ETVr mutants except those with mutations rtM(204V+L180M+V173L), and was similary in the ADVr mutants except rt(A121V+N236T). The virus replication was reduced after poly-IC treatment with LAMr and ADVr mutants similary to the wt virus. In contrast, ETVr mutants were resistant to the poly-IC treatment. In conclusion, the capacity of HBV replication and the sensitivity to IFN therapy are influenced by drug-resistant mutations. The IFN therapy may effectively inhibit HBV replication in particular in patients with LAMr or ADVr mutations but not in patients with ETVr mutations.

Subretinal transfection of chitosan-loaded TLR3-siRNA for the treatment of experimental autoimmune uveitis

The local interaction between retinal pigment epithelium (RPE) and immigrated effector T cells is crucial for the pathogenesis of autoimmune uveitis. After being activated by the pattern recognition receptors (PRRs) signaling pathway, RPE can present the antigen reactivated invading autoreactive T cells, resulting in uveitis. In the present study, we showed that the transfection of chitosan-loaded TLR3-siRNA toward RPE could effectively remit experimental autoimmune uveitis (EAU) in B10RIII mice. Initially, we verified the constitutive expression of Tlr3 in RPE at high levels, which was not altered in response to TNFα, IFNγ and IL-17A treatments. Compared with other TLRs, the activation of TLR3 signaling following polyIC treatment resulted in increased IL-6 and IFNγ secretion from and MHCII expression on RPE. It is polyIC-, but not other TLR ligands, treated RPE showed significant synergetic effect with IL-17 on stimulating RPE secreting CXCL8 and CCl2, which might be resulted from elevated Il17ra expression in RPE following polyIC treatment. Furthermore, polyIC-treated RPE caused a robust stimulation of differentiation of CD4 cell toward Th1 or Th17 cells, in addition to the secretion of the cytokines IFNγ and IL-17. The in vitro knockdown of TLR3 expression in RPE by chitosan/TLR3-siRNA transfection could effectively block polyIC-induced MHCII expression, pro-inflammatory cytokine secretion and autoreactive CD4 cell activation. Studies conducted in firefly luciferase gene transgenic mice demonstrated that the subretinal CS/Luci-siRNA transfection specifically reduced the luciferase activity in RPE but not in the liver and spleen. Finally, the CS/TLR3-siRNA was locally administered in the EAU induced B10RIII mice. The results revealed that chitosan-mediated TLR3-siRNA transfection had a significant therapeutic effect on either delaying the outbreak or remitting the severity of uveitis.

Melanoma Differentiation-Associated Gene 5 Regulates the Expression of a Chemokine CXCL10 in Human Mesangial Cells: Implications for Chronic Inflammatory Renal Diseases

Mesangial cells play an important role in inflammatory reactions in kidney. Although viral infections often trigger the worsening of chronic inflammatory renal diseases, the mechanisms are largely unknown. Melanoma differentiation-associated gene 5 (MDA5) is a member of RNA helicase family with a conserved Asp-Glu-x-His (DExH) box. In the present study, we examined the effect of polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA (dsRNA) that mimics viral dsRNAs, on MDA5 expression using primary culture of human mesangial cells. The cells were simply treated or transfected with poly IC; the former procedure is a model of cells exposed to viral dsRNA released from dying cells, and the latter is a model of entry of RNA virus into the cytoplasm. Expression levels of MDA5 mRNA in mesangial cells were increased about 70-100 fold in response to either treatment or transfection with poly IC. MDA5 protein expression was significantly induced as well. RNA interference experiments revealed that poly IC treatment induced MDA5 expression via Toll-like receptor 3 (TLR3) and interferon (IFN)-β, and that poly IC trasnfection induced MDA5 expression via another DExH box RNA helicase, retinoic acid-inducible gene-I (RIG-I), and IFN-β. Moreover, MDA5 induced by poly IC, in turn, increased the expression of a chemokine CXCL10. In addition, immunohistochemical staining demonstrated a high level of MDA5 expression in glomeruli, mainly in mesangial cells, of patients with severe lupus nephritis or proteinuric IgA nephropathy. MDA5 may be involved not only in physiological antiviral reactions but also in chronic inflammation in glomerular mesangial cells.

Intranasal Poly-IC treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population

Type I IFN has been demonstrated to have major regulatory effects on the outcome of bacterial infections. To assess the effects of exogenously induced type I IFN on the outcome of Mycobacterium tuberculosis infection, we treated pathogen-exposed mice intranasally with polyinosinic-polycytidylic acid condensed with poly-l-lysine and carboxymethylcellulose (Poly-ICLC), an agent designed to stimulate prolonged, high-level production of type I IFN. Drug-treated, M. tuberculosis-infected WT mice, but not mice lacking IFN-alphabeta receptor 1 (IFNalphabetaR; also known as IFNAR1), displayed marked elevations in lung bacillary loads, accompanied by widespread pulmonary necrosis without detectable impairment of Th1 effector function. Importantly, lungs from Poly-ICLC-treated M. tuberculosis-infected mice exhibited a striking increase in CD11b+F4/80+Gr1int cells that displayed decreased MHC II expression and enhanced bacterial levels relative to the same subset of cells purified from infected, untreated controls. Moreover, both the Poly-ICLC-triggered pulmonary recruitment of the CD11b+F4/80+Gr1int population and the accompanying exacerbation of infection correlated with type I IFN-induced upregulation of the chemokine-encoding gene Ccl2 and were dependent on host expression of the chemokine receptor CCR2. The above findings suggest that Poly-ICLC treatment can detrimentally affect the outcome of M. tuberculosis infection, by promoting the accumulation of a permissive myeloid population in the lung. In addition, these data suggest that agents that stimulate type I IFN should be used with caution in patients exposed to this pathogen.

Regulation of TLR2 Expression and Function in Human Airway Epithelial Cells

Toll-like receptor (TLR1-6) mRNAs are expressed in normal human bronchial epithelial cells with higher basal levels of TLR3. TLR2 mRNA and plasma membrane protein expression was enhanced by pretreatment with Poly IC, a synthetic double-stranded RNA (dsRNA) known to activate TLR3. Poly IC also enhanced mRNA expression of adaptor molecules (MyD88 and TIRAP) and coreceptors (Dectin-1 and CD14) involved in TLR2 signaling. Additionally, mRNA expression of TLR3 and dsRNA-sensing proteins MDA5 and RIG-I increased following Poly IC treatment. In contrast, basal mRNA expression of TLR5 and TLR2 coreceptor CD36 was reduced by 77% and 62%, respectively. ELISA of apical and basolateral solutions from Poly IC-stimulated monolayers revealed significantly higher levels of IL-6 and GM-CSF compared with the TLR2 ligand PAM(3)CSK(4). Pretreatment with anti-TLR2 blocking antibody inhibited the PAM(3)CSK(4)-induced increase in IL-6 secretion after Poly IC exposure. An increase in IL-6 secretion was also observed in cells stimulated with Alternaria extract after pretreatment with Poly IC. However, IL-6 secretion was not stimulated by zymosan or lipothechoic acid (LTA). These data demonstrated that upregulation of TLR2 following exposure to dsRNA enhances functional responses of the airway epithelium to certain (PAM(3)CSK(4)), but not all (zymosan, LTA) TLR2 ligands and that this is likely due to differences in coreceptor expression.

Double-stranded RNA enhances the expression of galectin-9 in vascular endothelial cells.

Treatment of cells with double-stranded RNA (dsRNA) in vitro mimics viral infection and regulates expression of various genes. We addressed the mechanisms of leucocyte traffic across the vascular endothelium induced by dsRNA. The present study focused on the expression of galectin-9, which is one of key molecules in the regulation of the interaction between vascular wall and white blood cells. Human umbilical vein endothelial cells (HUVEC) in culture were treated with polyinosinic-polycytidylic acid (poly IC), and expression of mRNA and protein of galectin-9 was analysed by reverse transcription polymerase-chain reaction (RT-PCR) and Western blotting. Poly IC enhanced the expression of galectin-9 mRNA and protein in concentration- and time-dependent manners. This effect of poly IC was almost completely suppressed by the pretreatment with 2-aminopurine, an inhibitor of dsRNA-dependent kinase. Poly IC treatment of HUVEC also enhanced the adherence of EoL-1 cells to the cells, which was inhibited by co-treatment with lactose. We conclude that poly IC upregulates galectin-9 expression in the vascular endothelium and this may explain part of the mechanism for leucocyte traffic through the vascular wall.

Characterization of the mouse olfactory glutathione S-transferases during the acute phase response.

The acute phase response (APR) has been shown to alter expression and activity of biotransformation enzymes, such as the phase I cytochromes p450 and phase II glutathione S-transferases (GSTs). The cytochromes p450 and GSTs are expressed abundantly and colocalized to non-neuronal cells of the olfactory mucosa. Previous studies indicate that olfactory cytochromes p450 expression and activity is altered during periods of localized inflammation and infection. Little is understood, however, about the influence of the APR on olfactory GST enzymes. This study investigated effects of the APR on olfactory GST isozymes expression and activity in mouse olfactory mucosa after 24-hr treatment with the acute phase inducer, polyinosinic: polycytidylic acid (polyIC). Western blot analysis using antibodies directed against specific GST isoforms alpha (A1-1), micro (M1-1), and pi (P1-1) demonstrated that their expression was unaltered by polyIC treatment. In contrast, olfactory p450 2E1 expression was significantly decreased. Enzymatic activity of the olfactory GSTs toward the general substrate, 1-chloro-2,4-dinitrobenzene (CDNB) was unchanged during the APR. Analysis of olfactory glutathione content during the APR showed that it was also unaffected by polyIC. The insensitivity of these olfactory GST isoforms during the APR may play a significant role toward limiting the impact of infection and inflammation on the olfactory system.

Influence of aging and polyIC treatment on xenobiotic metabolism and free radical generation in rats

Influence of aging and polyIC treatment on xenobiotic metabolism and free radical generation in rats