Inhibition of Polyinosinic-Polycytidylic Acid-Induced Acute Pulmonary Inflammation and NF-κB Activation in Mice by a Banana Plant Extract.

NF-κB activation is pivotal for the excess inflammation causing the critical condition and mortality of respiratory viral infection patients. This study was aimed to evaluate the effect of a banana plant extract (BPE) on suppressing NF-κB activity and acute lung inflammatory responses in mice induced by a synthetic double-stranded RNA viral mimetic, polyinosinic-polycytidylic acid (poly (I:C)). The inflammatory responses were analyzed by immunohistochemistry and HE stains and ELISA. The NF-κB activities were detected by immunohistochemistry in vivo and immunofluorescence and Western blot in vitro. Results showed that BPE significantly decreased influx of immune cells (neutrophils, lymphocytes, and total WBC), markedly suppressed the elevation of pro-inflammatory cytokines and chemokines (IL-6, RANTES, IFN-γ, MCP-1, keratinocyte-derived chemokine, and IL-17), and restored the diminished anti-inflammatory IL-10 in the bronchoalveolar lavage fluid (BALF) of poly (I:C)-stimulated mice. Accordingly, HE staining revealed that BPE treatment alleviated poly (I:C)-induced inflammatory cell infiltration and histopathologic changes in mice lungs. Moreover, immunohistochemical analysis showed that BPE reduced the pulmonary IL-6, CD11b (macrophage marker), and nuclear NF-κB p65 staining intensities, whilst restored that of IL-10 in poly (I:C)-stimulated mice. In vitro, BPE antagonized poly(I:C)-induced elevation of IL-6, nitric oxide, reactive oxygen species, NF-κB p65 signaling, and transient activation of p38 MAPK in human lung epithelial-like A549 cells. Taken together, BPE ameliorated viral mimic poly(I:C)-induced acute pulmonary inflammation in mice, evidenced by reduced inflammatory cell infiltration and regulation of both pro- and anti-inflammatory cytokines. The mechanism of action might closely associate with NF-κB signaling inhibition.

Poly-ICLC, a multi-functional immune modulator for treating cancer.

Immunotherapies have become the first line of treatment for many cancer types. Unfortunately, only a small fraction of patients benefits from these therapies. This low rate of success can be attributed to 3 main barriers: 1) low frequency of anti-tumor specific T cells; 2) lack of infiltration of the anti-tumor specific T cells into the tumor parenchyma and 3) accumulation of highly suppressive cells in the tumor mass that inhibit the effector function of the anti-tumor specific T cells. Thus, the identification of immunomodulators that can increase the frequency and/or the infiltration of antitumor specific T cells while reducing the suppressive capacity of the tumor microenvironment is necessary to ensure the effectiveness of T cell immunotherapies. In this review, we discuss the potential of poly-ICLC as a multi-functional immune modulator for treating cancer and its impact on the 3 above mentioned barriers. We describe the unique capacity of poly-ICLC in stimulating 2 separate pattern recognition receptors, TLR3 and cytosolic MDA5 and the consequences of these activations on cytokines and chemokines production. We emphasize the role of poly-ICLC as an adjuvant in the setting of peptide-based cancer vaccines and in situ tumor vaccination by mimicking natural immune responses to infections. Finally, we summarize the impact of poly-ICLC in enhancing T infiltration into the tumor parenchyma and address the implication of this finding in the clinic.

Enhancing the therapeutic efficacy of programmed death ligand 1 antibody for metastasized liver cancer by overcoming hepatic immunotolerance in mice.

BACKGROUND AND AIMS: Immunotherapy with programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) blockade has shown low response rates in liver cancer patients, with the underlying mechanisms unclear. To decipher a specific impact of the liver microenvironment, we compared the effects of anti-PD-L1 antibody (αPD-L1) blockade on the same tumor grown s.c. or in the liver. APPROACH AND RESULTS: We generated s.c. tumors in mice by inoculating MC38 colorectal cancer (CRC) cells under the skin and metastatic liver tumors by portal vein or splenic injection of CRC cells. Tumor-bearing mice were treated by i.p. injection of αPD-L1, polyinosinic:polycytidylic acid (poly[I:C]), or both. αPD-L1 monotherapy significantly suppressed s.c. tumor growth, but showed no effect on metastatic liver tumors. However, the combination of αPD-L1 with poly(I:C), an innate immunity-stimulating reagent, robustly inhibited tumor progression in liver. The combination therapy effectively down-regulated myeloid-derived suppressor cells (MDSCs), but up-regulated ratios of M1/M2 macrophages, CD8/CD4, and CD8/regulatory T (Treg) cells infiltrated into liver tumors and whole liver. A group of long-lasting T-bet+ Eomes- PD-1- cytotoxic T cells was maintained in the combo-treated liver, leading to resistance to tumor recurrence. Depleting macrophages or blocking type Ⅰ interferon signaling abrogated the synergistic antitumor effect of αPD-L1 and poly(I:C), indicating a requirement of boosting innate immunity for optimized activation of cytotoxic T cells by PD-1/PD-L1 blockade. CONCLUSIONS: The poor response of liver cancers to αPD-L1 therapy is largely attributable to a unique hepatic immunotolerant microenvironment, independent of tumor origins or types. The success of a combinatorial immunotherapy relies on coordinated inhibition or activation of various innate and adaptive immune cell activities.

Safety and antibody immune response of CHP-NY-ESO-1 vaccine combined with poly-ICLC in advanced or recurrent esophageal cancer patients.

The nanoparticle complex of cholesteryl pullulan (CHP) and NY-ESO-1 antigen protein (CHP-NY-ESO-1) presents multiple epitope peptides to MHC class I and II pathways, leading to CD8+ and CD4+ T cell responses. Poly-ICLC is a synthetic, double-stranded RNA, an agonist of toll-like receptor (TLR)-3, and a cytoplasmic receptor of melanoma differentiation-associated gene (MDA)-5. It should be a suitable immune adjuvant of cancer vaccine to overcome the inhibitory tumor microenvironment. We conducted a phase 1 clinical trial of CHP-NY-ESO-1 with poly-ICLC in patients with advanced or recurrent esophageal cancer. CHP-NY-ESO-1/poly-ICLC (µg/mg) was administered at a dose of 200/0.5 or 200/1.0 (cohorts 1 and 2, respectively) every 2 weeks for a total of six doses. The primary endpoints were safety and immune response. The secondary endpoint was tumor response. In total, 16 patients were enrolled, and six patients in each cohort completed the trial. The most common adverse event (AE) was injection site skin reaction (86.7%). No grade 3 or higher drug-related AEs were observed. No tumor responses were observed, and three patients (30%) had stable disease. The immune response was comparable between the two cohorts, and all patients (100%) achieved antibody responses with a median of 2.5 vaccinations. Comparing CHP-NY-ESO-1 alone to the poly-ICLC combination, all patients in both groups exhibited antibody responses, but the titers were higher in the combination group. In a mouse model, adding anti-PD-1 antibody to the combination of CHP-NY-ESO-1/poly-ICLC suppressed the growth of NY-ESO-1-expressing tumors. Combining the vaccine with PD-1 blockade holds promise in human trials.

The Microbiota Contributes to the Control of Highly Pathogenic H5N9 Influenza Virus Replication in Ducks.

Ducks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze whether the microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and nontreated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any significant difference in virus titers neither in the respiratory tract nor in the brain nor spleen. However, we found that antibiotic-treated H5N9 virus-infected ducks had significantly increased intestinal virus excretion at days 3 and 5 postinfection. This was associated with a significantly decreased antiviral immune response in the intestine of antibiotic-treated ducks. Our findings highlight the importance of an intact microbiota for an efficient control of avian influenza virus replication in ducks.IMPORTANCE Ducks are frequently infected with avian influenza viruses belonging to multiple subtypes. They represent an important reservoir species of avian influenza viruses, which can occasionally be transmitted to other bird species or mammals, including humans. Ducks thus have a central role in the epidemiology of influenza virus infection. Importantly, ducks usually show little or no clinical signs even following infection with a highly pathogenic avian influenza virus. We provide evidence that the microbiota contributes to the control of influenza virus replication in ducks by modulating the antiviral immune response. Ducks are able to control influenza virus replication more efficiently when they have an intact intestinal microbiota. Therefore, maintaining a healthy microbiota by limiting perturbations to its composition should contribute to the prevention of avian influenza virus spread from the duck reservoir.

Adenovirus-Mediated Inducible Expression of a PD-L1 Blocking Antibody in Combination with Macrophage Depletion Improves Survival in a Mouse Model of Peritoneal Carcinomatosis.

Immune checkpoint inhibitors (ICIs) have demonstrated remarkable efficacy in a growing number of malignancies. However, overcoming primary or secondary resistances is difficult due to pharmacokinetics issues and side effects associated with high systemic exposure. Local or regional expression of monoclonal antibodies (mAbs) using gene therapy vectors can alleviate this problem. In this work, we describe a high-capacity adenoviral vector (HCA-EFZP-aPDL1) equipped with a mifepristone-inducible system for the controlled expression of an anti-programmed death ligand 1 (PD-L1) blocking antibody. The vector was tested in an immune-competent mouse model of colorectal cancer based on implantation of MC38 cells. A single local administration of HCA-EFZP-aPDL1 in subcutaneous lesions led to a significant reduction in tumor growth with minimal release of the antibody in the circulation. When the vector was tested in a more stringent setting (rapidly progressing peritoneal carcinomatosis), the antitumor effect was marginal even in combination with other immune-stimulatory agents such as polyinosinic-polycytidylic acid (pI:C), blocking mAbs for T cell immunoglobulin, mucin-domain containing-3 (TIM-3) or agonistic mAbs for 4-1BB (CD137). In contrast, macrophage depletion by clodronate liposomes enhanced the efficacy of HCA-EFZP-aPDL1. These results highlight the importance of addressing macrophage-associated immunoregulatory mechanisms to overcome resistance to ICIs in the context of colorectal cancer.

Poly I:C-based rHBVvac therapeutic vaccine eliminates HBV via generation of HBV-specific CD8+ effector memory T cells.

OBJECTIVE: Chronic hepatitis B (CHB) virus infection is a global health problem. Finding a cure for CHB remains a challenging task. DESIGN: In this study, poly I:C was employed as an adjuvant for HBV therapeutic vaccine (referred to as pHBV-vaccine) and the feasibility and efficiency of pHBV-vaccine in CHB treatment were evaluated in HBV-carrier mice. RESULTS: We found that pHBV-vaccine decreased HBsAg and HBV DNA efficiently and safely in HBV-carrier mice. Further investigation showed that pHBV-vaccine promoted maturation and antigen presentation ability of dendritic cells in vivo and in vitro. This vaccine successfully restored the exhaustion of antigen-specific CD8+ T cells and partly broke the immune tolerance established in HBV-carrier mice. pHBV-vaccine also enhanced the proliferation and polyfunctionality of HBV-specific CD11ahi CD8αlo cells. Importantly, we observed that T cell activation molecule KLRG1 was only expressed on HBV specific CD11ahi CD8αlo cells. Furthermore, pHBV-vaccine reduced the expression of Eomes and increased the serum IL-12 levels, which in turn promoted the generation of effector memory short-lived effector cells (SLECs) to exhibit a critical role in HBV clearance. SLECs induced by pHBV-vaccine might play a crucial role in protecting from HBV reinfection. CONCLUSIONS: Findings from this study provide a new basis for the development of therapeutic pHBV-vaccine, which might be a potential candidate for clinical CHB therapy.

Poly (I:C) alleviates obesity related pro-inflammatory status and promotes glucose homeostasis.

Obesity associated insulin resistance (IR) is implicated in chronic inflammation that mediated by the immune system. Imbalance between anti-inflammatory and pro-inflammatory response contributes to the origins and drivers of IR. However, cells of innate and adaptive immune system participate in the pathogenesis of IR, while glucose homeostasis related immune tolerance could be compromised high fat diet (HFD) reduced metabolic disorder. Although previous studies have demonstrated that anti-inflammatory therapy has a protective role in alleviating the pro-inflammatory status in HFD induced IR, the precise mechanism is still unclear. Ploy (I:C) is a synthetic double-stranded RNA that activates innate and/or adaptive immune response via retinoic acid-inducible gene-I (RIG-I), toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein 5 (MDA5). In the present study, we initially perform a novel research on the relationship between Poly (I:C) preconditioning and improved glucose metabolism in obesity related IR. Interestingly, Poly (I:C) treatment has alleviated the pro-inflammatory status and promoted glucose homeostasis during a HFD feeding. Improved insulin sensitivity is consistent with enhanced immune tolerance, which accompanied with increased Foxp3+ regulatory T cells (Tregs). Of note, Tregs have a pivotal role in orchestrating the self-balance between autoimmunity and inflammation reaction. Thus, our findings reveal that Ploy (I:C) preconditioning prevents HFD induced glucose intolerance, which may be recognized as vaccination by the host. Overall, selectively targeting precise immune regulators may lead to new classes of potentially meaningful therapies for IR in the clinical trials.

Cytosolic Receptor Melanoma Differentiation-Associated Protein 5 Mediates Preconditioning-Induced Neuroprotection Against Cerebral Ischemic Injury.

BACKGROUND AND PURPOSE: Preconditioning with poly-l-lysine and carboxymethylcellulose (ICLC) provides robust neuroprotection from cerebral ischemia in a mouse stroke model. However, the receptor that mediates neuroprotection is unknown. As a synthetic double-stranded RNA, poly-ICLC may bind endosomal Toll-like receptor 3 or one of the cytosolic retinoic acid-inducible gene-I-like receptor family members, retinoic acid-inducible gene-I, or melanoma differentiation-associated protein 5. Activation of these receptors culminates in type I interferons (IFN-α/ß) induction-a response required for poly-ICLC-induced neuroprotection. In this study, we investigate the receptor required for poly-ICLC-induced neuroprotection. METHODS: Toll-like receptor 3, melanoma differentiation-associated protein 5-, and IFN-promoter stimulator 1-deficient mice were treated with poly-ICLC 24 hours before middle cerebral artery occlusion. Infarct volume was measured 24 hours after stroke to identify the receptor signaling pathways involved in protection. IFN-α/ß induction was measured in plasma samples collected 6 hours after poly-ICLC treatment. IFN-ß-deficient mice were used to test the requirement of IFN-ß for poly-ICLC-induced neuroprotection. Mice were treated with recombinant IFN-α-A to test the role of IFN-α as a potential mediator of neuroprotection. RESULTS: Poly-ICLC induction of both neuroprotection and systemic IFN-α/ß requires the cytosolic receptor melanoma differentiation-associated protein 5 and the adapter molecule IFN-promoter stimulator 1, whereas it is independent of Toll-like receptor 3. IFN-ß is not required for poly-ICLC-induced neuroprotection. IFN-α treatment protects against stroke. CONCLUSIONS: Poly-ICLC preconditioning is mediated by melanoma differentiation-associated protein 5 and its adaptor molecule IFN-promoter stimulator 1. This is the first evidence that a cytosolic receptor can mediate neuroprotection, providing a new target for the development of therapeutic agents to protect the brain from ischemic injury.

Live imaging of transforming growth factor-ß activated kinase 1 activation in Lewis lung carcinoma 3LL cells implanted into syngeneic mice and treated with polyinosinic:polycytidylic acid.

Transforming growth factor-ß activated kinase 1 (TAK1) has been shown to play a crucial role in cell death, differentiation, and inflammation. Here, we live-imaged robust TAK1 activation in Lewis lung carcinoma 3LL cells implanted into the s.c. tissue of syngeneic C57BL/6 mice and treated with polyinosinic:polycytidylic acid (PolyI:C). First, we developed and characterized a Förster resonance energy transfer-based biosensor for TAK1 activity. The TAK1 biosensor, named Eevee-TAK1, responded to stress-inducing reagents such as anisomycin, tumor necrosis factor-α, and interleukin1-ß. The anisomycin-induced increase in Förster resonance energy transfer was abolished by the TAK1 inhibitor (5z)-7-oxozeaenol. Activity of TAK1 in 3LL cells was markedly increased by PolyI:C in the presence of macrophages. 3LL cells expressing Eevee-TAK1 were implanted into mice and observed through imaging window by two-photon excitation microscopy. During the growth of tumor, the 3LL cells at the periphery of the tumor showed higher TAK1 activity than the 3LL cells located at the center of the tumor, suggesting that cells at the periphery of the tumor mass were under stronger stress. Injection of PolyI:C, which is known to induce regression of the implanted tumors, induced marked and homogenous TAK1 activation within the tumor tissues. The effect of PolyI:C faded within 4 days. These observations suggest that Eevee-TAK1 is a versatile tool to monitor cellular stress in cancer tissues.

Antiviral effects of liposome-encapsulated PolyICLC against Dengue virus in a mouse model.

This study presents the first investigation of the antiviral effects of the liposome-encapsulated PolyICLC (LE-PolyICLC) on Dengue virus (DENV) in a mouse model. In vivo efficacy studies showed that LE-PolyICLC acted to increase antiviral mechanisms mainly through promoting cytokine expression associated with innate immunity, such as IFN-γ. In addition, the pro-inflammatory cytokine TNF-α was also increased, while IL-6 level was decreased in serum. The titers of total antibodies against DENV2 in mice were also elevated. Administration of LE-PolyICLC not only alleviated the loss of body weight, degree of morbidity, and pathological damage in brains, but also reduced the viral titers and expression of viral E protein in the brain. Notably, the effectiveness of LE-PolyICLC was better than PolyICLC on the basis of the data presented in this study. These results, therefore, set a foundation for further development of LE-PolyICLC as an attractive candidate of antiviral agents to be used in both prophylactic and therapeutic settings in DENV diseases.

Fcγ receptor dependency of agonistic CD40 antibody in lymphoma therapy can be overcome through antibody multimerization.

Immunomodulatory mAbs, led by the anti-CTLA4 mAb ipilimumab, are an exciting new class of drugs capable of promoting anticancer immunity and providing durable control of some tumors. Close analysis of a number of agents has revealed a critical yet variable role for Fcγ receptors in their efficacy. In this article, we reveal that agonistic anti-CD40 mAbs have an absolute requirement for cross-linking by inhibitory FcγRIIB when used systemically to treat established BCL1 syngeneic lymphoma, and therapy is lost when using a mouse IgG2a mAb not cross-linked by FcγRIIB. Furthermore, in FcγRIIB-deficient mice the lymphoma itself can provide FcγRIIB to cross-link anti-CD40 on neighboring cells, and only when this is blocked does therapy fail. The dependence on FcγRIIB for immunostimulatory activity was not absolute, however, because when anti-CD40 mAbs were administered systemically with the TLR3 agonist polyinosinic:polycytidylic acid or were given subcutaneously, activatory FcγR could also provide cross-linking. Using this mechanistic insight, we designed multimeric forms of anti-CD40 mAb with intrinsic FcγR-independent activity that were highly effective in the treatment of lymphoma-bearing mice. In conclusion, FcγR-independent anti-CD40 activation is a viable strategy in vivo. These findings have important translational implications, as humans, unlike mice, do not have IgG that binds strongly to FcγRIIB; therefore FcγR-independent derivatives represent an attractive therapeutic option.

Mucosal polyinosinic-polycytidylic acid improves protection elicited by replicating influenza vaccines via enhanced dendritic cell function and T cell immunity.

Live-attenuated influenza vaccines (LAIVs) have the potential to generate CD8 T cell immunity that may limit the virulence of an antigenically shifted influenza strain in a population lacking protective Abs. However, current LAIVs exert limited T cell immunity restricted to the vaccine strains. One approach to improve LAIV-induced T cell responses is the use of specific adjuvants to enhance T cell priming by respiratory dendritic cells, but this hypothesis has not been addressed. In this study, we assessed the effect of the TLR3 ligand polyinosinic-polycytidylic acid (poly IC) on CD8 T cell immunity and protection elicited by LAIVs. Mucosal treatment with poly IC shortly after vaccination enhanced respiratory dendritic cell function, CD8 T cell formation, and production of neutralizing Abs. This adjuvant effect of poly IC was dependent on amplification of TLR3 signaling by nonhematopoietic radioresistant cells and enhanced mouse protection to homosubtypic, as well as heterosubtypic, virus challenge. Our findings indicate that mucosal TLR3 ligation may be used to improve CD8 T cell responses to replicating vaccines, which has implications for protection in the absence of pre-existing Ab immunity.

Treatment of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Systematic Review for a National Institutes of Health Pathways to Prevention Workshop.

BACKGROUND: Myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS) is a debilitating multisystem condition affecting more than 1 million adults in the United States. PURPOSE: To determine benefits and harms of treatments for adults with ME/CFS and identify future research needs. DATA SOURCES: MEDLINE, PsycINFO, and Cochrane databases (January 1988 to September 2014); clinical trial registries; reference lists; and manufacturer information. STUDY SELECTION: English-language randomized trials of the effectiveness and adverse effects of ME/CFS treatments. DATA EXTRACTION: Data on participants, study design, analysis, follow-up, and results were extracted and confirmed. Study quality was dual-rated by using prespecified criteria; discrepancies were resolved through consensus. DATA SYNTHESIS: Among 35 treatment trials enrolling participants primarily meeting the 1994 Centers for Disease Control and Prevention and Oxford case definitions of CFS, the immune modulator rintatolimod improved some measures of exercise performance compared with placebo in 2 trials (low strength of evidence). Trials of galantamine, hydrocortisone, IgG, valganciclovir, isoprinosine, fluoxetine, and various complementary medicines were inconclusive (insufficient evidence). Counseling therapies and graded exercise therapy compared with no treatment, relaxation, or support improved fatigue, function, global improvement, and work impairment in some trials; counseling therapies also improved quality of life (low to moderate strength of evidence). Harms were rarely reported across studies (insufficient evidence). LIMITATION: Trials were heterogeneous and were limited by size, number, duration, applicability, and methodological quality. CONCLUSION: Trials of rintatolimod, counseling therapies, and graded exercise therapy suggest benefit for some patients meeting case definitions for CFS, whereas evidence for other treatments and harms is insufficient. More definitive studies comparing participants meeting different case definitions, including ME, and providing subgroup analysis are needed to fill research gaps.

Poly (I:C) therapy decreases cerebral ischaemia/reperfusion injury via TLR3-mediated prevention of Fas/FADD interaction.

Toll-like receptor (TLR)-mediated signalling plays a role in cerebral ischaemia/reperfusion (I/R) injury. Modulation of TLRs has been reported to protect against cerebral I/R injury. This study examined whether modulation of TLR3 with poly (I:C) will induce protection against cerebral I/R injury. Mice were treated with or without Poly (I:C) (n = 8/group) 1 hr prior to cerebral ischaemia (60 min.) followed by reperfusion (24 hrs). Poly (I:C) pre-treatment significantly reduced the infarct volume by 57.2% compared with untreated I/R mice. Therapeutic administration of Poly (I:C), administered 30 min. after cerebral ischaemia, markedly decreased infarct volume by 34.9%. However, Poly (I:C)-induced protection was lost in TLR3 knockout mice. In poly (I:C)-treated mice, there was less neuronal damage in the hippocampus compared with untreated I/R mice. Poly (I:C) treatment induced IRF3 phosphorylation, but it inhibited NF-κB activation in the brain. Poly (I:C) also decreased I/R-induced apoptosis by attenuation of Fas/FasL-mediated apoptotic signalling. In addition, Poly (I:C) treatment decreased microglial cell caspase-3 activity. In vitro data showed that Poly (I:C) prevented hypoxia/reoxygenation (H/R)-induced interaction between Fas and FADD as well as caspase-3 and -8 activation in microglial cells. Importantly, Poly (I:C) treatment induced co-association between TLR3 and Fas. Our data suggest that Poly (I:C) decreases in cerebral I/R injury via TLR3 which associates with Fas, thereby preventing the interaction of Fas and FADD, as well as microglial cell caspase-3 and -8 activities. We conclude that TLR3 modulation by Poly (I:C) could be a potential approach for protection against ischaemic stroke.

Polyinosinic-polycytidylic acid is the most effective TLR adjuvant for SIV Gag protein-induced T cell responses in nonhuman primates.

Prime-boost immunization with heterologous vaccines elicits potent cellular immunity. In this study, we assessed the influence of various TLR ligands on SIV Gag-specific T cell immunity and protection following prime-boost immunization. Rhesus macaques (RMs) were primed with SIV Gag protein emulsified in Montanide ISA51 with or without TLR3 (polyinosinic-polycytidylic acid [poly-IC]), TLR4 (monophosphoryl lipid A), TLR7/8 (3M-012), TLR9 (CpG), or TLR3 (poly-IC) combined with TLR7/8 ligands, then boosted with replication defective adenovirus 5 expressing SIV Gag (rAd5-Gag). After priming, RMs that received SIV Gag protein plus poly-IC developed significantly higher frequencies of SIV Gag-specific CD4(+) Th1 responses in blood and bronchoalveolar lavage (BAL) fluid lymphocytes compared with all other adjuvants, and low-level SIV Gag-specific CD8(+) T cell responses. After the rAd5-Gag boost, the magnitude and breadth of SIV Gag-specific CD8(+) T cell responses were significantly increased in RM primed with SIV Gag protein plus poly-IC, with or without the TLR7/8 ligand, or CpG. However, the anamnestic, SIV Gag-specific CD8(+) T cell response to SIVmac251 challenge was not significantly enhanced by SIV Gag protein priming with any of the adjuvants. In contrast, the anamnestic SIV Gag-specific CD4(+) T cell response in BAL was enhanced by SIV Gag protein priming with poly-IC or CpG, which correlated with partial control of early viral replication after SIVmac251 challenge. These results demonstrate that prime-boost vaccination with SIV Gag protein/poly-IC improves magnitude, breadth, and durability of CD4(+) T cell immune responses, which could have a role in the control of SIV viral replication.

Increased numbers of monocyte-derived dendritic cells during successful tumor immunotherapy with immune-activating agents.

Local treatment with selected TLR ligands or bacteria such as bacillus Calmette-Guérin increases antitumor immune responses and delays tumor growth. It is thought that these treatments may act by activating tumor-associated dendritic cells (DCs), thereby supporting the induction of antitumor immune responses. However, common parameters of successful immune activation have not been identified. We used mouse models to compare treatments with different immune-activating agents for the ability to delay tumor growth, improve priming of tumor-specific T cells, and induce early cytokine production and DC activation. Treatment with polyinosinic-polycytidylic acid or a combination of monosodium urate crystals and Mycobacterium smegmatis was effective at delaying the growth of s.c. B16 melanomas, orthotopic 4T1 mammary carcinomas, and reducing 4T1 lung metastases. In contrast, LPS, monosodium urate crystals, or M. smegmatis alone had no activity. Effective treatments required both NK1.1(+) and CD8(+) cells, and resulted in increased T cell priming and the infiltration of NK cells and CD8(+) T cells in tumors. Unexpectedly, both effective and ineffective treatments increased DC numbers and the expression of costimulatory molecules in the tumor-draining lymph node. However, only effective treatments induced the rapid appearance of a population of monocyte-derived DCs in the draining lymph node, early release of IL-12p70 and IFN-γ, and low IL-10 in the serum. These results suggest that the activation of existing DC subsets is not sufficient for the induction of antitumor immune responses, whereas early induction of Th1 cytokines and monocyte-derived DCs are features of successful activation of antitumor immunity.

Polyinosinic-polycytidylic acid limits tumor outgrowth in a mouse model of metastatic lung cancer.

Polyinosinic-polycytidylic acid (poly I:C), a TLR3 ligand, is currently being tested in human clinical trials as an adjuvant to anti-cancer vaccines and in combination with other therapies. However, little is known about its activity in established pulmonary metastasis. The aim of our study was to elucidate the effect of poly I:C (1, 10, or 100 µg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth was arrested after a single administration of poly I:C. This was associated with higher influx of mature dendritic cells (DCs), which drove toward a Th1-like, Th17-like, and cytotoxic immune environment. The interference with IFN type I receptor signaling by means of a specific mAb reversed poly I:C-mediated tumor regression due to lower presence of myeloid DCs, cytotoxic DCs (CD11c(+)CD8(+)), NKT cells, CD8(+) T cells, and Th1-like cytokines. Moreover, the adoptive transfer of poly I:C-activated bone marrow-derived DCs into tumor-bearing mice resulted in activities similar to those of the systemic administration of poly I:C on lung tumor burden. In conclusion, our data prove that poly I:C has potential anti-tumor activity in a mouse model of established pulmonary metastasis. The activation of DCs and the production of IFN type I are responsible for an effective T cytotoxic immune response against metastatic lung cancer progression after poly I:C treatment.

Activation of toll-like receptor 3 attenuates alcoholic liver injury by stimulating Kupffer cells and stellate cells to produce interleukin-10 in mice.

BACKGROUND & AIMS: The important function of toll-like receptor (TLR) 4 in Kupffer cells and hepatic stellate cells (HSCs) has been well documented in alcoholic liver injury. However, little is known about the role of TLR3. Thus, we tested whether TLR3 activation in HSCs and Kupffer cells could attenuate alcoholic liver injury in vivo, and investigated its possible mechanism in vitro. METHODS: Alcoholic liver injury was achieved by feeding wild type (WT), TLR3 knockout (TLR3(-/-)) and interleukin (IL)-10(-/-) mice with high-fat diet plus binge ethanol drinking for 2 weeks. To activate TLR3, polyinosinic-polycytidylic acid (poly I:C) was injected into mice. For in vitro studies, HSCs and Kupffer cells were isolated and treated with poly I:C. RESULTS: In WT mice, poly I:C treatment reduced alcoholic liver injury and fat accumulation by suppressing nuclear factor-κB activation and sterol response element-binding protein 1c expression in the liver. In addition, freshly isolated HSCs and Kupffer cells from poly I:C-treated mice showed enhanced expression of IL-10 compared to controls. Infiltrated macrophage numbers and the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1 and IL-6 on these cells were decreased after poly I:C treatment. In vitro, poly I:C treatment enhanced the expression of IL-10 via a TLR3-dependent mechanism in HSCs and Kupffer cells. Finally, the protective effects of poly I:C on alcoholic liver injury were diminished in TLR3(-/-) and IL-10(-/-) mice. CONCLUSIONS: TLR3 activation ameliorates alcoholic liver injury via the stimulation of IL-10 production in HSCs and Kupffer cells. TLR3 could be a novel therapeutic target for the treatment of alcoholic liver injury.

TLR-3 receptor activation protects the very immature brain from ischemic injury.

BACKGROUND: We have shown that preconditioning by lipopolysaccharide (LPS) will result in 90% reduction in ischemic brain damage in P7 rats. This robust LPS neuroprotection was not observed in P3 or P5 pups (corresponding to human premature infant). LPS is a known Toll-like receptor 4 (TLR-4) ligand. We hypothesized that TLRs other than TLR-4 may mediate preconditioning against cerebral ischemic injury in the developing brain. METHODS: TLR-2, TLR-3, TLR-4, and TLR-9 expression was detected in brain sections from P3, P5, and P7 rats by immuno-staining. In subsequent experiments, P5 rats were randomly assigned to TLR-3 specific agonist, poly I:C, or saline treated group. At 48 h after the injections, hypoxic-ischemic (HI) injury was induced by unilateral carotid artery ligation followed by hypoxia for 65 min. Brains were removed 1 week after HI injury and infarct volumes were compared in H&E stained sections between the two groups. RESULTS: TLR-2 and TLR-3 were highly expressed in brains of P3 and P5 but not in P7 rats. The number of TLR-4 positive cells was lower in P3 and P5 compared to P7 brains (P <0.05). TLR-3 was predominately expressed in P5 pups (P <0.05). There was no significant difference in TLR-9 expression in the three age groups. There was a significant reduction in infarct volume (P=0.01) in poly I:C compared to saline pre-treated P5 pups. Pre-treatment with poly I:C downregulated NF-κB and upregulated IRF3 expression in P5 rat ischemic brains. Pre-treatment with poly I:C did not offer neuroprotection in P7 rat brains. CONCLUSION: TLRs expression and function is developmentally determined. Poly I:C-induced preconditioning against ischemic injury may be mediated by modulation of TLR-3 signaling pathways. This is the first study to show that TLR-3 is expressed in the immature brain and mediates preconditioning against ischemic injury.