Abstract
The present invention relates to the use of leukotriene B.sub.4 to enhance the response of Toll-like receptor (TLR), RIG-I-like receptor (RLR), and NOD-like receptor (NLR) when stimulated simultaneously with respective proper ligands. The use in combination of LTB.sub.4 with those ligands is useful to potentiate immune response for the treatment of autoimmune diseases, immunosuppressive diseases, as well as immunological disorders.
Claims
1. A method for triggering an innate immune response in a patient comprising administering to said patient a pharmacologically acceptable effective amount of leukotriene B.sub.4 (LTB.sub.4) in combination with at least one modulator of a receptor selected from the group consisting of a Toll-like receptor (TLR), a RIG-I-like receptor (RLR), and a NOD-like receptor (NLR).
2. The method according to claim 1, wherein said Toll-like receptor is selected from the group consisting of TLR1 to TLR10.
3. The method according to claim 1, wherein said Toll-like receptor is a TLR1/2 complex.
4. The method according to claim 1, wherein said Toll-like receptor is a TLR2/6 complex.
5. The method according to claim 1, wherein said Toll-like receptor is a TLR1, TLR2, TLR4, TLR5 or TLR6.
6. The method according to claim 1, wherein said Toll-like receptor is a TLR3.
7. The method according to claim 1, wherein the modulator of said Toll-like receptor is a TLR2 ligand, lipoteichoic acid (LTA), a synthetic tripalmitoylated lipopeptide (PAM3CSK4), zymosan, a lipoglycan, a lipoarabinomannan, a lipomannan, a peptidoglycan, a diacylated lipoprotein MALP-2, a synthetic diacylated lipoprotein FSL-1, a heat shock protein HSP60, a heat shock protein HSP70, a heat shock protein HSP96, a high-mobility-group protein 1 (HMG-1), a TLR3 ligand, a double-stranded RNA, a necrotic cell mRNA, a polyinosine-polycytidylic acid (poly I:C), a TLR4 ligand, a lipopolysaccharide (LPS), a monophosphoryl lipid A, a heat-shock protein HSP22, a fibrinogen, a fibronectin, a hyaluronan fragment, a heparan sulphate, a TLR5 ligand, flagellin, a TLR7 ligand, a TLR8 ligand, a single-stranded RNA, an imidazoquinoline compound, a guanosine analogue loxoribine, a thiazoloquinolone compound, a thymidine homopolymer phosphorothioate oligodeoxynucleotide (Poly(dT)), a TLR9 ligand, a double-stranded DNA, a cytosine guanine dinucleotide-containing oligodeoxynucleotides (CpG ODN) or a combination thereof.
8. The method according to claim 7, wherein said imidazoquinoline compound is imiquimod, gardiquimod or resiquimod.
9. The method according to claim 7, wherein said CpG ODN is SEQ ID NO:1.
10. The method according to claim 1, wherein the modulator of said RIG-I-like receptor is a retinoic acid-inducible gene-I (RIG-I) ligand, a melanoma differentiation-associated gene (Mda5) ligand, a LGP2 ligand, a single-stranded RNA, a double-stranded RNA, a 5-triphosphate RNA or a combination thereof.
11. The method according to claim 1, wherein the modulator of said NOD-like receptor is IPAF, Nalp1, Cryopirin/Nalp3 ligand or a combination thereof.
12. The method according to claim 1, wherein the modulator of said NOD-like receptor is meso-diaminopimelic acid, muramyl dipeptide (MDP) or a derivative thereof, flagellin or a combination thereof.
13. A method for stimulating neutrophils in a patient comprising administering to said patient a pharmacologically acceptable effective amount of leukotriene B.sub.4 (LTB.sub.4) in combination with at least one modulator of a receptor selected from the group consisting of a Toll-like receptor (TLR), a RIG-I-like receptor (RLR), and a NOD-like receptor (NLR).
14. A method for stimulating secretion of a pro-inflammatory cytokine in a patient comprising administering to said patient a pharmacologically acceptable effective amount of leukotriene B.sub.4 (LTB.sub.4) in combination with at least one modulator of a receptor selected from the group consisting of a Toll-like receptor (TLR), a RIG-I-like receptor (RLR), and a NOD-like receptor (NLR).
15. A method for stimulating intracellular kinase activation in a patient comprising administering to said patient a pharmacologically acceptable effective amount of leukotriene B.sub.4 (LTB.sub.4) in combination with at least one modulator of a receptor selected from the group consisting of a Toll-like receptor (TLR), a RIG-I-like receptor (RLR), and a NOD-like receptor (NLR).
16. The method of claim 15, wherein said kinase is TAK-1, p38, a c-Jun N-terminal kinase (JNK kinase) or a combination thereof.
17. A method for stimulating release of Tumor necrosis factor a (TNF-) in a patient comprising administering to said patient a pharmacologically acceptable effective amount of leukotriene B.sub.4 (LTB.sub.4) in combination with at least one modulator of a receptor selected from the group consisting of a Toll-like receptor (TLR), a RIG-I-like receptor (RLR), and a NOD-like receptor (NLR).
18. A method for treating a viral infection in a patient comprising administering to said patient a pharmacologically acceptable effective amount of leukotriene B.sub.4 (LTB.sub.4) in combination with at least one modulator of a receptor selected from the group consisting of a Toll-like receptor (TLR), a RIG-I-like receptor (RLR), and a NOD-like receptor (NLR).
19. The method according to claim 18, wherein said viral infection is from cytomegalovirus.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
[0034] FIG. 1 illustrates the effect of LTB.sub.4 on TLR2 mRNA expression by human neutrophils;
[0035] FIG. 2 illustrates the effect of LTB.sub.4 on TLR2 protein expression on either untreated human neutrophils (FIG. 2A) or stimulated with LTB.sub.4 (100 nM) for 15 (2), 30 (3), 60 (4) or 120 min (5) (FIG. 2B), LTB.sub.4 alone or in combination with LTA was used;
[0036] FIG. 3 illustrates the effect of LTB.sub.4 on TLR7, 8, and 9 mRNA expression by human neutrophils;
[0037] FIG. 4 illustrates the effect of LTB.sub.4 on TLR9 protein expression by human neutrophils in a time-dependent manner and compared to other neutrophil ligands, in FIG. 4A, cells were left untreated (dotted line) or were stimulated with LTB.sub.4 (100 nM) for 15, 30, 60 or 120 min; in FIG. 4B, cells were pre-treated for 30 min with the high affinity LTB.sub.4 receptor antagonist U75302 (10 M) prior to stimulation with LTB.sub.4 for 60 min; in FIGS. 4C-D, cells were stimulated with LTB.sub.4 or leukotrienes C4 (LTC.sub.4), leukotriene D4 (LTD.sub.4), the complement protein C5a or IL-8 for 60 min; in FIG. 4E neutrophils were pretreated with LTB.sub.4 for 60 min and incubated in the presence of CpG-ODN 2216 sequences (SEQ ID NO:1) that were tagged with cyanine-3 (Cy3) fluorochrome; and in FIG. 4F neutrophils were preincubated with LTB.sub.4 for 60 min in the presence of inhibitory CpG-ODN sequences;
[0038] FIG. 5 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and LTA on lipoteichoic acid-mediated TNF- secretion by human neutrophils using different LTA doses;
[0039] FIG. 6 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and LTA on lipoteichoic acid-mediated TNF- secretion by human neutrophils using a single LTB.sub.4 dose;
[0040] FIG. 7 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and LTA on lipoteichoic acid-mediated IL-8 secretion by human neutrophils;
[0041] FIG. 8 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and LTA on lipoteichoic acid-mediated TNF- secretion by human neutrophils in a time-dependent manner;
[0042] FIG. 9 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and LTA on lipoteichoic acid-mediated TNF- secretion by human neutrophils as dependent on the dose of LTB.sub.4 used;
[0043] FIG. 10 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and PAM.sub.3CSK.sub.4 on PAM.sub.3CSK.sub.4-mediated TNF- secretion by human neutrophils;
[0044] FIG. 11 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and PAM.sub.3CSK.sub.4 on PAM.sub.3CSK.sub.4-mediated IL-8 secretion by human neutrophils;
[0045] FIG. 12 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and LPS on LPS-mediated TNF- and IL-8 secretion by human neutrophils, wherein cell-free supernatants were collected and the quantitation of TNF- (FIG. 12A) or IL-8 (FIG. 12B) was performed using commercially available ELISA assays;
[0046] FIG. 13 illustrates the importance of IRAK1- and p38-mediated signaling in the enhanced effect of a combination therapy as described herein using LTB.sub.4 and LTA on LTA-mediated TNF- secretion by human neutrophils;
[0047] FIG. 14 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 with CpG-ODN on CpG-mediated TNF- secretion by human neutrophils;
[0048] FIG. 15 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 with CpG-ODN on CpG-mediated IL-8 secretion by human neutrophils;
[0049] FIG. 16 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 with CpG-ODN on CpG-mediated TNF- secretion by human neutrophils in a time-dependent manner;
[0050] FIG. 17 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 on CpG-mediated TNF- secretion by human neutrophils as dependent on the dose of LTB.sub.4 used;
[0051] FIG. 18 illustrates the implication of the high affinity LTB.sub.4 receptor, BLT1, in the enhanced effect of a combination therapy as described herein using LTB.sub.4 with CpG-ODN on CpG-mediated TNF- secretion by human neutrophils;
[0052] FIG. 19 illustrates that simultaneous stimulation with LTB.sub.4 and LTA is a more potent combination therapy than a therapy using LTB.sub.4 in pre-treatment following LTA stimulation on LTA-mediated TNF- secretion by human neutrophils;
[0053] FIG. 20 illustrates the independence of endosomial signaling in the enhanced effect of a combination therapy as described herein using LTB.sub.4 with CpG-ODN on CpG-mediated TNF- secretion by human neutrophils;
[0054] FIG. 21 illustrates the dependence of endosomial signaling in the enhanced effect of a combination therapy as described herein using LTB.sub.4 with CpG-ODN on CpG-mediated TNF- secretion by peripheral blood mononuclear cells (PMBC);
[0055] FIG. 22 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and LTA on LTA-mediated TAK-1 and p38 activation in human neutrophils in a dose-dependent manner;
[0056] FIG. 23 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and LTA on LTA-mediated IRAK-1 kinase activity in human neutrophils;
[0057] FIG. 24 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 and either LTA or Herpes simplex-1 virus (HSV-1) on TNF- secretion by murine peripheral mononuclear cells, wherein cells were stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), LTA (0.1 g/ml) (FIG. 24A) or the prototypical viral activator or TLR2 herpes simplex virus-1 (HSV-1, 510.sup.4 viral particles) (FIG. 24B) or a combination of either LTB.sub.4 (100 nM in placebo solution) and LTA (0.1 g/ml) or HSV-1 (510.sup.4 viral particles);
[0058] FIG. 25 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 on CpG-mediated TAK-1, p38, and Jun kinase activation by human neutrophils and peripheral blood mononuclear cells, wherein human neutrophils (FIG. 25A) or PBMCs (FIG. 25B) (510.sup.6 cells) were stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), CpG-ODN 2216 (10 g/ml) or a combination of LTB.sub.4 (100 nM in placebo solution) and CpG-ODN 2216 (10 g/ml);
[0059] FIG. 26 illustrates the independence of endosomial signaling in the enhanced effect of a combination therapy as described herein using LTB.sub.4 on CpG-mediated TAK-1, p38, and Jun kinase activation by human neutrophils and peripheral blood mononuclear cells, wherein a combination therapy using LTB.sub.4 in the presence of CpG-ODN enhances intracellular signalling implicating TAK-1, p38, and JNK kinases in both human neutrophils (FIG. 26A) and PBMCs (FIG. 26B) via an endosome-independent pathway; and
[0060] FIG. 27 illustrates the enhanced effect of a combination therapy as described herein using LTB.sub.4 on CpG-mediated in vivo antiviral activity directed against murine cytomegalovirus in salivary glands of infected Balb/c mice.
[0061] FIG. 28 illustrates the enhanced effect of LTB.sub.4-mediated, RIG-I-dependent NFB promoter activity in HEK-293T cells stably expressing BLT1 Cells were transiently transfected with a combination of RIG-I-expressing vector and luciferase reporter gene under NFB promoter control. Promoter activity was recorded following a 24 h stimulation with LTB.sub.4 (1 M).
[0062] FIG. 29 illustrates the enhanced effect of LTB.sub.4-mediated, RIG-I-dependent IFN- promoter activity in HEK-293T cells stably expressing BLT1. Cells were transiently transfected with a combination of RIG-I-expressing vector and luciferase reporter gene under IFN- promoter control. Promoter activity was recorded following a 24 h stimulation with LTB.sub.4 (1 M).
[0063] FIG. 30 illustrates the enhanced effect of LTB.sub.4-mediated, IPS-I-dependent NFB promoter activity in HEK-293T cells stably expressing BLT1. Cells were transiently transfected with a combination of IPS-I-expressing vector and luciferase reporter gene under NFB promoter control. Promoter activity was recorded following a 24 h stimulation with LTB.sub.4 (1 M).
[0064] FIG. 31 illustrates the enhanced effect of LTB.sub.4-mediated, IPS-I-dependent IFN- promoter activity in HEK-293T cells stably expressing BLT1. Cells were transiently transfected with a combination of IPS-I-expressing vector and luciferase reporter gene under IFN- promoter control. Promoter activity was recorded following a 24 h stimulation with LTB.sub.4 (1 M).
[0065] FIG. 32 illustrates that the combined treatment with LTB.sub.4 and the RLR agonist poly I:C potentiates synthesis of IFN mRNA levels in A549 cells.
[0066] FIG. 33 illustrates that the combined treatment with LTB.sub.4 and the RLR agonist poly I:C potentiates secretion of IFN by A549 cells.
[0067] FIG. 34 illustrates that combined treatment with LTB.sub.4 and the RLR agonists 3P-RNA or Sendai virus increases synthesis of IFN mRNA levels in A549 cells.
[0068] FIG. 35 illustrates that combined treatment with LTB.sub.4 and 3P-RNA or Sendai virus potentiates secretion of IFN in A549 cells.
[0069] FIG. 36 illustrates that treatment with LTB.sub.4 in combination with Sendai virus enhances release of IFN by wild type MEFs.
[0070] FIG. 37 illustrates that the combination therapy as described herein using LTB.sub.4 and the NLR agonist N-acetyl MDP (NacMDP) synergistically enhances IL-6 secretion by mouse embryogenic fibroblasts (MEF).
[0071] FIG. 38 illustrates that the combination therapy as described herein using LTB.sub.4 and the NLR agonist NacMDP enhances IL-6 secretion by IAV-infected MEF.
[0072] FIG. 39 illustrates that the combination therapy as described herein using LTB.sub.4 and the NLR agonist NacMDP causes an improved reduction in IAV viral load in infected MEF.
[0073] FIG. 40 illustrates that the combination therapy as described herein using LTB.sub.4 and the NLR agonist NacMDP causes an improved reduction in IAV viral load in the lungs of infected mice.
[0074] FIG. 41 illustrates that the combination therapy as described herein using LTB.sub.4 and the NLR agonist NacMDP reduces inflammation, as measured by IL-6 secretion, in the lungs of IAV-infected mice.
[0075] FIG. 42 illustrates that the combination therapy as described herein using LTB.sub.4 and the NLR agonist NacMDP restores normal lung architecture in IAV-infected mice. Examples of a bronchiole (B) and alveoli (A) are shown. Arrows identify inflammatory cell infiltration.
DETAILED DESCRIPTION
[0076] It is provided herein a new combination therapy using LTB.sub.4 with at least one of a Toll-like receptor (TLR) ligand, a RIG-I-like receptor (RLR) ligand or a NOD-like receptor (NLR) ligand and a new composition for enhancing an immune response response in immunosuppressed patients or in patients suffering from immunological disorders.
LTB.SUB.4
[0077] The leukotriene B4 (LTB.sub.4) agent as described herein refers to LTB.sub.4 [5S,12R-6,8,10,14(Z,E,E,Z)-eicosatetraenoic acid] or a functional variant or analog thereof.
Toll-Like Receptor Ligand
[0078] Toll-like receptor ligand, which are known in the art, refers to activators of TLR1/2, TLR2, TLR2/6, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, and TLR10 signalling. These include, but are not restricted to, lipoteichoic acid, tripalmitoylated lipopeptide (PAM.sub.3CSK.sub.4), zymosan, peptidoglycan, diacylated lipoprotein MALP-2, diacylated lipoprotein FSL-1, heat-shock proteins 22, 60, 70, and 96, HMG-1, double-stranded RNA, polyinosine-polycytidylic acid (poly I:C), lipopolysaccharide, lipid A, fibronectin, fibrinogen, hyaluronan fragments, flagellin, single-stranded RNA, imiquimod, gardiquimod, and resiquimod, loxoribine, thiazoloquinolone compounds, double-stranded DNA, and cytosine guanine dinucleotide-containing oligodeoxynucleotides (CpG ODN). Toll-like receptor ligand also refers to any modified molecules from the aforementioned TLR ligands that can bind to any one of TLR1-10 and lead to intracellular transduced signalling resulting in biological activities. Toll-like receptor ligand also refers to antibodies raised against any one of TLR1-10 that may induce biological activities following binding to their respective TLR molecule. Data are being presented herein that demonstrate the potential for LTB.sub.4 in modulating mRNA levels of TLR2, 7, 8, and 9 in human neutrophils. Moreover, a stimulation with LTB.sub.4 leads to upregulated TLR2 and TLR9 protein expression by human neutrophils.
RIG-I-like Receptor Ligand
[0079] RIG-I-like receptor (RLR) ligand, which are known in the art, refers to activator of RIG-I, Mda5, as well as LGP2 signalling. These ligands include, but are not restricted to, single-stranded RNA, double-stranded RNA, and 5-triphosphate RNA. RIG-I-like receptor ligand also refers to any modification introduced in an RNA molecule that can lead to binding and activation of RIG-I, Mda5, and LGP2 leading to RLR-like biological activities.
NOD-like Receptor Ligand
[0080] NOD-like receptor (NLR) ligand, which are known in the art, refers to activator of NOD1, NOD2, IPAF, Nalp1b, and Nalp3 signalling. These ligands include, but are not restricted to, meso-diaminopimelic acid, muramyl dipeptide, and flagellin. NOD-like receptor ligand also refers to any modified molecules from the aforementioned NOD-like receptor ligands that can bind to the different members of the NLR family and lead to intracellular transduced signalling resulting in biological activities.
Combination Therapy
[0081] Combination therapy refers to the use of LTB.sub.4 in combination with a specific TLR, RLR or NLR ligand for an enhanced immune response. In human neutrophils, in vitro stimulation with LTB.sub.4 in combination with the TLR2 ligands lipoteichoic acid or PAM.sub.3CSK.sub.4 for 6 h lead to upregulated secretion of the pro-inflammatory cytokines TNF- and IL-8 when compared to single stimulation with LTB.sub.4 or lipoteichoic acid or PAM.sub.3CSK.sub.4. This enhanced cytokine secretion by combination stimulation was also found to be time- and dose-dependent. The same effect, although to a more impressive extent, is observed in neutrophils, when cells are stimulated with a combination of CpG-ODN and LTB.sub.4. Combination therapy using LTB.sub.4 with LTA or CpG also lead to an upregulation in the activation of TAK-1 kinase. These results demonstrate a potential for the combination therapy for adequate modulation of the immune system. By modulating the immune response, a combination therapy using LTB.sub.4 with a particular TLR, RLR or NLR ligand could be found useful to enhance an innate immune response.
Pathology
[0082] The pathologies that may be treated by combination therapy consisting of LTB.sub.4 agent and either TLR, RLR or NLR ligand can be divided in three main axes namely: suppressed immunity, inflammatory processes and autoimmune cancer.
[0083] Innate immunity refers to immunological defence triggered by the non-specific immune response. Many natural TLR agonists such as nucleic acids have been described as possessing immunomodulatory properties. While being potent activators of the innate immunity, treatments with TLR agonists alone partially restore an adequate immune response. For that reason, it becomes highly desirable to be provided with a component showing greater efficacy, and a combination of LTB.sub.4 with any one of a TLR agonist, a RLR agonist, and a NLR agonist is such a candidate.
[0084] Inflammatory processes refers to situations where inflammation is uncontrolled leading to the development of pathologies. As mentioned earlier, TLR5 stimulation with flagellin can control gut inflammation mediated by opportunistic infection as well as unbalanced microbial activities of the gut microflora. Combination therapy combining LTB.sub.4 with flagellin therefore provide a new immunomodulatory strategy for the control of gut inflammation and is of determinant efficacy in the control of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, and Helicobacter pylori-mediated gastric ulcers. TLR signalling has also been associated with the protection of the nervous system. When appropriately controlled, TLR signalling is crucial for preserving central nervous system structure and function whether infection is present or not. Combination therapy with LTB.sub.4 and such TLR ligands is therefore promising for the development of a neuroprotective drug strategy. TLRs are playing an important role in the inflammatory lung disease including acute respiratory distress syndrome, asthma, and chronic obstructive pulmonary disease (COPD). For example, administration of CpG immunostimulatory ODN can directly inhibit allergic response in the lung. The use of TLR ligands has also been proposed to induce protective immunity that may reduce the risk of developing infective exacerbations of COPD. Combination therapy using LTB.sub.4 and TLR ligands is therefore promising in the treatment of lung diseases.
[0085] Cancer is the third area where combination therapy has beneficial effects. TLR signalling has been associated with the control of tumor development and metastasis. For example, the TLR2 agonist MALP-2 induces in vitro tumoricidal activity by macrophages. In a pancreatic cancer mouse model, MALP-2 reduced metastases formation in the lung. The TLR4 agonist ONO-4007 (Ono Pharmaceutical Co., Osaka, Japan) has a remarkable and selective efficacy on Tumor necrosis factor (TNF-)-sensitive tumours. Imidazoquinoline compounds have also shown therapeutic potential as agents against bladder cancer, leukemia, as well as benign and malignant skin lesions. TLR9 agonists are also in development for cancer therapy. The combination and synergistic therapies is now being proposed in the use of CpG-ODN in order to reach the full clinical potential of such strategies. Therefore, a therapy using LTB.sub.4 and TLR ligands is now being proposed for enhanced cancer regression.
[0086] In the present invention, the combination drug therapy described herein utilizes LTB.sub.4 with at least one of a Toll-like receptor (TLR) ligand, a RIG-I-like receptor (RLR) ligand or a NOD-like receptor (NLR) ligand.
[0087] In one aspect, the composition of LTB.sub.4 with one of a Toll-like receptor (TLR) ligand, a RIG-I-like receptor (RLR) ligand or a NOD-like receptor (NLR) ligand can be made under different forms such as tablet, capsules, suspension, solution or powder, known to the person of ordinary skilled in the art.
[0088] The present invention will be more readily understood by referring to the following examples.
EXAMPLE I
Stimulation of Human Neutrophils with LTB.SUB.4 .Leads to an Upregulation in TLR mRNA and Protein Levels in Neutrophils
[0089] Peripheral blood neutrophils or peripheral blood mononuclear cells were isolated from healthy donors and were purified by centrifugation over Ficoll separation medium gradient as already described (Larochelle B, Blood. 1998;92:291-299). Human neutrophils (210.sup.6) were resuspended in culture medium and stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS) or LTB.sub.4 (100 nM or 1 M in placebo solution) for two hours. Following stimulation, total RNA was extracted and RT-PCR was performed using specific primers for human TLR2 (FIG. 1) and TLR7-9 (FIG. 3). As can be seen from FIG. 1, expression of TLR2 mRNA is stimulated by LTB.sub.4 in a dose-dependent form (concentration of LTB.sub.4).
[0090] In FIG. 2A, cells were left untreated (1) or were stimulated with LTB.sub.4 (100 nM) for 15 (2), 30 (3), 60 (4) or 120 min (5). Cells were then fixed in paraformaldehyde and TLR2 expression was assessed by flow cytometry. Background cellular autofluorescence is represented by the dotted line (6). In FIG. 2B, LTB.sub.4 alone or in combination with LTA enhanced TLR2 expression. Similarly, in FIG. 3, it can be seen that the mRNA expression of TLR7-9 in cells is stimulated in a dose-dependent form as well by LTB.sub.4.
[0091] In FIG. 4A, cells were left untreated (dotted line) or were stimulated with LTB.sub.4 (100 nM) for 15, 30, 60 or 120 min. Background cellular autofluorescence is represented by shaded histogram. Cells were then fixed in paraformaldehyde, permeabilized in methanol, and TLR9 expression was assessed by flow cytometry. In FIG. 4B, cells were pre-treated for 30 min with the high affinity LTB.sub.4 receptor antagonist U75302 (10 M) prior to stimulation with LTB.sub.4 for 60 min. Cells were then fixed in paraformaldehyde, permeabilized in methanol, and TLR9 expression was assessed by flow cytometry. In FIGS. 4C-D, cells were stimulated with LTB.sub.4 or leukotrienes C4 (LTC.sub.4), leukotriene D4 (LTD.sub.4), the complement protein C5a or IL-8 for 60 min. Cells were then fixed in paraformaldehyde, permeabilized in methanol, and TLR9 expression was assessed by flow cytometry. In FIG. 4E, neutrophils were pretreated with LTB.sub.4 for 60 min. Following this pretreatment, neutrophils were incubated in the presence of CpG-ODN 2216 sequence (5-G*G*GGGACGATCGTCG*G* G*G*G*G*-3) (SEQ ID NO:1) that were tagged with cyanine-3 (Cy3) fluorochrome. After an incubation for 4 h with Cy3-CpG-ODN sequences, neutrophils were extensively washed with phosphate buffer and Cy3 expression (corresponding to the degree in CpG-ODN binding on human neutrophils) was assessed by flow cytometry. In FIG. 4F, neutrophils were preincubated with LTB.sub.4 for 60 min in the presence of inhibitory CpG-ODN sequence 5-TTTAGGGTTAGGGTTAGGGTTAGGG-3 (phosphothioate as backbone) (SEQ ID NO:3). Following preincubation, Cy3-CpG-ODN binding assay was performed as in FIG. 4E. As can be seen from FIG. 4, LTB.sub.4 induces TLR9 expression on human neutrophils in a time-dependent manner via the high affinity LTB.sub.4 receptor BLT1. LTB.sub.4 is also a potent inducer of TLR9 expression in human neutrophils when compared to other leukotrienes (LTC.sub.4 and LTD.sub.4) or other potent neutrophil agonists such as C5a and IL-8. Moreover, LTB.sub.4 enhances CpG-ODN binding on human neutrophils in a TLR9-dependent manner.
EXAMPLE II
Stimulation of Human Neutrophils with a Combination of LTB.SUB.4 .with TLR2 Ligands Lipoteichoic Acid (LTA) and PAM.SUB.3.CSK.SUB.4 .and TLR4 Ligand Lipopolysaccharide (LPS) Leads to an Upregulated Secretion of Pro-inflammatory Cytokines
[0092] As described in Example I, peripheral blood neutrophils were isolated from healthy donors and were purified by centrifugation over Ficoll separation medium gradient. Cells (510.sup.6) were resuspended in culture medium and stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), different concentrations of LTA (0.1-50 g/ml) or a combination of LTA and LTB.sub.4 at their aforementioned respective concentrations. Six hours post-stimulation, cell-free supernatants were collected and the quantitation of TNF- was performed by ELISA (FIG. 5). As shown in FIGS. 6 and 7, neutrophils were isolated from healthy donors and were purified by centrifugation over Ficoll separation medium gradient. Cells (510.sup.6) were resuspended in culture medium and stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), LTA at the fixed concentration of 10 g/ml or a combination of LTA and LTB.sub.4 at their aforementioned respective concentrations. Six hours post-stimulation, cell-free supernatants were collected and the quantification of TNF- (FIG. 6) or IL-8 (FIG. 7) was performed by ELISA. As can be seen from FIGS. 5-7, LTB.sub.4 can potentiate pro-inflammatory cytokine secretion by human neutrophils stimulated with LTA.
[0093] In FIG. 8, cells were prepared as in FIG. 6 and stimulated with LTA for 2 h, 6 h or 12 h before cell-free supernatants were collected for TNF- detection. In FIG. 9, cells were prepared as in FIG. 6 and were stimulated with LTA in the presence of 0, 1, 10, 100 or 1000 nM of LTB.sub.4. Six hours post-stimulation, cell-free supernatants were collected and the quantification of TNF- was performed. As can be seen in FIGS. 8-9, LTB.sub.4 can potentiate LTA-mediated TNF- secretion by human neutrophils in a time- and dose-dependent manner. In FIGS. 10-11, cells (510.sup.6) were resuspended in culture medium and stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), PAM.sub.3CSK.sub.4 (10 g/ml) or a combination of LTB.sub.4 (100 nM in placebo solution) and PAM.sub.3CSK.sub.4 (10 g/ml). Six hours post-stimulation, cell-free supernatants were collected and the quantification of TNF- (FIG. 10) or IL-8 (FIG. 11) was performed using commercially available ELISA assays. The symbol * represents a probability of p<0.05 when analyzed by one-tailed ANOVA followed by a Newman-Keuls post hoc test using PRISM3 software.
[0094] In FIG. 12, neutrophils were prepared as in FIG. 6, and were stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), LTA (10 g/ml), the TLR4 ligand LPS (100 ng/ml) or a combination of LTB.sub.4 (100 nM in placebo solution) and either LTA (10 g/ml) or LPS (100 ng/ml). Following cell incubation with single ligands or the different ligand combinations for six hours, cell-free supernatants were collected and the quantification of TNF- (FIG. 12A) or IL-8 (FIG. 12B) was performed using commercially available ELISA assays. The symbol * represents a probability of p<0.05 when analyzed by one-tailed ANOVA followed by a Newman-Keuls post hoc test using PRISM3 software. As can be seen in FIGS. 10-12, LTB.sub.4, when administered in combination with different TLR agonists, potentiated TLR-mediated pro-inflammatory cytokine secretion by human neutrophils. In FIG. 13, human neutrophils were pretreated or not with pharmacological inhibitors of p38 kinase (SB203580, 10 M). IRAK 1/2 kinase inhibitor (IRAK inhibitor, 10 M) for one hour prior to stimulation with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), LTA (10 g/ml) or a combination of LTB.sub.4 in the presence of LTA for 6 hours. Following the incubation with indicated single or combined agonists, cell-free supernatants were collected and the quantification of IL-8 was performed using commercially available ELISA assay. FIG. 13 shows the implication of IRAK1 as well as p38-mediated signalling in LTB.sub.4-induced LTA-mediated cytokine secretion by human neutrophils.
EXAMPLE III
Stimulation of Human Neutrophils with a Combination of LTB.SUB.4 .and TLR9 Ligand CpG Oligodeoxynucleotide (ODN) Leads to an Upregulated Secretion of Pro-inflammatory Cytokines
[0095] Peripheral blood neutrophils were isolated as already described in Example I. Cells (1510.sup.6) were resuspended in culture medium and stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), CpG-ODN 2216 (5-G*G*GGGACGATCGTCG*G*G*G*G*G*-3) (SEQ ID NO:1), where * represents a phosphorothioate linkage) (10 g/ml) or a combination of LTB.sub.4 (100 nM in placebo solution) and CpG-ODN 2216 (10 g/ml). Six hours post-stimulation, cell-free supernatants were collected and the quantification of TNF- (FIG. 14) or IL-8 (FIG. 15) was performed using commercially available ELISA assays. In FIG. 16, cells were prepared as in FIG. 11 and stimulated for 2 h, 6 h or 12 h before cell-free supernatants were collected for TNF- detection.
[0096] In FIG. 17, cells were prepared as in FIG. 14 and were stimulated with CpG-ODN 2216 (10 g/ml) in the presence of 0, 1, 10, 100 or 1000 nM of LTB.sub.4. Six hours post-stimulation, cell-free supernatants were collected and the quantification of TNF- was performed. The symbol * represents a probability of p<0.05 analyzed by one-tailed ANOVA followed by a Newman-Keuls post hoc test using PRISM3 software. In FIG. 18, neutrophils were pre-treated for 30 min with the high affinity LTB.sub.4 receptor antagonist U75302 (Cayman, Ann Arbor, Mich.) (10 M) prior to stimulation with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), CpG-ODN 2216 (10 g/ml) or a combination of LTB.sub.4 (100 nM in placebo solution) and CpG-ODN 2216 (10 g/ml). Six hours post-stimulation, cell-free supernatants were collected and the quantification of TNF- was assessed by performing a standardized commercially available ELISA assay. In FIG. 19, neutrophils were either pre-treated with LTB.sub.4 prior to CpG-ODN stimulation (10 g/ml) or cells were simultaneously stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), CpG-ODN 2216 (10 g/ml) or a combination of LTB.sub.4 (100 nM in placebo solution) and CpG-ODN 2216 (10 g/ml). Six hours post-stimulation, cell-free supernatants were collected and the quantification of TNF- was assessed by commercial ELISA assay. The symbol * represents a probability of p<0.05 analyzed by one-tailed ANOVA followed by a Newman-Keuls post hoc test using PRISM3 software. As seen in FIGS. 14-19, a combined stimulation with LTB.sub.4 and the TLR9 agonist CpG-ODN induces an enhanced time- and concentration-dependent pro-inflammatory cytokine secretion by human neutrophils involving the high affinity LTB.sub.4 receptor BLT1.
EXAMPLE IV
LTB.SUB.4.-mediated Potentiation in CpG Signaling Occurs via Endosomal-dependent Signaling in PBMC, but not in Human Neutrophils
[0097] Peripheral blood neutrophils (FIG. 20) or peripheral blood mononuclear cells (PBMC) (FIG. 21) were isolated as already described in Example I. Cells (1510.sup.6) were resuspended in culture medium and incubated with chloroquine (CQ), an inhibitor of endosomal activation, for 30 minutes. Following inhibitor incubation, cells were extensively washed with phosphate buffer and were stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), CpG-ODN 2216 (10 g/ml) or a combination of LTB.sub.4 (100 nM in placebo solution) and CpG-ODN 2216 (10 g/ml). After stimulation for six hours, cell-free supernatants were collected and the quantification of TNF- was assessed by commercial ELISA assay. The symbol * represents a probability of p<0.05 analyzed by one-tailed ANOVA followed by a Newman-Keuls post hoc test using PRISM3 software. As seen in FIGS. 20-21, the combination therapy using LTB.sub.4 and CpG-ODN implicates endosomal signalling in PBMCs (FIG. 21), but not in human neutrophils (FIG. 20).
EXAMPLE V
Stimulation of Human Neutrophils with a Combination of LTB.SUB.4 .and TLR Ligands Leads to an Upregulation in Intracellular Kinase Activation
[0098] Peripheral blood neutrophils or PBMCs were isolated as already described in Example I. Human neutrophils (510.sup.6) were resuspended in culture medium and stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), increasing concentrations of LTB.sub.4 (in placebo solution), LTA (10 g/ml) or a combination of either LTB.sub.4 and LTA (10 g/ml) for 30 minutes (FIG. 22). Cell lysates were subjected to Western blotting and levels of phosphorylated TAK1 and p38 were determined. Total p38 levels were also determined as a loading control. In FIG. 23, in vitro IRAK-1 kinase assay was performed. In short, human neutrophils (510.sup.7 cells/ml) were incubated or not with a specific IRAK-1/4 inhibitor (In) for 30 minutes. Following the inhibitor incubation, cells were stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), LTA (10 g/ml) or a combination of either LTB.sub.4 (100 nM in placebo solution) and LTA (10 g/ml) or with LPS (100 ng/ml) as a positive control.
[0099] After 2 or 5 minutes (indicated 2 and 5 on the figure), cells were lysed in cold lysis buffer (20 mM Tris pH 7.5, 1% Igepal, 1 mM EDTA, 150 mM NaCl, 10 mM -glycerophosphate and protease inhibitor cocktail) by an incubation for 30 minutes on ice. Following lysis, cell lysates were pre-cleared overnight with protein A/G agarose beads and 1 g of rabbit anti-rat IgG. Free lysates were immunoprecipitated with IRAK-1 antibody or unspecific rabbit IgG and protein A/G agarose beads for 2 h on ice. Beads were washed three times with lysis buffer and three additional times with a kinase assay buffer (20 mM HEPES pH 7.5, 20 mM MgCl2, 3 mM MnCl.sub.2, and 10 mM -glycerophosphate). A portion of the beads were kept for Western blotting analysis of the quantity of immunoprecipitate IRAK-1 proteins. The remaining of the beads was submitted to IRAK-1 in vitro kinase assay. Beads were incubated for 30 min at 37 C. in kinase assay buffer (50 l) containing 4 g of myelin basic protein, a phosphorylation substrate for IRAK-1, 4 Ci of -.sup.32P, and 5 M of ATP for 30 minutes. The reaction was stopped by the addition of 20 l of sample loading buffer (5 of 50 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 2.5% 13-mercaptoethanol, 0.05% bromophenol blue). Samples were boiled and ran on SDS-PAGE western blot gels.
[0100] Phosphorylated MBP (myelin basic protein) bands were detected using X-ray Kodak films or by image analyzer. Total immunoprecipitated IRAK-1 protein was detected by Western blot (WB). FIGS. 22-23 illustrate that a combination therapy using LTB.sub.4 with LTA enhances intracellular signaling pathways implicating IRAK-1, TAK-1, as well as p38 kinases in human neutrophils. In FIG. 24, murine splenocytes were isolated from Balb/c mouse spleens (n=5). Cells (510.sup.6 cells) were incubated with p38 kinase (SB203580) and IRAK-1/4 kinase inhibitors (IRAK-1/4 kinase inhibitor) for 60 minutes. Following incubation with inhibitors, cells were stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), LTA (0.1 g/ml) (FIG. 24A) or the prototypical viral activator or TLR2 herpes simplex virus-1 (HSV-1, 510.sup.4 viral particles) (FIG. 24B) or a combination of either LTB.sub.4 (100 nM in placebo solution) and LTA (0.1 g/ml) or HSV-1 (510.sup.4 viral particles) and LTB.sub.4 for 24 h. Cell-free supernatants were harvested and murine TNF- concentrations were evaluated using commercially available ELISA assay. The symbol * represents a probability of p<0.05 analyzed by one-tailed ANOVA followed by a Newman-Keuls post hoc test using PRISM3 software. As seen in FIGS. 24A and 24B, a combination therapy with LTB.sub.4 and TLR2 agonists enhances TNF- release by murine cells.
[0101] In FIG. 25, human neutrophils (FIG. 25A) or PBMCs (FIG. 25B) (510.sup.6 cells) were stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), CpG-ODN 2216 (10 g/ml) or a combination of LTB.sub.4 (100 nM in placebo solution) and CpG-ODN 2216 (10 g/ml) for 30 minutes. Following stimulation, cells were lysed and proteins were subjected to Western blotting for determination of phosphorylation levels for TAK1, p38, and Jun kinase (JNK). Total p38 levels were also determined as a loading control. In FIG. 26, cells were prepared as in FIG. 25 and incubated with chloroquine (20 M) for 30 minutes. Following inhibitor incubation, cells were subjected to a stimulation with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS), LTB.sub.4 (100 nM in placebo solution), CpG-ODN 2216 (10 g/ml) or a combination of LTB.sub.4 (100 nM in placebo solution) and CpG-ODN 2216 (10 g/ml) for 30 minutes. Following stimulation, cells were lysed and proteins were subjected to Western blotting for determination of phosphorylation levels for TAK1, p38, and Jun kinase (JNK). Total p38 levels were also determined as a loading control.
[0102] As seen in FIGS. 25-26, a combination therapy using LTB.sub.4 in the presence of CpG-ODN enhances intracellular signaling implicating TAK-1, p38, and JNK kinases in both human neutrophils and PBMCs via an endosome-independent pathway.
EXAMPLE VI
Administration of a Combination Therapy Including LTB.SUB.4 .with CpG-ODN Enhances Antiviral Defense in Mice Infected with Murine Cytomegalovirus
[0103] In FIG. 27, four-five week-old female Balb/c mice (15 mice/group) were infected by intraveinous (i.v.) injection of a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose) containing murine cytomegalovirus (mCMV) (400 plaque-forming units/mouse). On day 3 post-infection, mice were administered i.v. 100 l of placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose), LTB.sub.4 (1000 ng/kg in placebo solution), CpG-ODN 1826 (100 g in placebo solution) (5-TCCATGACGTTCCGACGTT-3; SEQ ID NO:2) or a combination of LTB.sub.4 (1000 ng/kg in placebo solution) and CpG-ODN 2216 (100 g in placebo solution). From day 4 to day 7 post-infection, daily doses of LTB.sub.4 were injected i.v. to groups receiving either LTB.sub.4 alone or LTB.sub.4 in combination with CpG-ODN 1826. Mice were sacrificed by isoflurane overdose on day 8 for salivary gland extraction. Salivary gland murine cytomegalovirus titers were evaluated by a standard plaque assay on mouse embryonic fibroblasts as already reported (Gosselin, J. Immunol., 2005, 174(3): 1587-1593). As seen in FIG. 26, a combination therapy including LTB.sub.4 with CpG-ODN 1826 (SEQ ID NO:2) resulted in enhanced control of murine cytomegalovirus replication when compared to mice treated with a placebo solution, LTB.sub.4 alone or with CpG-ODN alone.
EXAMPLE VII
Combination Therapy Including LTB4 and RLR-expressing Vectors or RLR Agonists Enhances RLR-mediated Signaling
[0104] In FIG. 28-31, HEK-293T cell line transfected with BLT1 cDNA was used as a cellular model for promoter activation assays. In FIG. 28-29, BLT1-expressing HEK-293T cells were first co-transfected with control vector or RIG-I-expressing vector (100 ng) along with a vector containing a luciferase gene reporter under NF-B promoter control (100 ng) (FIG. 28) or IFN- promoter control (100 ng) (FIG. 29) using Escort V transfection reagent. Briefly, cells were seeded at 510.sup.4 cells per well in a 24-well plate one day prior to transfection. Twenty-four hours post-transfection, cells were stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS) or LTB.sub.4 (100 nM in placebo solution) for 24 h. Following stimulation, cells were lysed in luciferase buffer (1% triton, 10% glycerol, 20 mM tris phosphate, pH 7.8). Luciferase activity was measured by luminometry and relative light units (RLU) were normalized by protein dosage using BCA protein assay kit. The symbol * represents a probability of p<0.05 analyzed by one-tailed ANOVA followed by a Newman-Keuls post hoc test using PRISM3 software. As can be seen in FIG. 28-29, the expression of an active RIG-I form in combination with LTB.sub.4 stimulation enhances NF-B as well as IFN- promoter activity. In FIG. 30-31, BLT1-expressing HEK-293T cells were first co-transfected with control vector or IPS-1-expressing vector (100 ng) along with a vector containing a luciferase gene reporter under NF-B promoter control (100 ng) (FIG. 30) or IFN- promoter control (100 ng) (FIG. 31) using Escort V transfection reagent. It has to be noted that IPS-1 is a key accessory protein involved in RLR-mediated signaling. Briefly, cells were seeded at 510.sup.4 cells per well in a 24-well plate one day prior to transfection. Twenty-four hours post-transfection, cells were stimulated with a placebo solution (0.45% w/v NaCl containing 0.25% w/v dextrose, NS) or LTB.sub.4 (100 nM in placebo solution) for 24 h. Following stimulation, cells were lysed in luciferase buffer (1% triton, 10% glycerol, 20 mM tris phosphate, pH 7.8). Luciferase activity was measured by luminometry and relative light units (RLU) were normalized by protein dosage using BCA protein assay kit. The symbol * represents a probability of p<0.05 analyzed by one-tailed ANOVA followed by a Newman-Keuls post hoc test using PRISM3 software. FIG. 30-31 show that the presence of an active IPS-1 protein in BLT1-expressing cells stimulated with LTB.sub.4 induces NF-B as well as IFN- promoter activity, key elements in RLR-mediated signaling. In FIG. 32, lung carcinoma A549 cells (ATCC #CCL-185) were seeded at 110.sup.5 cells per well in a 24-well plate and grown in Ham culture medium supplemented with 10% heat-inactivated fetal bovine serum. Cells were stimulated with synthetic dsRNA poly I:C (1 g/ml and 10 g/ml) or with LTB.sub.4 (100 nM) alone or with a combination of synthetic RNA with LTB.sub.4 for 5 hours. Poly I:C was partially digested with dsRNA-specific endonuclease RNase III prior its use. Non stimulated cells (NS) referred to as cells stimulated with a placebo solution (0.45% W/V NaCl containing 0.25% W/V dextrose). The symbol * represents a probability of p<0.05 analyzed by one-tailed ANOVA followed by a Newman-Keuls post hoc test using PRISM3 software. After 5 hours of stimulation, total RNA was extracted from samples using TRIZol reagents (Invitrogen) following the manufacturer's instructions. DNase-treated RNA (1 g) was reverse transcribed using SuperScript reverse transcriptase (Invitrogen) and IFN PCR was performed using the specific primers: 5-GAACTTTGAC ATCCCTGAGG AGATTAAGCA GC-3 (SEQ ID NO:4) and 5-GTTCCTTAGG ATTTCCACTC TGACTATGGT CC-3 (SEQ ID NO:5). GAPDH specific primers 5-CCACCCATGG CAAATTCCAT GGCA-3 (SEQ ID NO:6) and 5-TCTAGACGGC AGGTCAGGTC CACC-3 (SEQ ID NO:7) were used as internal control. The results presented herein clearly show when cells were stimulated with a combination of RLR agonist (namely the synthetic dsRNA poly I:C) and LTB.sub.4, that mRNA levels of IFN were found to be significantly increased (FIG. 32), indicating that LTB.sub.4 potentiates the effects of poly I:C on RLR signaling pathway. In 18 hours stimulated cell samples, cell-free supernatants were harvested and assayed for IFN determinations by ELISA (PBL Interferon Source, NJ, USA). As showed in FIG. 33, stimulation with LTB.sub.4 led to an increase in IFN levels produced by poly I:C-treated cells. Similarly, mRNA levels of IFN as well as secretion of IFN by A549 cells was also found to be increased when cells were treated with a combination of LTB.sub.4 and 3P-RNA (1 g/ml) or with a combination of LTB4 and Sendai virus (FIG. 34-35). These results clearly indicate that LTB.sub.4 potentiates type 1 IFN secretion following stimulation with RLR ligands. Finally, using mouse embryonic fibroblasts (MEFs) isolated from wild type mice and stimulated with LTB.sub.4 alone or in combination with Sendai virus, it is observed that LTB.sub.4 induces secretion of IFN but also potentiates secretion of IFN in MEFs stimulated with Sendai virus (FIG. 36), indicating that LTB.sub.4 can exert its action through RLR signaling pathway.
EXAMPLE VIII
Synergistic Pro-inflammatory Cytokine Secretion by LTB4 and N-acetyl Muramyl Dipeptide in Mouse Embryogenic Fibroblasts
[0105] To illustrate the effect of the combination of LTB.sub.4 with a NLR agonist, the NOD2 NLR agonist N-acetyl muramyl dipeptide (NacMDP) was used. For in vitro experiments, mouse embryogenic fibroblasts (MEF) were derived from 14-day C57131/6 mice embryos and expanded in Minimum Essential Medium Eagle (MEM) containing 10% Fetal Bovine Serum (FBS). Cells were then resuspended in culture medium and allowed to adhere overnight in 24-well plates (110.sup.5 cells/well). Following incubation, cells were washed once with HBSS and were either left unstimulated in culture medium or stimulated for 6 hours with increasing concentrations of NacMDP alone (0.1 g/ml, 1 g/ml and 10 g/ml), LTB.sub.4 alone (100 nM) or a combination of LTB.sub.4 and increasing concentrations of NacMDP (FIG. 37). Following stimulation, cell culture supernatants were harvested and assayed for IL-6 determination by ELISA. As can be seen from FIG. 37, NacMDP alone and LTB.sub.4 alone do not induce significant IL-6 secretion compared to background, however, when used in combination, they cause a significant synergistic increase in IL-6 secretion at 1 and 10 g/ml NacMDP. In FIG. 38, cells were either left uninfected or infected for 16 hours with Influenza A/PR/8/34 H1N1 (IAV) alone (0.1 m.o.i.) or in the presence of either NacMDP alone (0.1 g/ml), LTB.sub.4 alone (100 nM) or a combination of NacMDP and LTB.sub.4. Following stimulation/infection, cell culture supernatants were harvested and assayed for IL-6 determination by ELISA. In IAV-infected cells (FIG. 38), both NacMDP alone and LTB4 alone significantly enhance IL-6 secretion compared to infected but untreated cells. This synergistic enhancement becomes additive when both compounds are used in combination. In FIG. 37, * p0.05 compared to unstimulated cells or cells stimulated with increasing concentrations of NacMDP alone or LTB.sub.4 alone. In FIG. 38, * p0.05 compared to cells infected with IAV alone and ** p0.05 compared to cells infected with IAV alone or in the presence of NacMDP alone or LTB.sub.4 alone.
EXAMPLE IX
Synergistic Antiviral Activity of LTB4 and N-acetyl Muramyl Dipeptide Against Influenza Virus in vitro and in vivo
[0106] In FIG. 39, MEFs were generated and plated as described in Example VII. Cells were infected with IAV (0.1 m.o.i.) alone (), LTB.sub.4 alone (100 nM) or with a combination of LTB.sub.4 and NacMDP (0.1 g/ml) for 24 hours. Following infection, cell culture supernatants were harvested and assayed for viral load determination by standard plaque assay in MDCK cells. For in vivo assessment of the antiviral activity of LTB.sub.4 and NacMDP, 4-6 weeks old C57131/6 female mice (n=4/group) were infected intranasally (in.) with 50 plaque-forming units (pfu) of IAV (FIG. 40). Starting at day one post-infection, mice were treated daily for three days with either saline, LTB.sub.4 alone (1 g/kg) or with a combination of LTB.sub.4 and NacMDP (2 mg/kg), via the intravenous (iv.) route. Four days post-infection, animals were sacrificed and viral loads were determined from triturated lungs by standard plaque assay in MDCK cells. Both in vitro (FIG. 39) and in vivo (FIG. 40), LTB.sub.4 treatment causes a significant decrease in viral load, which is synergistically enhanced upon combination with NacMDP. In FIG. 39, * p0.05. In FIG. 40, * p0.05 compared IAV-infected mice receiving saline as treatment.
EXAMPLE X
Reduced Inflammation in the Lungs of Influenza-infected Mice Treated with a Combination of LTB4 and N-acetyl MOP
[0107] To further investigate the in vivo consequences associated with the treatment of IAV-infected mice with a combination of LTB.sub.4 and the NOD2 agonist NacMDP, 4-6 weeks old C57131/6 female mice (n=4/group) were infected as described in Example IX and treated with either saline or a combination of LTB.sub.4 and NacMDP also as described. In FIG. 41, four-day infected animals were sacrificed and IL-6 titers were determined from triturated lungs by ELISA. In FIG. 42, hematoxylin & eosin stains were performed on pulmonary lobes from four-day infected mice treated daily with saline or a combination of LTB.sub.4 and NacMDP as described. As can be seen in FIGS. 41 and 42 the antiviral activity of the combined LTB.sub.4/NacMDP treatment shown in Example IX is accompanied by a significant reduction in pro-inflammatory IL-6 secretion and was also found to restore normal lung architecture of IAV-infected mice at day three post-treatment initiation. In FIG. 41, * p0.05 compared to IAV-infected mice receiving saline as treatment.
[0108] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.
