A role for IRF3-dependent RXRalpha repression in hepatotoxicity associated with viral infections.

Viral infections and antiviral responses have been linked to several metabolic diseases, including Reye's syndrome, which is aspirin-induced hepatotoxicity in the context of a viral infection. We identify an interferon regulatory factor 3 (IRF3)-dependent but type I interferon-independent pathway that strongly inhibits the expression of retinoid X receptor alpha (RXRalpha) and suppresses the induction of its downstream target genes, including those involved in hepatic detoxification. Activation of IRF3 by viral infection in vivo greatly enhances bile acid- and aspirin-induced hepatotoxicity. Our results provide a critical link between the innate immune response and host metabolism, identifying IRF3-mediated down-regulation of RXRalpha as a molecular mechanism for pathogen-associated metabolic diseases.

Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response.

Type I interferon (IFN) production is a critical component of the innate defence against viral infections. Viral products induce strong type I IFN responses through the activation of Toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as protein kinase R (PKR). Here we demonstrate that cells lacking TRAF3, a member of the TNF receptor-associated factor family, are defective in type I IFN responses activated by several different TLRs. Furthermore, we show that TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKK-epsilon, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. In addition to TLR stimulation, we also show that TRAF3-deficient fibroblasts are defective in their type I IFN response to direct infection with vesicular stomatitis virus, indicating that TRAF3 is also an important component of TLR-independent viral recognition pathways. Our data demonstrate that TRAF3 is a major regulator of type I IFN production and the innate antiviral response.

TANK-binding kinase-1 plays an important role during in vitro and in vivo type I IFN responses to DNA virus infections.

TANK-binding kinase-1 (TBK1) and the inducible IkappaB kinase (IKK-i) have recently been shown to activate type I IFN responses elicited by intracellular detection of RNA or DNA from infecting viruses. Detection of viral RNA is mediated by retinoic acid inducible gene-I or melanoma differentiation-associated gene-5 pathways in which TBK1 and IKK-i have been demonstrated to play redundant roles in IFN activation. In this study, we have examined whether such redundancy occurs in the type I IFN response to DNA viral challenges by examining induction of IFNs and IFN-mediated signaling and gene programs in TBK1(-/-) macrophages. In contrast to the normal IFN responses in TBK1(-/-) macrophages infected with an RNA virus, IFN responses were severely abrogated during DNA virus infections in TBK1(-/-) macrophages. Because both TBK1 and IKK-i are expressed in macrophages, our studies suggest that TBK1 and IKK-i differ functionally in DNA virus-mediated IFN responses; however, they are redundant in RNA virus-mediated IFN responses. Confirmatively, reconstitution of TBK1(-/-)IKK-i(-/-) fibroblasts revealed that TBK1 rescued IFN responses to transfected B-DNA to a much stronger degree than IKK-i. Finally, we demonstrate the requirement for the TBK1-IFN regulatory factor-3 pathway in host defense against a DNA virus infection in vivo.

Closed-loop control of cellular functions using combinatory drugs guided by a stochastic search algorithm.

A mixture of drugs is often more effective than using a single effector. However, it is extremely challenging to identify potent drug combinations by trial and error because of the large number of possible combinations and the inherent complexity of the underlying biological network. With a closed-loop optimization modality, we experimentally demonstrate effective searching for potent drug combinations for controlling cellular functions through a large parametric space. Only tens of iterations out of one hundred thousand possible trials were needed to determine a potent combination of drugs for inhibiting vesicular stomatitis virus infection of NIH 3T3 fibroblasts. In addition, the drug combination reduced the required dosage by approximately 10-fold compared with individual drugs. In another example, a potent mixture was identified in thirty iterations out of a possible million combinations of six cytokines that regulate the activity of nuclear factor kappa B in 293T cells. The closed-loop optimization approach possesses the potential of being an effective approach for manipulating a wide class of biological systems.

A repetitive region of gammaherpesvirus genomic DNA is a ligand for induction of type I interferon.

Innate immune responses against viral infection, especially the induction of type I interferon, are critical for limiting the replication of the virus. Although it has been shown that DNA can induce type I interferon, to date no natural DNA ligand of a virus that induces type I interferon has been described. Here we screened the genome of murine gammaherpesvirus 68 with mutations at various genomic locations to map the region of DNA that induces type I interferon. A repetitive region termed the 100-base-pair repeat region is a ligand that is both necessary and sufficient for the viral genomic DNA to induce type I interferon. A region colinear with this ligand in the genome of Kaposi's sarcoma-associated herpesvirus also induces type I interferon. We have thus defined a repetitive region of the genomes of gammaherpesviruses as the first natural DNA virus ligand that induces type I interferon.

Successful repair of intraoperative cerebrospinal fluid leaks improves outcomes in endoscopic skull base surgery.

BACKGROUND: The impact of failed cerebrospinal fluid leak (CSF) leak repair in endoscopic skull base surgery has not been adequately studied. METHODS: In this investigation we reviewed patients who had undergone endoscopic skull base surgery between 2002 and 2014 at 7 international centers. Demographic variables, comorbidities, tumor characteristics, and repair techniques were evaluated to determine association with successful repair of CSF leak. Postoperative complications and length of stay were compared among groups. RESULTS: Data were collected on 2097 patients who were divided into 3 groups: (1) those with no intraoperative leak (n = 1533); (2) those with successful repair of their intraoperative leak (n = 452); and (3) those with failed repair (n = 112). Compared with successful repair, failed repair was associated with an increased risk of intracranial infection (odds ratio [OR], 5.6; 95% confidence interval [CI], 5.3-13.15), pneumocephalus (OR, 16; 95% CI, 5.8-44.4), 30-day readmission (OR, 8.4; 95% CI, 5.3-13.5), reoperation (OR, 115.4; 95% CI, 56.3-236.8), and prolonged hospital stay (14.9 vs 7.0 days, p < 0.01). Outcomes in patients who had successful repairs of intraoperative leaks were similar to those who never had leakage. Intraoperative use of pedicled nasoseptal flaps was associated with successful repair (OR, 0.60; 95% CI, 0.34-0.92). CONCLUSION: Intraoperative CSF leaks are a frequent and expected occurrence during endoscopic skull base surgery. Failed CSF leak repair has a significant impact on patient outcomes, with increased rates of postoperative pneumocephalus, intracranial infections, reoperation, deep vein thrombosis, readmission, and prolonged hospital stay. Recognition and repair of intraoperative CSF leaks reduces postoperative complications. Use of pedicled nasoseptal flaps improves outcomes in reconstructing defects at higher risk for postoperative leak.

Role of Vitamin D in Pathogenesis of Chronic Sinusitis with Nasal Polyposis.

Vitamin D3 (VD3) is a potent immunomodulatory steroid hormone involved in the regulation of numerous host immune cells. Additionally, it may have an important role in host immunity against chronic bacterial infections, as well as in chronic rhinosinusitis. Many factors modulate expression of various enzymes important for production of VD3, local concentration of which may be important for its complex and diverse functions. Here, we explore some of the contributions of VD3 to chronic rhinosinusitis with nasal polyposis and its role as a disease-modifying agent.

Impact of postoperative endoscopy upon clinical outcomes after endoscopic sinus surgery.

BACKGROUND: After endoscopic sinus surgery (ESS), endoscopy is used to gauge surgical success and clinical outcomes. Prior studies have not examined this topic prospectively using validated outcome metrics across multiple institutions. METHODS: A multi-institutional, prospective study of patients with chronic rhinosinusitis (CRS) who underwent ESS completed the 22-item Sino-Nasal Outcome Test (SNOT-22), missed productivity, and medication usage questionnaires 6 months postoperatively. Lund-Kennedy endoscopy scoring (LKES) was performed with reviewers blinded to patient-reported data. A control cohort of non-CRS patients was recruited for comparison. RESULTS: Complete data was available on 183 CRS patients and 48 non-CRS control patients. Approximately 50% of patients achieve perfect or near perfect endoscopy (LKES 0 to 2) after ESS. Postoperative endoscopy correlated with total SNOT-22 scores (r = 0.278, p < 0.001), with the strongest correlations to rhinologic and extranasal subdomains in the nasal polyp cohort. Improved postoperative endoscopy was associated with decreased antibiotic and oral steroid usage, but had little association with missed productivity. Among patients who achieved near perfect postoperative endoscopy, those with nasal polyps had SNOT-22 scores that were similar to non-CRS control patients (mean SNOT-22 scores 17.7 and 16.3, respectively). However, CRS patients without nasal polyps remained more symptomatic than non-CRS controls and CRS with nasal polyps patients despite nearly perfect endoscopy (mean SNOT-22 score 21.6). CONCLUSION: Postoperative endoscopy correlates with SNOT-22 and medication usage in CRS patients. Polyp patients who achieve near perfect endoscopy have similar symptoms to healthy controls; however, nonpolyp patients with near perfect endoscopy still have rhinologic and extranasal symptoms that are worse than healthy controls.

Targeting Toll-like receptor 2 inhibits growth of head and neck squamous cell carcinoma.

Infection-driven inflammation has been proposed to be involved in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). Oral HNSCC is often colonized with microbes such as gram-positive bacteria and yeast, where ligands derived from their wall components have been shown to specifically bind to Toll-like receptor 2 (TLR2). Although TLR2 has been described to be expressed in oral HNSCC, its function has not been well characterized. Here, we show the expression of TLR2 in both HNSCC cell lines and primary patient-derived HNSCC xenograft tumors. Activation of TLR2 with a yeast-derived ligand of TLR2, zymosan, promoted organoid formation in an ex vivo model of tumor growth, while blockade with anti-TLR2 antibodies inhibited organoid formation. Zymosan also induced phosphorylation of ERK and the p65 subunit of NF-κB, which was inhibited in the presence of anti-TLR2 antibodies, indicating that this receptor is functional in HNSCC and that the signaling through these pathways is intact. TLR2 blockade also inhibited growth of human xenografted tumors in immunodeficient mice. In summary, our data show that TLR2 is a functional receptor expressed in human HNSCC that plays a direct pro-tumorigenic role, and that it can be therapeutically targeted with blocking antibodies to reduce tumor growth.

T3JAM, a novel protein that specifically interacts with TRAF3 and promotes the activation of JNK(1).

Previous studies suggest that localization of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family members is important for regulating their signal transduction. During a screen for TRAF3-associated proteins that potentially alter TRAF3 subcellular localization and enable signal transduction, we identified a novel protein, T3JAM (TRAF3-interacting Jun N-terminal kinase (JNK)-activating modulator). This protein associates specifically with TRAF3 but not other TRAF family members. Coexpression of T3JAM with TRAF3 recruits TRAF3 to the detergent-insoluble fraction. More importantly, T3JAM and TRAF3 synergistically activate JNK but not nuclear factor (NF)-kappaB. Our studies indicate that T3JAM may function as an adapter molecule that specifically regulates TRAF3-mediated JNK activation.

Cascade search for HSV-1 combinatorial drugs with high antiviral efficacy and low toxicity.

BACKGROUND: Infectious diseases cause many molecular assemblies and pathways within cellular signaling networks to function aberrantly. The most effective way to treat complex, diseased cellular networks is to apply multiple drugs that attack the problem from many fronts. However, determining the optimal combination of several drugs at specific dosages to reach an endpoint objective is a daunting task. METHODS: In this study, we applied an experimental feedback system control (FSC) method and rapidly identified optimal drug combinations that inhibit herpes simplex virus-1 infection, by only testing less than 0.1% of the total possible drug combinations. RESULTS: Using antiviral efficacy as the criterion, FSC quickly identified a highly efficacious drug cocktail. This cocktail contained high dose ribavirin. Ribavirin, while being an effective antiviral drug, often induces toxic side effects that are not desirable in a therapeutic drug combination. To screen for less toxic drug combinations, we applied a second FSC search in cascade and used both high antiviral efficacy and low toxicity as criteria. Surprisingly, the new drug combination eliminated the need for ribavirin, but still blocked viral infection in nearly 100% of cases. CONCLUSION: This cascade search provides a versatile platform for rapid discovery of new drug combinations that satisfy multiple criteria.

Type I IFNs mediate development of postinfluenza bacterial pneumonia in mice.

Influenza-related complications continue to be a major cause of mortality worldwide. Due to unclear mechanisms, a substantial number of influenza-related deaths result from bacterial superinfections, particularly secondary pneumococcal pneumonia. Here, we report what we believe to be a novel mechanism by which influenza-induced type I IFNs sensitize hosts to secondary bacterial infections. Influenza-infected mice deficient for type I IFN-alpha/beta receptor signaling (Ifnar-/- mice) had improved survival and clearance of secondary Streptococcus pneumoniae infection from the lungs and blood, as compared with similarly infected wild-type animals. The less effective response in wild-type mice seemed to be attributable to impaired production of neutrophil chemoattractants KC (also known as Cxcl1) and Mip2 (also known as Cxcl2) following secondary challenge with S. pneumoniae. This resulted in inadequate neutrophil responses during the early phase of host defense against secondary bacterial infection. Indeed, influenza-infected wild-type mice cleared secondary pneumococcal pneumonia after pulmonary administration of exogenous KC and Mip2, whereas neutralization of Cxcr2, the common receptor for KC and Mip2, reversed the protective phenotype observed in Ifnar-/- mice. These data may underscore the importance of the type I IFN inhibitory pathway on CXC chemokine production. Collectively, these findings highlight what we believe to be a novel mechanism by which the antiviral response to influenza sensitizes hosts to secondary bacterial pneumonia.

Regulation of antiviral responses by a direct and specific interaction between TRAF3 and Cardif.

Upon recognition of viral infection, RIG-I and Helicard recruit a newly identified adapter termed Cardif, which induces type I interferon (IFN)-mediated antiviral responses through an unknown mechanism. Here, we demonstrate that TRAF3, like Cardif, is required for type I interferon production in response to intracellular double-stranded RNA. Cardif-mediated IFNalpha induction occurs through a direct interaction between the TRAF domain of TRAF3 and a TRAF-interaction motif (TIM) within Cardif. Interestingly, while the entire N-terminus of TRAF3 was functionally interchangeable with that of TRAF5, the TRAF domain of TRAF3 was not. Our data suggest that this distinction is due to an inability of the TRAF domain of TRAF5 to bind the TIM of Cardif. Finally, we show that preventing association of TRAF3 with this TIM by mutating two critical amino acids in the TRAF domain also abolishes TRAF3-dependent IFN production following viral infection. Thus, our findings suggest that the direct and specific interaction between the TRAF domain of TRAF3 and the TIM of Cardif is required for optimal Cardif-mediated antiviral responses.

MyD88 mediates neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus.

MyD88 is an important signaling adaptor for both TLR and IL-1R family members. Here, we evaluated the role of TLR2/MyD88 and IL-1R/MyD88 signaling in host defense against S. aureus by using a cutaneous infection model in conjunction with bioluminescent bacteria. We found that lesions of S. aureus-infected MyD88- and IL-1R-deficient mice were substantially larger with higher bacterial counts compared with wild-type mice. In contrast, TLR2-deficient mice had lesions that were only moderately larger with minimally higher bacterial counts. In addition, MyD88- and IL-1R- but not TLR2-deficient mice had severely decreased recruitment of neutrophils to the site of infection. This neutrophil recruitment was not dependent upon IL-1R/MyD88 signaling by recruited bone marrow-derived cells, suggesting that resident skin cells utilize IL-1R/MyD88 signaling to promote neutrophil recruitment.