Innate immune responses against priority pathogens
Program leader: Jenny Ting
SE-RP-010: Novel roles of the NLR protein in host response against WNV and DENV
University of North Carolina
A major conceptual advance for innate immunity has been the discovery of Pathogen-associated Molecular Pattern recognition receptors or sensors, a prime example being the toll-like receptor (TLR). More recently, we and others discovered a large family of pathogen sensors in humans and mice, called the NLR (nucleotide-binding domain, leucine-rich repeat containing) family, known previously as CATERPILLER, NACHT-LRR, or NOD-like receptor. An important function of NLR proteins is the formation of a biochemically-defined complex, called the inflammasome, which processes pro-caspase-1 and pro-IL-1b/IL-18 to their mature forms. Additional studies indicate that NLR proteins mediate type I interferon (IFN) production in response to viruses through an interference of the intracellular pathway mediated by the mitochondrial anti-viral signaling molecule (MAVS, a.k.a. IPS, VISA and CARDIF). This proposal will examine the divergent roles of NLR during flavivirus infection. Flaviviruses such as Dengue (DENV) and West Nile (WNV) have surfaced at the forefront of biodefense and emerging infections. Type I IFN is pivotal in anti-flaviviral host defense, and the intracellular pathway of viral RNA recognition is crucial for this response during DENV and WNV viral infection. Of equal importance, IL-1 production has been observed by several groups as being a crucial outcome of human infection by flaviviruses. However, the link between NLR members to any of these anti-viral host responses has not been explored. During the last funding period, we have started to decipher if NLRP3 is important for host response to these two flaviviruses. For WNV, we have initiated in vivo experiments and the preliminary data suggest that NLRP3 does control IL-1b response to WNV. For DENV, we have spent a significant amount of time finding a cellular system where the virus induced the inflammasome and IFN-I as separate pathways. We have found that DENV infection of one macrophage line resulted in an antibody-dependent enhanced infection that is accompanied by inflammasome activation, while DENV infection of another line resulted in enhanced IFN-I induction. These experiments formed the basis for our plans to explore the roles of NLRs in host defense and inflammation upon DENV and WNV.
SE-RP-011: Regulation of antibody responses by toll-like receptors
Emory University
Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence suggests that they activate dendritic cells (DCs) via Toll-like receptors (TLRs). For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed, activates DCs via multiple TLRs to stimulate pro-inflammatory cytokines. Triggering specific combinations of TLRs in DCs can induce synergistic production of cytokines, which results in enhanced T cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that programs such antibody responses remains a major challenge in vaccinology. Here we demonstrate that immunization of mice with synthetic nanoparticles containing antigens plus Toll-like receptor (TLR) ligands 4 + 7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with a single TLR ligand. Consistent with this there was enhanced persistence of germinal centers (GCs), and of plasma cell responses, which persisted in the lymph nodes for >1.5 years. Surprisingly, there was no enhancement of the early short-lived plasma cell response, relative to that observed with single TLR ligands. Molecular profiling of activated B cells, isolated 7 days after immunization, indicated early programming towards B cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells (DCs), as well as on T-cell help. Immunization protected completely against lethal avian and swine influenza virus strains in mice, and induced robust immunity against pandemic H1N1 influenza in rhesus macaques.
SE-RP-012: TLR7 and responses to flaviviruses
North Carolina State University
Flaviviruses have evolved complex mechanisms to interact with the host immune response. The earliest immune responses to infection involve components of the innate immune system, beginning with the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), such as toll-like receptors (TLRs), and intracellular RNA helicases, such as RIG-I and Mda-5. PAMP recognition results in activation of specific signal transduction pathways that result in production of proinflammatory cytokines, such as type I interferon (IFN), TNFa and others. This project focuses on the role of TLRs and TLR7 in particular in the control of flavivirus pathogenesis. Using a footpad inoculation model of WNV encephalitis, a protective role of TLR7 was identified, with TLR7 ko mice demonstrating significantly higher mortality. Virologic analysis demonstrated marginally higher viral loads in peripheral tissues and the CNS and a reduction in WNV specific B cells. Cytokine responses were generally augmented in TLR7 ko mice, peaking around the time of onset of disease. In contrast, analysis of the primary cytokine response in the draining lymph node after footpad inoculation with single cycle infectious WNV replicon particles demonstrated reduced levels of IFNa mRNA as well as reduced levels of other proinflammatory cytokines. Despite this reduction compared to wt mice, IFNa was still induced significantly in TLR7 ko animals but not in mice deficient for the IFNa/b receptor, indicating that the majority of IFNa is produced through the IFNR-dependent JAK-STAT amplification loop. Taken together our findings indicate that TLR7 plays a protective role at a later stage of WNV pathogenesis but not at limiting virus replication in primary lymphoid tissues.
In addition to the analysis of TLR7’s role in WNV pathogenesis further progress was made into the investigation of mechanisms WNV uses to circumvent the innate immune response to infection. We have previously described the WNV NS1 protein as an inhibitor of TLR3 signal transduction and have now solid evidence that NS1 is a general inhibitor of TLR signal transduction. A cellular target of NS1 was identified whose activation is inhibited in the presence of NS1, and we are in the process of characterizing this inhibition further. To further define the molecular mechanisms of NS1 inhibition of TLRs, a mutational screen of NS1 was conducted to identify mutants that lose the ability to inhibit TLR signaling while at the same time preserving NS1’s function in viral RNA replication. Seven mutants with the desired characteristics were identified and are presently being characterized in detail. One of the most significant findings of our characterization of TLR inhibition by NS1 was the demonstration that secreted NS1 is able to inhibit TLRs in a concentration dependent manner. Since NS1 is secreted to a high level by infected cells this provides an intriguing mechanism of the virus manipulating innate immune responses on potential target cells prior to infecting them.
