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We have previously observed that IL-1β and type I IFN (IFNβ) cooperate to control viral replication within the CNS of infected animals and in viral target cells after challenge with West Nile virus (WNV). However, the mechanism by which these cytokine signaling pathways cooperate to elicit this immune control is not clearly defined. Using global transcriptome analysis, we have identified a distinct and targeted IL-1-driven “antiviral signature” comprised of known and novel host restriction factors involved in WNV control. Remarkably, co-treatment of myeloid cells with both IL-1β and IFNβ induced the expression of distinct subsets of this signature, revealing IL-1β-dependent, IFNβ-dependent, or synergistically driven gene induction profiles. Furthermore, within this signature we observed specific requirements for IRF3, IRF5, IRF7 and IFNAR activation, demonstrating that IL-1 driven host restriction factors may be regulated by distinct IRF interactions. Together these data demonstrate that inflammatory and antiviral signals integrate at the level of IL-1β and IFNβ to elicit IRF-specific targeted ISG expression and control of WNV. Furthermore, these data illuminate the possibility that specific host-driven viral signatures exist in a pathogen-dependent manner. Thus, strategies to co-opt these cytokine activated antiviral signatures may increase our ability to generate novel targeted therapeutic strategies tailored specifically to individual pathogens.

Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1β (IL-1β) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1β compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1β during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1β after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1β secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1β mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1β processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1β production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1β production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1β activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease., Author Summary Hepatitis C virus (HCV) causes chronic infection of the liver and is a leading cause of liver inflammation, cirrhosis and liver cancer in nearly 200 million people worldwide. Importantly, hepatic inflammation during chronic HCV infection is considered to be the primary catalyst for progressive liver disease and development of liver cancer. However, the underlying molecular mechanism(s) of HCV-mediated hepatic inflammation are not well understood. The goal of this study was to determine the mechanisms of HCV-induced inflammation. We found that serum IL-1β levels are elevated in chronic hepatitis C patients. Furthermore, we found that hepatic macrophages or Kupffer cells are the major IL-1β-producing cell population within HCV infected livers. Our studies, using the THP1 cell culture model of HCV exposure, reveal that exposure of macrophages to HCV induces IL-1β through a process of infection-independent phagocytic virus uptake that triggers signaling through MyD88/TLR7 and NLRP3 inflammasome pathways to drive IL-1β expression and maturation/secretion, respectively. RNA sequencing (RNA-seq) analysis of patient liver biopsies shows that viral triggering of these signaling pathways drives an inflammatory response linked with liver disease in patients with chronic hepatitis C. Our results identify HCV-induced IL-1β production by hepatic macrophages as a critical and central process that promotes liver inflammation and disease.

The actions of the RIG-I like receptor (RLR) and type I interferon (IFN) signaling pathways are essential for a protective innate immune response against the emerging flavivirus West Nile virus (WNV). In mice lacking RLR or IFN signaling pathways, WNV exhibits enhanced tissue tropism, indicating that specific host factors of innate immune defense restrict WNV infection and dissemination in peripheral tissues. However, the immune mechanisms by which the RLR and IFN pathways coordinate and function to impart restriction of WNV infection are not well defined. Using a systems biology approach, we defined the host innate immune response signature and actions that restrict WNV tissue tropism. Transcriptional profiling and pathway modeling to compare WNV-infected permissive (spleen) and nonpermissive (liver) tissues showed high enrichment for inflammatory responses, including pattern recognition receptors and IFN signaling pathways, that define restriction of WNV replication in the liver. Assessment of infected livers from Mavs−/−×Ifnar−/− mice revealed the loss of expression of several key components within the natural killer (NK) cell signaling pathway, including genes associated with NK cell activation, inflammatory cytokine production, and NK cell receptor signaling. In vivo analysis of hepatic immune cell infiltrates from WT mice demonstrated that WNV infection leads to an increase in NK cell numbers with enhanced proliferation, maturation, and effector action. In contrast, livers from Mavs−/−×Ifnar−/− infected mice displayed reduced immune cell infiltration, including a significant reduction in NK cell numbers. Analysis of cocultures of dendritic and NK cells revealed both cell-intrinsic and -extrinsic roles for the RLR and IFN signaling pathways to regulate NK cell effector activity. Taken together, these observations reveal a complex innate immune signaling network, regulated by the RLR and IFN signaling pathways, that drives tissue-specific antiviral effector gene expression and innate immune cellular processes that control tissue tropism to WNV infection., Author Summary West Nile virus (WNV), a mosquito-transmitted RNA flavivirus, is an NIAID Category B infectious agent that has emerged in the Western hemisphere as a serious public health threat. The innate immune effectors that impart restriction of WNV infection are not well defined. WNV infection is sensed by the host RIG-I like receptors (RLR), a class of pattern recognition receptors, to trigger type I interferon (IFN) and related innate immune defense programs. Using a systems biology approach, we evaluated the contribution of the RLR and type I IFN signaling pathways in controlling tissue tropism. WNV infection triggers tissue-specific innate immune responses, specifically antiviral effector genes and natural killer (NK) cell signaling related genes, which are directly regulated by the combined actions of the RLR and type I IFN signaling pathways. Cocultures of dendritic and NK cells revealed that RLR and type I IFN signaling pathways are essential in promoting NK cell activation during WNV infection. Our observations indicate that combined RLR- and type I IFN-dependent signaling programs drive specific antiviral effector gene expression and programs NK cell responses that, together, serve to restrict WNV tissue tropism.

The innate cytokines IL-12 and IFN-α/β act in concert to facilitate clearance of intracellular pathogens. However their precise role in promoting human T cell responses is still unclear. In this study, we demonstrate that these cytokines regulate distinct aspects of effector and memory human CD8+ T cell differentiation. We found that IL-12 programs the development of TEM cells which secrete high levels of IFN-γand TNF-α and attain strong cytolytic activity. Programming of TEM cells by IL-12 correlated to the induction of the IL-12 receptor β 2 (IL-2Rβ2) and was associated with cells which had undergone rapid division. Alternatively, IFN- α/β enhanced a population of TCM cells which expressed high levels of the lymph node homing receptor CCR7. This population did not display immediate effector activity however, upon secondary activation, were capable of rapid proliferation and acquisition of both memory and effector phenotypes. TCM development was regulated by IFN-αand proceeded by slowing the progression of cell division in a subpopulation of cells that selectively expressed elevated IFN- α/βreceptor-2 (IFNAR2). We further examined the effects of the strength of TCR signal on TEM and TCM development. In the presence of both IL-12 and IFN- α/β, these cytokine signals were amplified as the strength of the TCR signal was increased, promoting the simultaneous development of both TCM and TEM. Together, our results support a novel model in which IL-12 and IFN- α/βact in a non-redundant manner to regulate the co-linear generation of both effector and memory cells. This study is supported by a grant from the National Institutes of Health/National Institute of Allergy and Infectious Disease (AI56222) and by a predoctoral fellowship from the National Instititue of Health/Nation Institute of Allergy and Infectious Disease (AI68622).

Antigen presenting cells secrete distinct patterns of innate cytokines upon activation via Toll-like receptors (TLRs). These innate cytokines shape adaptive immune responses to pathogens. For example, interleukin 12 (IL-12) is produced in response to bacterial recognition via TLRs 2, 4, 5, and 6. IL-12 induces CD4+ T helper 1 (Th1) cell development through the activation of signal transducer and activator of transcription 4 (STAT4), leading to the secretion of interferon gamma (IFN-γ). Conversely, during viral infection, signaling through TLRs 3, 7, and 9 results in the secretion of both IL-12 and type I interferon (IFN-α/β). The effects of combined IL-12 and IFN-α/β stimulation on T helper development are not well-studied. We have demonstrated that activation of naïve CD4+ T cells in the presence of IFN-α/β alone fails to induce Th1 development. However, a combination of IL-12 and IFN-α/β induces phenotypic variegation to generate two populations of T helper cells. An effector population secretes both IFN-γ and IFN-α/β, and these cells are efficient in eliminating viral infections in vitro. A second subset displays memory properties and may be involved in long-term viral immunity. Furthermore, these two subsets can be distinguished by expression of chemokine receptors. These data have important implications for the understanding of viral infections as well as for the development of novel vaccines and therapies.