Adelle P. McFarland

The favorable IFNL3 genotype escapes mRNA decay mediated by AU-rich elements and hepatitis C virus-induced microRNAs

The IFNL3 (IL28B) gene has received immense attention in the hepatitis C virus (HCV) field as multiple independent genome-wide association studies identified a strong association between polymorphisms near the IFNL3 gene and HCV clearance. However, the mechanism underlying this association has remained elusive. In this study, we report the identification of a functional polymorphism (rs4803217) located in the 3′ untranslated region (3′ UTR) of the IFNL3 mRNA that dictates transcript stability. This polymorphism influences AU-rich element-mediated decay as well as the binding of HCV-induced microRNAs during infection. Together, these pathways mediate robust repression of the unfavorable IFNL3 genotype. These data reveal a novel mechanism by which HCV attenuates the antiviral response and uncover new potential therapeutic targets for HCV treatment.IFNL3, which encodes interferon-λ3 (IFN-λ3), has received considerable attention in the hepatitis C virus (HCV) field, as many independent genome-wide association studies have identified a strong association between polymorphisms near IFNL3 and clearance of HCV. However, the mechanism underlying this association has remained elusive. In this study, we report the identification of a functional polymorphism (rs4803217) in the 3′ untranslated region (UTR) of IFNL3 mRNA that dictated transcript stability. We found that this polymorphism influenced AU-rich element (ARE)-mediated decay (AMD) of IFNL3 mRNA, as well as the binding of HCV-induced microRNAs during infection. Together these pathways mediated robust repression of the unfavorable IFNL3 polymorphism. Our data reveal a previously unknown mechanism by which HCV attenuates the antiviral response and indicate new potential therapeutic targets for HCV treatment.

A novel role for IL-22R1 as a driver of inflammation

The interleukin (IL)-22R1 chain of the heterodimeric IL-22 receptor is not expressed on normal leukocytes, but this receptor is expressed on T cells from anaplastic lymphoma kinase-positive (ALK(+)) anaplastic large cell lymphoma (ALCL) patients. To investigate the consequences of aberrant expression of this receptor on lymphocytes, we generated transgenic mice that express IL-22R1 on lymphocytes. The health of these animals progressively deteriorated at 8 to 12 weeks of age, as they displayed respiratory distress, rough coat and sluggish movement, and subsequent lethality due to multiorgan inflammation. The IL-22R1 transgenic animals developed neutrophilia that correlated with increased levels of circulating IL-17 and granulocyte colony-stimulating factor. In addition, these mice had increased serum IL-22 levels, suggesting that T cells expressing IL-22R1 generate IL-22 in a positive autoregulatory loop. As a result of the mouse model findings, we analyzed circulating cytokine levels in ALK(+)ALCL patients and detected elevated levels of IL-22, IL-17, and IL-8 in untreated patient samples. Importantly, IL-22 and IL-17 were undetectable in all patients who were in complete remission after chemotherapy. This study documents a previously unknown role of IL-22R1 in inflammation and identifies the involvement of IL-22R1/IL-22 in ALK(+)ALCL.

IRF-5 and NF-κB p50 co-regulate IFN-β and IL-6 expression in TLR9-stimulated human plasmacytoid dendritic cells.

Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN‐β) and pro‐inflammatory (IL‐6) cytokines by CpG‐stimulated human pDCs and the human CAL‐1 pDC cell line. Results show that interferon regulatory factor‐5 (IRF‐5) and NF‐κB p50 are key co‐regulators of IFN‐β and IL‐6 expression following TLR9‐mediated activation of human pDCs. The nuclear accumulation of IRF‐1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF‐8 was identified as a novel negative regulator of gene activation in CpG‐stimulated pDCs. As variants of IRF‐5 and IRF‐8 were recently found to correlate with susceptibility to certain autoimmune diseases, these findings are relevant to our understanding of the pharmacologic effects of “K” ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions.

Localized Delivery of Interferon-β by Lactobacillus Exacerbates Experimental Colitis

Background There have been conflicting reports of the role of Type I interferons (IFN) in inflammatory bowel disease (IBD). Clinical trials have shown potent efficacy of systemic interferon-beta (IFN-β) in inducing remission of ulcerative colitis. Likewise, IFNAR1−/− mice display an increased sensitivity to dextran sulfate sodium (DSS)-induced colitis, suggesting Type I IFN play a protective role during inflammation of the gut. Curiously, however, there have also been reports detailing the spontaneous development of IBD in patients receiving systemic IFN-β therapy for multiple sclerosis or hepatitis. Methodology/Principal Findings To investigate the effects of local administration of IFN-β on a murine model of colitis, we developed a transgenic Lactobacillus acidophilus strain that constitutively expresses IFN-β (La-IFN-β). While pretreatment of mice with control Lactobacillus (La-EV) provided slight protective benefits, La-IFN-β increased sensitivity to DSS. Analysis showed colitic mice pretreated with La-IFN-β had increased production of TNF-α, IFN-γ, IL-17A and IL-13 by intestinal tissues and decreased regulatory T cells (Tregs) in their small intestine. Examination of CD103+ dendritic cells (DCs) in the Peyers patches revealed that IFNAR1 expression was dramatically reduced by La-IFN-β. Similarly, bone marrow-derived DCs matured with La-IFN-β experienced a 3-fold reduction of IFNAR1 and were impaired in their ability to induce Tregs. Conclusions/Significance Our IFNAR1 expression data identifies a correlation between the loss/downregulation of IFNAR1 on DCs and exacerbation of colitis. Our data show that Lactobacillus secreting IFN-β has an immunological effect that in our model results in the exacerbation of colitis. This study underscores that the selection of therapeutics delivered by a bacterial vehicle must take into consideration the simultaneous effects of the vehicle itself.

Hepatitis-C-virus-induced microRNAs dampen interferon-mediated antiviral signaling

Hepatitis C virus (HCV) infects 200 million people globally, and 60–80% of cases persist as a chronic infection that will progress to cirrhosis and liver cancer in 2–10% of patients. We recently demonstrated that HCV induces aberrant expression of two host microRNAs (miRNAs), miR-208b and miR-499a-5p, encoded by myosin genes in infected hepatocytes. These miRNAs, along with AU-rich-element-mediated decay, suppress IFNL2 and IFNL3, members of the type III interferon (IFN) gene family, to support viral persistence. In this study, we show that miR-208b and miR-499a-5p also dampen type I IFN signaling in HCV-infected hepatocytes by directly down-regulating expression of the type I IFN receptor chain, IFNAR1. Inhibition of these miRNAs by using miRNA inhibitors during HCV infection increased expression of IFNAR1. Additionally, inhibition rescued the antiviral response to exogenous type I IFN, as measured by a marked increase in IFN-stimulated genes and a decrease in HCV load. Treatment of HCV-infected hepatocytes with type I IFN increased expression of myosins over HCV infection alone. Since these miRNAs can suppress type III IFN family members, these data collectively define a novel cross-regulation between type I and III IFNs during HCV infection.

Sensing of Bacterial Cyclic Dinucleotides by the Oxidoreductase RECON Promotes NF-κB Activation and Shapes a Proinflammatory Antibacterial State

SUMMARY Bacterial and host cyclic dinucleotides (cdNs) mediate cytosolic immune responses through the STING signaling pathway, although evidence suggests that alternative pathways exist. We used cdN‐conjugated beads to biochemically isolate host receptors for bacterial cdNs, and we identified the oxidoreductase RECON. High‐affinity cdN binding inhibited RECON enzyme activity by simultaneously blocking the substrate and cosubstrate sites, as revealed by structural analyses. During bacterial infection of macrophages, RECON antagonized STING activation by acting as a molecular sink for cdNs. Bacterial infection of hepatocytes, which do not express STING, revealed that RECON negatively regulates NF‐&kgr;B activation. Loss of RECON activity, via genetic ablation or inhibition by cdNs, increased NF‐&kgr;B activation and reduced bacterial survival, suggesting that cdN inhibition of RECON promotes a proinflammatory, antibacterial state that is distinct from the antiviral state associated with STING activation. Thus, RECON functions as a cytosolic sensor for bacterial cdNs, shaping inflammatory gene activation via its effects on STING and NF‐&kgr;B. HIGHLIGHTSBacterial, but not eukaryotic, cyclic dinucleotides inhibit the oxidoreductase RECONRECON binding to bacterial cyclic dinucleotides antagonizes STING signalingLoss of RECON enzyme activity promotes inflammation by regulating NF‐&kgr;B activityRECON enzyme activity controls bacterial survival in immune and non‐immune cells &NA; Bacterial and host cyclic dinucleotides (cdNs) mediate cytosolic immune responses through the STING pathway. McFarland et al. find that the oxidoreductase RECON acts as a sensor for bacterial cdNs. cdN binding to RECON inhibits its enzymatic activity and promotes a proinflammatory, antibacterial state that is distinct from the antiviral state associated with STING activation.

RECON-Dependent Inflammation in Hepatocytes Enhances Listeria monocytogenes Cell-to-Cell Spread

ABSTRACT The oxidoreductase RECON is a high-affinity cytosolic sensor of bacterium-derived cyclic dinucleotides (CDNs). CDN binding inhibits RECON’s enzymatic activity and subsequently promotes inflammation. In this study, we sought to characterize the effects of RECON on the infection cycle of the intracellular bacterium Listeria monocytogenes, which secretes cyclic di-AMP (c-di-AMP) into the cytosol of infected host cells. Here, we report that during infection of RECON-deficient hepatocytes, which exhibit hyperinflammatory responses, L. monocytogenes exhibits significantly enhanced cell-to-cell spread. Enhanced bacterial spread could not be attributed to alterations in PrfA or ActA, two virulence factors critical for intracellular motility and intercellular spread. Detailed microscopic analyses revealed that in the absence of RECON, L. monocytogenes actin tail lengths were significantly longer and there was a larger number of faster-moving bacteria. Complementation experiments demonstrated that the effects of RECON on L. monocytogenes spread and actin tail lengths were linked to its enzymatic activity. RECON enzyme activity suppresses NF-κB activation and is inhibited by c-di-AMP. Consistent with these previous findings, we found that augmented NF-κB activation in the absence of RECON caused enhanced L. monocytogenes cell-to-cell spread and that L. monocytogenes spread correlated with c-di-AMP secretion. Finally, we discovered that, remarkably, increased NF-κB-dependent inducible nitric oxide synthase expression and nitric oxide production were responsible for promoting L. monocytogenes cell-to-cell spread. The work presented here supports a model whereby L. monocytogenes secretion of c-di-AMP inhibits RECON’s enzymatic activity, drives augmented NF-κB activation and nitric oxide production, and ultimately enhances intercellular spread. IMPORTANCE To date, bacterial CDNs in eukaryotes are solely appreciated for their capacity to activate cytosolic sensing pathways in innate immunity. However, it remains unclear whether pathogens that actively secrete CDNs benefit from this process. Here, we provide evidence that secretion of CDNs leads to enhancement of L. monocytogenes cell-to-cell spread. This is a heretofore-unknown role of these molecules and suggests L. monocytogenes may benefit from their secretion in certain contexts. Molecular characterization revealed that, surprisingly, nitric oxide was responsible for the enhanced spread. Pathogens act to prevent nitric oxide production or, like L. monocytogenes, they have evolved to resist its direct antimicrobial effects. This study provides evidence that intracellular bacterial pathogens not only tolerate nitric oxide, which is inevitably encountered during infection, but can also capitalize on the changes this pleiotropic molecule enacts on the host cell. IMPORTANCE To date, bacterial CDNs in eukaryotes are solely appreciated for their capacity to activate cytosolic sensing pathways in innate immunity. However, it remains unclear whether pathogens that actively secrete CDNs benefit from this process. Here, we provide evidence that secretion of CDNs leads to enhancement of L. monocytogenes cell-to-cell spread. This is a heretofore-unknown role of these molecules and suggests L. monocytogenes may benefit from their secretion in certain contexts. Molecular characterization revealed that, surprisingly, nitric oxide was responsible for the enhanced spread. Pathogens act to prevent nitric oxide production or, like L. monocytogenes, they have evolved to resist its direct antimicrobial effects. This study provides evidence that intracellular bacterial pathogens not only tolerate nitric oxide, which is inevitably encountered during infection, but can also capitalize on the changes this pleiotropic molecule enacts on the host cell.