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.

The annotation of the mammalian protein coding genome is incomplete. Arbitrary open reading frame (ORF) size restriction and the absolute requirement for a methionine codon as the sole initiator of translation, have constrained identification of potentially important transcripts with non-canonical protein coding potential1,2. Using unbiased transcriptomic approaches in macrophages responding to bacterial infection, we show widespread ribosome association with a large number of RNAs that were previously annotated as “non-protein coding”. Although the ability of such non-canonical ORFs to encode functional protein is controversial3,4, we identify a plethora of novel short and non-ATG initiated ORFs with the ability to generate stable and spatially distinct proteins. Importantly, we show that the translation of a novel ORF ‘hidden’ within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. Together this work expands our interpretation of the protein coding genome and demonstrates the critical nature of proteinaceous products generated from non-canonical ORFs to the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein coding genes in immunity and disease.

Highlights d Epithelial and immune cell IL-18 are not required to combat S. typhimurium d Enteric neurons express IL-18 d Enteric neuronal IL-18 controls goblet cell antimicrobial protein expression d Neuronal IL-18 directs killing of enteric bacterial pathogens

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. www.eji-journal.eu Eur. J. Immunol. 2013. 43: 1896-1906 Innate immunity Keywords 1897Several structurally distinct classes of CpG ODN have been described. Those of the "K" class (also referred to as "B" class) are characterized by their ability to stimulate human pDCs to secrete pro-inflammatory cytokines such as IL-6 and TNF-α. Clinical trials of "K" ODN show promise for the treatment of cancer, allergy, and infectious disease [4,5]. Identifying the signaling pathways triggered when human pDCs are stimulated by "K" ODN is, thus, of clinical relevance.pDCs are a major source of type I IFNs and various proinflammatory cytokines [6,7]. We recently showed that "K" ODN induced a rapid but short-lived pulse of IFN-β. This led to the upregulation of IFN-stimulated genes known to enhance host resistance to virus infection [8][9][10][11][12]. "K" ODN also upregulate the expression of IL-6, which contributes to the activation of multiple pro-inflammatory genes and the subsequent shift from innate to adaptive immunity [8][9][10][11][12]. The current study was designed to identify the key signaling pathway(s) responsible for the upregulation of IFN-β and IL-6, as these would provide important insights into the pattern of "K" ODN mediated activation of human pDCs.Previous efforts to examine the signaling cascade(s) triggered by the interaction of TLR9 with CpG DNA focused primarily on murine myeloid DCs (mDCs), monocytes, and macrophages [13]. Studies examining the regulation of IL-6 by "K" ODN in mice documented a role for interferon regulatory factor-5 (IRF-5) and the binding of the NF-κB transcription factors p50/p65/c-Rel to the IL-6 promoter [14,15], while IRF-1 was identified as a key mediator of IFN-β induction by "K" ODN [16]. Yet, there is reason to question whether those findings are applicable to human pDC, as there are fundamental differences in the signaling cascades utilized by mDCs versus pDCs and mice versus humans [...

Psoriasis pathology is driven by the type 3 cytokines IL-17 and Il-22, but little is understood about the dynamics that initiate alterations in tissue homeostasis. Here, we use mouse models, single-cell RNA-seq (scRNA-seq), computational inference and cell lineage mapping to show that psoriasis induction reconfigures the functionality of skin-resident ILCs to initiate disease. Tissue-resident ILCs amplified an initial IL-23 trigger and were sufficient, without circulatory ILCs, to drive pathology, indicating that ILC tissue remodeling initiates psoriasis. Skin ILCs expressed type 2 cytokines IL-5 and IL-13 in steady state, but were epigenetically poised to become ILC3-like cells. ScRNA-seq profiles of ILCs from psoriatic and naïve skin of wild type (WT) and Rag1 -/mice form a dense continuum, consistent with this model of fluid ILC states. We inferred biological "topics" underlying these states and their relative importance in each cell with a generative model of latent Dirichlet allocation, showing that ILCs from untreated skin span a spectrum of states, including a naïve/quiescent-like state and one expressing the Cd74 and Il13 but little Il5. Upon disease induction, this spectrum shifts, giving rise to a greater proportion of classical Il5-and Il13expressing "ILC2s" and a new, mixed ILC2/ILC3-like subset, expressing Il13, Il17, and Il22. Using these key topics, we related the cells through transitions, revealing a quiescence-ILC2-ILC3s state trajectory. We demonstrated this plasticity in vivo, combining an IL-5 fate mouse with IL-17A and IL-22 reporters, validating the transition of IL-5-producing ILC2s to IL-22-and IL-17A-producing cells during disease initiation. Thus, steady-state skin ILCs are actively repressed and cued for a plastic, type 2 response, which, upon induction, morphs into a type 3 response that drives psoriasis. This suggests a general model where specific immune activities are primed in healthy tissue, dynamically adapt to provocations, and left unchecked, drive pathological remodeling.

Hong et al. show that IFNλ4 exhibits similar antiviral activity to IFNλ3. Humans deploy several mechanisms to limit expression of functional IFNλ4 through noncoding splice variants and nonfunctional protein isoforms.

24 ^Correspondence to: A.R aregev@broadinstitute.com and R.A.F richard.flavell@yale.edu 25 Psoriasis pathology is driven by the type 3 cytokines IL-17 and Il-22, but little is understood 26 about the dynamics that initiate alterations in tissue homeostasis. Here, we use mouse 27 models, single-cell RNA-seq (scRNA-seq), computational inference and cell lineage mapping 28 to show that psoriasis induction reconfigures the functionality of skin-resident ILCs to 29 initiate disease. Tissue-resident ILCs amplified an initial IL-23 trigger and were sufficient, 30 without circulatory ILCs, to drive pathology, indicating that ILC tissue remodeling initiates 31 psoriasis. Skin ILCs expressed type 2 cytokines IL-5 and IL-13 in steady state, but were 32 epigenetically poised to become ILC3-like cells. ScRNA-seq profiles of ILCs from psoriatic 33 and naïve skin of wild type (WT) and Rag1 -/mice form a dense continuum, consistent with 34 this model of fluid ILC states. We inferred biological "topics" underlying these states and 35 their relative importance in each cell with a generative model of latent Dirichlet allocation, 36 showing that ILCs from untreated skin span a spectrum of states, including a 37 naïve/quiescent-like state and one expressing the Cd74 and Il13 but little Il5. Upon disease 38 induction, this spectrum shifts, giving rise to a greater proportion of classical Il5-and Il13-39 expressing "ILC2s" and a new, mixed ILC2/ILC3-like subset, expressing Il13, Il17, and Il22. 40 Using these key topics, we related the cells through transitions, revealing a quiescence-ILC2-41ILC3s state trajectory. We demonstrated this plasticity in vivo, combining an IL-5 fate mouse 42 with IL-17A and IL-22 reporters, validating the transition of IL-5-producing ILC2s to IL-43 22-and IL-17A-producing cells during disease initiation. Thus, steady-state skin ILCs are 44 actively repressed and cued for a plastic, type 2 response, which, upon induction, morphs 45 into a type 3 response that drives psoriasis. This suggests a general model where specific 46 immune activities are primed in healthy tissue, dynamically adapt to provocations, and left 47 G). This was also observed in mice treated with FTY720 during the primary injection (Fig. S1E), 129suggesting that the plastic psoriatic response is not due to ILC recruitment. 130 131We hypothesized that this plasticity may be encoded epigenetically. To test this hypothesis, we 132 profiled sorted total skin ILC populations from naïve mice by ATAC-seq. We observed the 133 expected open chromatin signature at the TSS of Gata3, Il5 and Il13 and not at the TSS of Tbx21 134or Rorc, which encode T-bet and Rorgt, the hallmark transcription factors (TFs) of ILC1s and 135

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 patients1–3. 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 hepatocytes4. 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.