Christian K. Holm

Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

The innate immune system senses infection by detecting either evolutionarily conserved molecules essential for the survival of microbes or the abnormal location of molecules. Here we demonstrate the existence of a previously unknown innate detection mechanism induced by fusion between viral envelopes and target cells. Virus-cell fusion specifically stimulated a type I interferon response with expression of interferon-stimulated genes, in vivo recruitment of leukocytes and potentiation of signaling via Toll-like receptor 7 (TLR7) and TLR9. The fusion-dependent response was dependent on the stimulator of interferon genes STING but was independent of DNA, RNA and viral capsid. We suggest that membrane fusion is sensed as a danger signal with potential implications for defense against enveloped viruses and various conditions of giant-cell formation.

Proteasomal Degradation of Herpes Simplex Virus Capsids in Macrophages Releases DNA to the Cytosol for Recognition by DNA Sensors

The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9 and that nonimmune tissue cells can sense herpesvirus DNA in the nucleus. However, it remains unknown how and where myeloid cells, such as macrophages and conventional dendritic cells, detect infections with herpesviruses. In this study, we demonstrate that the HSV-1 capsid was ubiquitinated in the cytosol and degraded by the proteasome, hence releasing genomic DNA into the cytoplasm for detection by DNA sensors. In this context, the DNA sensor IFN-γ–inducible 16 is important for induction of IFN-β in human macrophages postinfection with HSV-1 and CMV. Viral DNA localized to the same cytoplasmic regions as did IFN-γ–inducible 16, with DNA sensing being independent of viral nuclear entry. Thus, proteasomal degradation of herpesvirus capsids releases DNA to the cytoplasm for recognition by DNA sensors.

TLR3 deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of CNS infection in mice

Herpes simplex viruses (HSVs) are highly prevalent neurotropic viruses. While they can replicate lytically in cells of the epithelial lineage, causing lesions on mucocutaneous surfaces, HSVs also establish latent infections in neurons, which act as reservoirs of virus for subsequent reactivation events. Immunological control of HSV involves activation of innate immune pattern-recognition receptors such as TLR3, which detects double-stranded RNA and induces type I IFN expression. Humans with defects in the TLR3/IFN pathway have an elevated susceptibility to HSV infections of the CNS. However, it is not known what cell type mediates the role of TLR3 in the immunological control of HSV, and it is not known whether TLR3 sensing occurs prior to or after CNS entry. Here, we show that in mice TLR3 provides early control of HSV-2 infection immediately after entry into the CNS by mediating type I IFN responses in astrocytes. Tlr3-/- mice were hypersusceptible to HSV-2 infection in the CNS after vaginal inoculation. HSV-2 exhibited broader neurotropism in Tlr3-/- mice than it did in WT mice, with astrocytes being most abundantly infected. Tlr3-/- mice did not exhibit a global defect in innate immune responses to HSV, but astrocytes were defective in HSV-induced type I IFN production. Thus, TLR3 acts in astrocytes to sense HSV-2 infection immediately after entry into the CNS, possibly preventing HSV from spreading beyond the neurons mediating entry into the CNS.

Increased Interleukin 21 (IL-21) and IL-23 Are Associated with Increased Disease Activity and with Radiographic Status in Patients with Early Rheumatoid Arthritis

Objective. To investigate the levels of the T helper (Th)17-related cytokines interleukin 17A (IL-17A), IL-21, and IL-23 and their association with disease activity in rheumatoid arthritis (RA). Methods. In a longitudinal sample set from patients with early RA (< 6 months; n = 40), we measured the plasma cytokine levels of IL-17A, IL-21, and IL-23 and analyzed for correlation with disease activity in 28 joints (Disease Activity Score 28-joint count; DAS28), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and total Sharp score (TSS). In a transverse sample set of patients with chronic RA (> 8 years), using paired peripheral blood mononuclear cells and synovial fluid mononuclear cells, we investigated the cellular expression of IL-17A, IL-21, and IL-23R. Results. Patients with early-stage RA had significantly increased plasma levels of IL-21 and IL-23, but not IL-17A, compared to patients with chronic RA and healthy volunteer controls. Plasma levels of IL-21 and IL-23 after 12 months of treatment correlated with DAS28 and ESR, but not to TSS. Changes in IL-23 plasma levels from time of diagnosis to 12 months correlated with change in DAS28 and with TSS scores at 2 years. The numbers of CD4+ T cells producing IL-21 were significantly increased in the synovial fluid of patients with chronic RA, with only marginal coexpression of IL-21 and IL-17A. Conclusion. Our results show a significant association between plasma levels of IL-21 and IL-23 and disease activity in RA, supporting the hypothesis that IL-21 and IL-23 are important pathogenic factors of this disease.

Activation of Autophagy by α-Herpesviruses in Myeloid Cells Is Mediated by Cytoplasmic Viral DNA through a Mechanism Dependent on Stimulator of IFN Genes

Autophagy has been established as a player in host defense against viruses. The mechanisms by which the host induces autophagy during infection are diverse. In the case of HSV type 1 (HSV-1), dsRNA-dependent protein kinase is essential for induction of autophagy in fibroblasts through phosphorylation of eukaryotic initiation factor 2α (eIF2α). HSV-1 counteracts autophagy via ICP34.5, which dephosphorylates eIF2α and inhibits Beclin 1. Investigation of autophagy during HSV-1 infection has largely been conducted in permissive cells, but recent work suggests the existence of a eIF2α-independent autophagy-inducing pathway in nonpermissive cells. To clarify and further characterize the existence of a novel autophagy-inducing pathway in nonpermissive cells, we examined different HSV and cellular components in murine myeloid cells for their role in autophagy. We demonstrate that HSV-1–induced autophagy does not correlate with phosphorylation of eIF2α, is independent of functional dsRNA-dependent protein kinase, and is not antagonized by ICP34.5. Autophagy was activated independent of viral gene expression, but required viral entry. Importantly, we found that the presence of genomic DNA in the virion was essential for induction of autophagy and, conversely, that transfection of HSV-derived DNA induced microtubule-associated protein 1 L chain II formation, a marker of autophagy. This occurred through a mechanism dependent on stimulator of IFN genes, an essential component for the IFN response to intracellular DNA. Finally, we observed that HSV-1 DNA was present in the cytosol devoid of capsid material following HSV-1 infection of dendritic cells. Thus, our data suggest that HSV-1 genomic DNA induces autophagy in nonpermissive cells in a stimulator of IFN gene-dependent manner.

Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses

Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV.

IFI16 is required for DNA sensing in human macrophages by promoting production and function of cGAMP

Innate immune activation by macrophages is an essential part of host defence against infection. Cytosolic recognition of microbial DNA in macrophages leads to induction of interferons and cytokines through activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Other host factors, including interferon-gamma inducible factor 16 (IFI16), have been proposed to contribute to immune activation by DNA. However, their relation to the cGAS-STING pathway is not clear. Here, we show that IFI16 functions in the cGAS-STING pathway on two distinct levels. Depletion of IFI16 in macrophages impairs cGAMP production on DNA stimulation, whereas overexpression of IFI16 amplifies the function of cGAS. Furthermore, IFI16 is vital for the downstream signalling stimulated by cGAMP, facilitating recruitment and activation of TANK-binding kinase 1 in STING complex. Collectively, our results suggest that IFI16 is essential for efficient sensing and signalling upon DNA challenge in macrophages to promote interferons and antiviral responses.

TLR3 Ligand Polyinosinic:Polycytidylic Acid Induces IL-17A and IL-21 Synthesis in Human Th Cells

TLR3 and TLR9 recognize the pathogen-associated microbial patterns dsRNA and unmethylated DNA, respectively. The recent discovery that these receptors also recognize endogenous ligands from necrotic material has drawn increased attention to their involvement in autoimmunity. Th cell cytokines IL-17A and IL-21 have been assigned with pivotal roles in the regulation of such autoimmune diseases. IL-17A is the hallmark cytokine of the recently discovered proinflammatory Th cell subset TH17. By contrast, the expression of IL-21 does not seem to be limited to a single distinct Th cell subset. We investigated the expression of IL-17A and IL-21 in human CD4+ T cells in response to stimulation with the TLR3 ligand polyinosinic:polycytidylic acid (poly(I:C)) and the TLR9 ligand CpG. We discovered that poly(I:C) induced synthesis of both IL-17A and IL-21. Moreover, we found that poly(I:C) was able to drive the differentiation of naive Th cells into an IL-21 but not into an IL-17A-producing phenotype and did this without affecting the levels of transcription factors T-bet, GATA-3, or retinoic acid receptor-related orphan receptor C. Finally, we found that the IL-21-producing cells that were differentiated in response to poly(I:C) expressed the chemokine receptor CXCR3, which is important in the recruitment of T cells into inflamed joints in rheumatoid arthritis. This is the first report to show that the TLR3 ligand poly(I:C) can directly induce the synthesis of IL-17A and IL-21 and drive differentiation of human naive CD4+ T cells.

DNA recognition in Immunity and Disease

Great progress has been made in understanding how immune cells detect microbial pathogens. An area that has received particular attention is nucleic acid sensing where RNA and DNA sensing machineries have been uncovered. For DNA, TLR9 in endosomes and numerous cytoplasmic DNA binding proteins have been identified. Several of these have been proposed to couple DNA recognition to induction of type I IFNs, pro-inflammatory cytokines and/or caspase-1 activation. Given the ubiquitous expression of many of these DNA binding proteins and the significant potential for endogenous DNA to engage these molecules, it is important that DNA recognition is tightly regulated. A better understanding of DNA recognition pathways can provide new insights into infectious, inflammatory and autoimmune diseases.

T Cells Detect Intracellular DNA but Fail to Induce Type I IFN Responses: Implications for Restriction of HIV Replication

HIV infects key cell types of the immune system, most notably macrophages and CD4+ T cells. Whereas macrophages represent an important viral reservoir, activated CD4+ T cells are the most permissive cell types supporting high levels of viral replication. In recent years, it has been appreciated that the innate immune system plays an important role in controlling HIV replication, e.g. via interferon (IFN)-inducible restriction factors. Moreover, innate immune responses are involved in driving chronic immune activation and the pathogenesis of progressive immunodeficiency. Several pattern recognition receptors detecting HIV have been reported, including Toll-like receptor 7 and Retinoic-inducible gene-I, which detects viral RNA. Here we report that human primary T cells fail to induce strong IFN responses, despite the fact that this cell type does express key molecules involved in DNA signaling pathways. We demonstrate that the DNA sensor IFI16 migrates to sites of foreign DNA localization in the cytoplasm and recruits the signaling molecules stimulator of IFN genes and Tank-binding kinase, but this does not result in expression of IFN and IFN-stimulated genes. Importantly, we show that cytosolic DNA fails to affect HIV replication. However, exogenous treatment of activated T cells with type I IFN has the capacity to induce expression of IFN-stimulated genes and suppress HIV replication. Our data suggest the existence of an impaired DNA signaling machinery in T cells, which may prevent this cell type from activating cell-autonomous anti-HIV responses. This phenomenon could contribute to the high permissiveness of CD4+ T cells for HIV-1.