ISG15/USP18/STAT2 is a molecular hub regulating IFN I-mediated control of Dengue and Zika virus replication.

The establishment of a virus infection is the result of the pathogen's ability to replicate in a hostile environment generated by the host's immune system. Here, we found that ISG15 restricts Dengue and Zika viruses' replication through the stabilization of its binding partner USP18. ISG15 expression was necessary to control DV replication driven by both autocrine and paracrine type one interferon (IFN-I) signaling. Moreover, USP18 competes with NS5-mediated STAT2 degradation, a major mechanism for establishment of flavivirus infection. Strikingly, reconstitution of USP18 in ISG15-deficient cells was sufficient to restore the STAT2's stability and restrict virus growth, suggesting that the IFNAR-mediated ISG15 activity is also antiviral. Our results add a novel layer of complexity in the virus/host interaction interface and suggest that NS5 has a narrow window of opportunity to degrade STAT2, therefore suppressing host's IFN-I mediated response and promoting virus replication.

Cannabinoid CB2 receptor agonist reduces local and systemic inflammation associated with pneumonia-induced sepsis in mice.

Sepsis is a severe condition secondary to dysregulated host response to infection leading to tissue damage and organ dysfunction. Cannabinoid CB2 receptor has modulatory effects on the immune response. Therefore, this study investigated the effects of a cannabinoid CB2 receptor agonist on the local and systemic inflammatory process associated with pneumonia-induced sepsis. Pneumonia-induced sepsis was induced in mice by intratracheal inoculation of Klebsiella pneumoniae. Tissue and bronchoalveolar lavage (BAL) were collected 6, 24, or 48 h after surgery. Mice were treated with CB2 agonist (AM1241, 0.3 and 3 mg/kg, i.p.) and several parameters of inflammation were evaluated 24 h after sepsis induction. Polymorphonuclear cell migration to the infectious focus peaked 24 h after pneumonia-induced sepsis induction in male and female animals. Septic male mice presented a significant reduction of cannabinoid CB2 receptor density in the lung tissue after 24 h, which was not observed in females. CB2 expression in BAL macrophages was also reduced in septic animals. Treatment of septic mice with AM1241 reduced cell migration, local infection, myeloperoxidase activity, protein extravasation, and NOS-2 expression in the lungs. In addition, the treatment reduced plasma IL-1ß, increased IL-10 and reduced the severity and mortality of septic animals. These results suggest that AM1241 promotes an interesting balance in the inflammatory response, maintaining lung function and preventing organ injury. Therefore, cannabinoid CB2 receptors are potential targets to control the excessive inflammatory process that occurs in severe conditions, and agonists of these receptors can be considered promising adjuvants in pneumonia-induced sepsis treatment.

PKR-mediated stress response enhances dengue and Zika virus replication.

IMPORTANCE: One of the fundamental features that make viruses intracellular parasites is the necessity to use cellular translational machinery. Hence, this is a crucial checkpoint for controlling infections. Here, we show that dengue and Zika viruses, responsible for nearly 400 million infections every year worldwide, explore such control for optimal replication. Using immunocompetent cells, we demonstrate that arrest of protein translations happens after sensing of dsRNA and that the information required to avoid this blocking is contained in viral 5'-UTR. Our work, therefore, suggests that the non-canonical translation described for these viruses is engaged when the intracellular stress response is activated.

The Endogenous Retinoic Acid Receptor Pathway Is Exploited by Mycobacterium tuberculosis during Infection, Both In Vitro and In Vivo.

Retinoic acid (RA) is a fundamental vitamin A metabolite involved in regulating immune responses through the nuclear RA receptor (RAR) and retinoid X receptor. While performing experiments using THP-1 cells as a model for Mycobacterium tuberculosis infection, we observed that serum-supplemented cultures displayed high levels of baseline RAR activation in the presence of live, but not heat-killed, bacteria, suggesting that M. tuberculosis robustly induces the endogenous RAR pathway. Using in vitro and in vivo models, we have further explored the role of endogenous RAR activity in M. tuberculosis infection through pharmacological inhibition of RARs. We found that M. tuberculosis induces classical RA response element genes such as CD38 and DHRS3 in both THP-1 cells and human primary CD14+ monocytes via a RAR-dependent pathway. M. tuberculosis-stimulated RAR activation was observed with conditioned media and required nonproteinaceous factor(s) present in FBS. Importantly, RAR blockade by (4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthalen-2-yl]ethenyl]benzoic acid), a specific pan-RAR inverse agonist, in a low-dose murine model of tuberculosis significantly reduced SIGLEC-F+CD64+CD11c+high alveolar macrophages in the lungs, which correlated with 2× reduction in tissue mycobacterial burden. These results suggest that the endogenous RAR activation axis contributes to M. tuberculosis infection both in vitro and in vivo and reveal an opportunity for further investigation of new antituberculosis therapies.

DNA-PKcs restricts Zika virus spreading and is required for effective antiviral response.

Zika virus (ZIKV) is a single-strand RNA mosquito-borne flavivirus with significant public health impact. ZIKV infection induces double-strand DNA breaks (DSBs) in human neural progenitor cells that may contribute to severe neuronal manifestations in newborns. The DNA-PK complex plays a critical role in repairing DSBs and in the innate immune response to infection. It is unknown, however, whether DNA-PK regulates ZIKV infection. Here we investigated the role of DNA-PKcs, the catalytic subunit of DNA-PK, during ZIKV infection. We demonstrate that DNA-PKcs restricts the spread of ZIKV infection in human epithelial cells. Increased ZIKV replication and spread in DNA-PKcs deficient cells is related to a notable decrease in transcription of type I and III interferons as well as IFIT1, IFIT2, and IL6. This was shown to be independent of IRF1, IRF3, or p65, canonical transcription factors necessary for activation of both type I and III interferon promoters. The mechanism of DNA-PKcs to restrict ZIKV infection is independent of DSB. Thus, these data suggest a non-canonical role for DNA-PK during Zika virus infection, acting downstream of IFNs transcription factors for an efficient antiviral immune response.

Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges.

Tissue-resident macrophages are the most abundant immune cell population in healthy adipose tissue. Adipose tissue macrophages (ATMs) change during metabolic stress and are thought to contribute to metabolic syndrome. Here, we studied ATM subpopulations in steady state and in response to nutritional and infectious challenges. We found that tissue-resident macrophages from healthy epididymal white adipose tissue (eWAT) tightly associate with blood vessels, displaying very high endocytic capacity. We refer to these cells as vasculature-associated ATMs (VAMs). Chronic high-fat diet (HFD) results in the accumulation of a monocyte-derived CD11c+CD64+ double-positive (DP) macrophage eWAT population with a predominant anti-inflammatory/detoxifying gene profile, but reduced endocytic function. In contrast, fasting rapidly and reversibly leads to VAM depletion, while acute inflammatory stress induced by pathogens transiently depletes VAMs and simultaneously boosts DP macrophage accumulation. Our results indicate that ATM populations dynamically adapt to metabolic stress and inflammation, suggesting an important role for these cells in maintaining tissue homeostasis.

Liver Immune Cells Release Type 1 Interferon Due to DNA Sensing and Amplify Liver Injury from Acetaminophen Overdose.

Hepatocytes may rupture after a drug overdose, and their intracellular contents act as damage-associated molecular patterns (DAMPs) that lead to additional leukocyte infiltration, amplifying the original injury. Necrosis-derived DNA can be recognized as a DAMP, activating liver non-parenchymal cells (NPCs). We hypothesized that NPCs react to DNA by releasing interferon (IFN)-1, which amplifies acetaminophen (APAP)-triggered liver necrosis. We orally overdosed different knockout mouse strains to investigate the pathways involved in DNA-mediated amplification of APAP-induced necrosis. Mice were imaged under intravital confocal microscopy to estimate injury progression, and hepatocytes and liver NPCs were differentially isolated for gene expression assays. Flow cytometry (FACS) using a fluorescent reporter mouse estimated the interferon-beta production by liver leukocytes under different injury conditions. We also treated mice with DNase to investigate the role of necrosis DNA signaling in IFN-1 production. Hepatocytes released a large amount of DNA after APAP overdose, which was not primarily sensed by these cells. However, liver NPCs promptly sensed such environmental disturbances and activated several DNA sensing pathways. Liver NPCs synthesized and released IFN-1, which was associated with concomitant hepatocyte necrosis. Ablation of IFN-1 recognition in interferon α/ß receptor (IFNAR-/-) mice delayed APAP-mediated liver necrosis and dampened IFN-1 sensing pathways. We demonstrated a novel loop involving DNA recognition by hepatic NPCs and additional IFN-1 mediated hepatocyte death.

ISG15-Induced IL-10 Is a Novel Anti-Inflammatory Myeloid Axis Disrupted during Active Tuberculosis.

IFN-stimulated gene 15 (ISG15) deficiency in humans leads to severe IFNopathies and mycobacterial disease, the latter being previously attributed to its extracellular cytokine-like activity. In this study, we demonstrate a novel role for secreted ISG15 as an IL-10 inducer, unique to primary human monocytes. A balanced ISG15-induced monocyte/IL-10 versus lymphoid/IFN-γ expression, correlating with p38 MAPK and PI3K signaling, was found using targeted in vitro and ex vivo systems analysis of human transcriptomic datasets. The specificity and MAPK/PI3K-dependence of ISG15-induced monocyte IL-10 production was confirmed in vitro using CRISPR/Cas9 knockout and pharmacological inhibitors. Moreover, this ISG15/IL-10 axis was amplified in leprosy but disrupted in human active tuberculosis (TB) patients. Importantly, ISG15 strongly correlated with inflammation and disease severity during active TB, suggesting its potential use as a biomarker, awaiting clinical validation. In conclusion, this study identifies a novel anti-inflammatory ISG15/IL-10 myeloid axis that is disrupted in active TB.

Beyond ISGlylation: Functions of Free Intracellular and Extracellular ISG15.

ISG15 is a ubiquitin-like type I IFN-stimulated protein of 15 kDa and is one of the most prominently expressed proteins in viral infections. ISG15 is widely known to be involved in a process called ISGylation, where it binds to over 150 targets from a variety of classes of proteins including central immune signaling pathways such as those mediated by NFκB, JNK, and IRF-3. However, ISG15 also exists in a free form that can act intra- or extracellularly. In vitro and in vivo evidences suggest that free ISG15 play different roles in several cellular processes, from cancer and defense against viral infections to activation of immune cells such as lymphocytes, monocytes, and NK cells. This review discusses the roles of free intracellular and secreted ISG15 approaching questions yet to be answered about the mechanism of action of this protein.

Tests in mice of a dengue vaccine candidate made of chimeric Junin virus-like particles and conserved dengue virus envelope sequences.

Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (≥6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties.

Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.

UNLABELLED: Drug-induced liver injury (DILI) is an important cause of acute liver failure, with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Among these molecules, self-DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, whether DNA released from damaged hepatocytes accumulates into necrotic liver and the impact of its recognition by the immune system remains elusive. Here we show that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release into the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within liver necrotic areas, together with an intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to acetaminophen overdose led to a directional migration of neutrophils to DNA-rich areas, where they exhibit an active patrolling behavior. DNA removal by intravenous DNASE1 injection or ablation of Toll-like receptor 9 (TLR9)-mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment, and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils up-regulated TLR9 expression during acetaminophen-mediated necrosis, and these cells sensed and reacted to extracellular DNA by activating the TLR9/NF-κB pathway. Likewise, adoptive transfer of wild-type neutrophils to TLR9(-/-) mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence. CONCLUSION: Hepatic DNA accumulation is a novel feature of DILI pathogenesis. Blockage of DNA recognition by the innate immune system may constitute a promising therapeutic venue.

Role of IL-4 in an experimental model of encephalitis induced by intracranial inoculation of herpes simplex virus-1 (HSV-1).

Herpes simplex virus-1 (HSV-1) is a pathogen that may cause severe encephalitis in humans. In this study, we aimed to investigate the role of interleukin-4 (IL-4) in a model of HSV-1 brain infection. IL-4 knockout (IL-4-/-) and wild type (WT) C57BL/6 mice were inoculated with 10(4) plaque-forming units of HSV-1 by the intracranial route. Histopathologic analysis revealed a distinct profile of infiltrating cells at 3 days post-infection (dpi). Infected WT mice presented mononuclear inflammatory cells while IL-4-/- mice developed meningoencephalitis with predominance of neutrophils. IL-4-/- mice had diminished leukocyte adhesion at 3 dpi when compared to infected WT animals in intravital microscopy study. Conversely no differences were found in cerebral levels of CXCL1, CXCL9, CCL3, CCL5 and TNF-α between WT and IL-4-/- infected mice. IL-4 may play a role in the recruitment of cells into central nervous system in this acute model of severe encephalitis caused by HSV-1.

Role of IL-4 in an experimental model of encephalitis induced by intracranial inoculation of herpes simplex virus-1 (HSV-1) / Papel da IL-4 em modelo experimental de encefalite induzida pela inoculação intracraniana do herpes simplex vírus-1 (HSV-1)

Herpes simplex virus-1 (HSV-1) is a pathogen that may cause severe encephalitis in humans. In this study, we aimed to investigate the role of interleukin-4 (IL-4) in a model of HSV-1 brain infection. IL-4 knockout (IL-4-/-) and wild type (WT) C57BL/6 mice were inoculated with 10(4) plaque-forming units of HSV-1 by the intracranial route. Histopathologic analysis revealed a distinct profile of infiltrating cells at 3 days post-infection (dpi). Infected WT mice presented mononuclear inflammatory cells while IL-4-/- mice developed meningoencephalitis with predominance of neutrophils. IL-4-/- mice had diminished leukocyte adhesion at 3 dpi when compared to infected WT animals in intravital microscopy study. Conversely no differences were found in cerebral levels of CXCL1, CXCL9, CCL3, CCL5 and TNF-α between WT and IL-4-/- infected mice. IL-4 may play a role in the recruitment of cells into central nervous system in this acute model of severe encephalitis caused by HSV-1.

O vírus herpes simplex-1 (HSV-1) é um patógeno que pode causar encefalite grave em humanos. Neste estudo, buscamos investigar o papel da interleucina-4 (IL-4) no modelo de infecção intracerebral por HSV-1. Camundongos C57BL/6 selvagens (WT) e deficientes no gene IL-4 (IL-4-/-) foram inoculados com 10(4) unidades formadoras de placas de HSV-1 por via intracraniana. A análise histopatológica revelou um padrão distinto de infiltrado leucocitário. Camundongos WT infectados apresentaram infiltrado de células mononucleares, enquanto camundongos IL-4-/- desenvolveram meningoencefalite com predomínio de neutrófilos 3 dias pós-infecção (dpi). Animais IL-4-/- tiveram menor adesão de leucócitos 3 dpi quando comparados aos animais WT infectados à microscopia intravital. Em contrapartida, não foram encontradas diferenças nos níveis cerebrais de CXCL1, CXCL9, CCL3, CCL5 e TNF-α entre camundongos WT e IL-4-/- infectados. Esses resultados sugerem que IL-4 pode desempenhar um papel no recrutamento de células no sistema nervoso central neste modelo agudo de encefalite grave causada pelo HSV-1.

Toll-like receptor (TLR) 2 and TLR9 expressed in trigeminal ganglia are critical to viral control during herpes simplex virus 1 infection.

Herpes simplex virus 1 (HSV-1) is a neurotropic DNA virus that is responsible for several clinical manifestations in humans, including encephalitis. HSV-1 triggers toll-like receptors (TLRs), which elicit cytokine production. Viral multiplication and cytokine expression in C57BL/6 wild-type (WT) mice infected with HSV-1 were evaluated. Virus was found in the trigeminal ganglia (TG), but not in the brains of animals without signs of encephalitis, between 2 and 6 days postinfection (d.p.i.). Cytokine expression in the TG peaked at 5 d.p.i. TLR9-/- and TLR2/9-/- mice were more susceptible to the virus, with 60% and 100% mortality, respectively, as opposed to 10% in the WT and TLR2-/- mice. Increased levels of both CXCL10/IP-10 and CCL2/MCP-1, as well as reduced levels of interferon-γ and interleukin 1-ß transcripts, measured in both the TG and brains at 5 d.p.i., and the presence of virus in the brain were correlated with total mortality in TLR2/9-/- mice. Cytokine alterations in TLR2/9-/- mice coincided with histopathological changes in their brains, which did not occur in WT and TLR2-/- mice and occurred only slightly in TLR9-/- mouse brain. Increased cellularity, macrophages, CD8 T cells producing interferon-γ, and expression levels of TLR2 and TLR9 were detected in the TG of WT-infected mice. We hypothesize that HSV-1 infection is controlled by TLR-dependent immune responses in the TG, which prevent HSV-1 encephalitis.

TNFR1 plays a critical role in the control of severe HSV-1 encephalitis.

Herpes simplex virus-1 (HSV-1) is a pathogen for humans that may cause severe encephalitis. Tumor necrosis factor alpha (TNF-alpha) plays a role in several viral diseases of the central nervous system (CNS). The classic proinflammatory activities of TNF-alpha are mediated mainly through activation of the receptor 1 for TNF-alpha (TNFR1). However, when HSV-1 is inoculated in the periphery, TNF-alpha seems to protect C57Bl/6 mice against encephalitis by a mechanism independent of TNFR1. This study aims to investigate the role of TNFR1 in HSV-1 encephalitis induced by the inoculation of the virus into the brain. Wild-type C57BL/6 (WT) and TNFR1(-/-) were inoculated with 10(2) plaque-forming units of HSV-1 by the intracranial route. Infection with HSV-1 was lethal in TNFR1(-/-) mice in early times after infection. TNFR1(-/-) mice had reduced expression of the chemokines CCL3 and CCL5, and decreased leukocyte adhesion in the brain vasculature compared to WT mice 4 days post-infection (dpi). At this time point TNFR1(-/-) infected mice also had higher HSV-1 viral replication and more injuries in the brain, especially in the hippocampus. In conclusion, TNFR1 seems to play a relevant role in the control of viral replication in the CNS when HSV-1 is inoculated by intracranial route.

The chemokine CCL5 is essential for leukocyte recruitment in a model of severe Herpes simplex encephalitis.

The Herpes simplex virus-1 (HSV-1) is responsible for several clinical manifestations in humans, including encephalitis. To induce encephalitis, C57BL/6 mice were inoculated with 10(4) plaque-forming cells of HSV-1 by the intracranial route. Met-RANTES (regulated upon activation, normal T cell expressed and presumably secreted) (10 microg/mouse), a CC chemokine family receptor (CCR)1 and CCR5 antagonist, was given subcutaneously the day before, immediately after, and at days 1, 2, and 3 after infection. Treatment with Met-RANTES had no effect on the viral titers. In contrast, intravital microscopy revealed that treatment with Met-RANTES decreased the number of leukocytes adherent to the pial microvasculature at days 1 and 3 after infection. The levels of the chemokines CCL3, CCL5, CXCL1, and CXCL9 increased after infection and were enhanced further by the treatment with Met-RANTES. Treatment with a polyclonal anti-CCL5 antibody 2 h before the intravital microscopy decreased leukocyte adhesion in the microcirculation of infected mice. In conclusion, CCL5, a chemokine that binds to CCR1 and CCR5, is essential for leukocyte adhesion during HSV-1 encephalitis. However, blocking of CCR1 and CCR5 did not affect HSV-1 replication, suggesting that other immune mechanisms are involved in the process of infection control.

Traffic of leukocytes in the central nervous system is associated with chemokine up-regulation in a severe model of herpes simplex encephalitis: an intravital microscopy study.

Herpes simplex virus type 1 (HSV-1) is a human pathogen that may cause severe encephalitis. The development of experimental models of HSV-1 encephalitis is relevant for the comprehension of the immune mechanisms involved in this infection. C57BL/6 mice were inoculated intracranially with 10(4) PFU of neurotropic HSV-1. All animals developed signs of encephalitis and died until day 6 post-infection (pi). Using intravital microscopy, we demonstrated increased leukocyte rolling and adhesion in the brain microvasculature of infected mice at days 1, 3 and 5 pi. The infection was followed by a significant increase in chemokine levels, including CCL2, CCL3, CCL5, CXCL1 and CXCL9. TNF-alpha also showed a significant increase at day 3 pi. Histological analyses demonstrated diffuse meningoencephalitis characterized mainly by mononuclear cell infiltrates. The present model of HSV-1 encephalitis exhibits high mortality in the very first days of infection. Accordingly, there were increased rolling and adhesion of leukocytes along the brain endothelium wall and a high expression of chemokines in the central nervous system. These results corroborate the role of chemokines in leukocyte recruitment following HSV-1 infection in the central nervous system.

Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii.

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.