Immune Profiling of Vulvar Squamous Cell Cancer Discovers a Macrophage-rich Subtype Associated with Poor Prognosis.

The incidence rates of vulvar squamous cell cancer (VSCC) have increased over the past decades, requiring personalized oncologic approaches. Currently, lymph node involvement is a key factor in determining prognosis and treatment options. However, there is a need for additional immune-related biomarkers to provide more precise treatment and prognostic information. Here, we used IHC and expression data to characterize immune cells and their spatial distribution in VSCC. Hierarchical clustering analysis identified distinct immune subtypes, of which the macrophage-rich subtype was associated with adverse outcome. This is consistent with our findings of increased lymphogenesis, lymphatic invasion, and lymph node involvement associated with high macrophage infiltration. Further in vitro studies showed that VSCC-associated macrophages expressed VEGF-A and subsequently induced VEGF-A in the VSCC cell line A-431, providing experimental support for a pro-lymphangiogenic role of macrophages in VSCC. Taken together, immune profiling in VSCC revealed tumor processes, identified a subset of patients with adverse outcome, and provided a valuable biomarker for risk stratification and therapeutic decision making for anti-VEGF treatment, ultimately contributing to the advancement of precision medicine in VSCC. SIGNIFICANCE: Immunoprofiling in VSCC reveals subtypes with distinct clinical and biological behavior. Of these, the macrophage-rich VSCC subtype is characterized by poor clinical outcome and increased VEGF-A expression, providing a biomarker for risk stratification and therapeutic sensitivity.

Chronic endoplasmic reticulum stress in myotonic dystrophy type 2 promotes autoimmunity via mitochondrial DNA release.

Myotonic dystrophy type 2 (DM2) is a tetranucleotide CCTG repeat expansion disease associated with an increased prevalence of autoimmunity. Here, we identified an elevated type I interferon (IFN) signature in peripheral blood mononuclear cells and primary fibroblasts of DM2 patients as a trigger of chronic immune stimulation. Although RNA-repeat accumulation was prevalent in the cytosol of DM2-patient fibroblasts, type-I IFN release did not depend on innate RNA immune sensors but rather the DNA sensor cGAS and the prevalence of mitochondrial DNA (mtDNA) in the cytoplasm. Sublethal mtDNA release was promoted by a chronic activation of the ATF6 branch of the unfolded protein response (UPR) in reaction to RNA-repeat accumulation and non-AUG translated tetrapeptide expansion proteins. ATF6-dependent mtDNA release and resulting cGAS/STING activation could also be recapitulated in human THP-1 monocytes exposed to chronic endoplasmic reticulum (ER) stress. Altogether, our study demonstrates a novel mechanism by which large repeat expansions cause chronic endoplasmic reticulum stress and associated mtDNA leakage. This mtDNA is, in turn, sensed by the cGAS/STING pathway and induces a type-I IFN response predisposing to autoimmunity. Elucidating this pathway reveals new potential therapeutic targets for autoimmune disorders associated with repeat expansion diseases.

A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA.

RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.

C-terminal variants in CDC42 drive type I interferon-dependent autoinflammation in NOCARH syndrome reversible by ruxolitinib.

C-terminal variants in CDC42 encoding cell division control protein 42 homolog underlie neonatal-onset cytopenia, autoinflammation, rash, and hemophagocytic lymphohistiocytosis (NOCARH). Pyrin inflammasome hyperactivation has been shown to contribute to disease pathophysiology. However, mortality of NOCARH patients remains high despite inflammasome-focused treatments. Here, we demonstrate in four NOCARH patients from three families that cell-intrinsic activation of type I interferon (IFN) is a previously unrecognized driver of autoinflammation in NOCARH. Our data show that aberrant innate immune activation is caused by sensing of cytosolic nucleic acids released from mitochondria, which exhibit disturbances in integrity and dynamics due to CDC42 dysfunction. In one of our patients, treatment with the Janus kinase inhibitor ruxolitinib led to complete remission, indicating that inhibition of type I IFN signaling may have an important role in the management of autoinflammation in patients with NOCARH.

Inhibition of cellular RNA methyltransferase abrogates influenza virus capping and replication.

Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

Complexing CpG adjuvants with cationic liposomes enhances vaccine-induced formation of liver TRM cells.

Tissue resident memory T cells (TRM cells) can provide effective tissue surveillance and can respond rapidly to infection. Vaccination strategies aimed at generating TRM cells have shown promise against a range of pathogens. We have previously shown that the choice of adjuvant critically influences CD8+ TRM cell formation in the liver. However, the range of adjuvants tested was limited. Here, we assessed the ability of a broad range of adjuvants stimulating membrane (TLR4), endosomal (TLR3, TLR7 and TLR9) and cytosolic (cGAS, RIG-I) pathogen recognition receptors for their capacity to induce CD8+ TRM formation in a subunit vaccination model. We show that CpG oligodeoxynucleotides (ODN) remain the most efficient inducers of liver TRM cells among all adjuvants tested. Moreover, their combination with the cationic liposome DOTAP further enhances the potency, particularly of the class B ODN CpG 1668 and the human TLR9 ligand CpG 2006 (CpG 7909). This study informs the design of efficient liver TRM-based vaccines for their potential translation.

Deficiency for SAMHD1 activates MDA5 in a cGAS/STING-dependent manner.

Defects in nucleic acid metabolizing enzymes can lead to spontaneous but selective activation of either cGAS/STING or RIG-like receptor (RLR) signaling, causing type I interferon-driven inflammatory diseases. In these pathophysiological conditions, activation of the DNA sensor cGAS and IFN production are linked to spontaneous DNA damage. Physiological, or tonic, IFN signaling on the other hand is essential to functionally prime nucleic acid sensing pathways. Here, we show that low-level chronic DNA damage in mice lacking the Aicardi-Goutières syndrome gene SAMHD1 reduced tumor-free survival when crossed to a p53-deficient, but not to a DNA mismatch repair-deficient background. Increased DNA damage did not result in higher levels of type I interferon. Instead, we found that the chronic interferon response in SAMHD1-deficient mice was driven by the MDA5/MAVS pathway but required functional priming through the cGAS/STING pathway. Our work positions cGAS/STING upstream of tonic IFN signaling in Samhd1-deficient mice and highlights an important role of the pathway in physiological and pathophysiological innate immune priming.

SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA.

Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here, we show that human SAM-domain- and HD-domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3'exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic-acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.

Monocyte-derived macrophages aggravate pulmonary vasculitis via cGAS/STING/IFN-mediated nucleic acid sensing.

Autoimmune vasculitis is a group of life-threatening diseases, whose underlying pathogenic mechanisms are incompletely understood, hampering development of targeted therapies. Here, we demonstrate that patients suffering from anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) showed increased levels of cGAMP and enhanced IFN-I signature. To identify disease mechanisms and potential therapeutic targets, we developed a mouse model for pulmonary AAV that mimics severe disease in patients. Immunogenic DNA accumulated during disease onset, triggering cGAS/STING/IRF3-dependent IFN-I release that promoted endothelial damage, pulmonary hemorrhages, and lung dysfunction. Macrophage subsets played dichotomic roles in disease. While recruited monocyte-derived macrophages were major disease drivers by producing most IFN-ß, resident alveolar macrophages contributed to tissue homeostasis by clearing red blood cells and limiting infiltration of IFN-ß-producing macrophages. Moreover, pharmacological inhibition of STING, IFNAR-I, or its downstream JAK/STAT signaling reduced disease severity and accelerated recovery. Our study unveils the importance of STING/IFN-I axis in promoting pulmonary AAV progression and identifies cellular and molecular targets to ameliorate disease outcomes.

Induction of Interferon-Stimulated Genes Correlates with Reduced Growth of Influenza A Virus in Lungs after RIG-I Agonist Treatment of Ferrets.

Intracellular RIG-I receptors represent key innate sensors of RNA virus infection, and RIG-I activation results in the induction of hundreds of host effector genes, including interferon-stimulated genes (ISGs). Synthetic RNA agonists targeting RIG-I have shown promise as antivirals against a broad spectrum of viruses, including influenza A virus (IAV), in both in vitro and mouse models of infection. Herein, we demonstrate that treatment of a ferret airway epithelial (FRL) cell line with a RIG-I agonist rapidly and potently induced expression of a broad range of ISGs and resulted in potent inhibition of growth of different IAV strains. In ferrets, a single intravenous injection of RIG-I agonist was associated with upregulated ISG expression in peripheral blood mononuclear cells and lung tissue, but not in nasal tissues. In a ferret model of viral contact transmission, a single treatment of recipient animals 24 h prior to cohousing with IAV-infected donors did not reduce virus transmission and shedding but did result in reduced lung virus titers 6 days after treatment. A single treatment of the IAV-infected donor animals also resulted in reduced virus titers in the lungs 2 days later. Thus, a single intravenous treatment with RIG-I agonist prior to infection or to ferrets with an established IAV infection can reduce virus growth in the lungs. These findings support further development of RIG-I agonists as effective antiviral treatments to limit the impact of IAV infections, particularly in reducing virus replication in the lower airways. IMPORTANCE RIG-I agonists have shown potential as broad-spectrum antivirals in vitro and in mouse models of infection. However, their antiviral potential has not been reported in outbred animals such as ferrets, which are widely regarded as the gold standard small animal model for human IAV infections. Herein, we demonstrate that RIG-I agonist treatment of a ferret airway cell line resulted in ISG induction and inhibition of a broad range of human influenza viruses. A single intravenous treatment of ferrets also resulted in systemic induction of ISGs, including in lung tissue, and when delivered to animals prior to IAV exposure or to animals with established IAV infection treatment resulted in reduced virus replication in the lungs. These data demonstrate the effectiveness of single RIG-I treatment against IAV in the ferret model and highlight the importance of future studies to optimize treatment regimens and delivery routes to maximize their ability to ameliorate IAV infections.

Retinoic Acid-Inducible Gene I Activation Inhibits Human Respiratory Syncytial Virus Replication in Mammalian Cells and in Mouse and Ferret Models of Infection.

Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid-inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an "antiviral state." In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represent a promising antiviral drug for RSV prophylaxis.

Expression of a Functional Mx1 Protein Is Essential for the Ability of RIG-I Agonist Prophylaxis to Provide Potent and Long-Lasting Protection in a Mouse Model of Influenza A Virus Infection.

RIG-I is an innate sensor of RNA virus infection and its activation induces interferon-stimulated genes (ISGs). In vitro studies using human cells have demonstrated the ability of synthetic RIG-I agonists (3pRNA) to inhibit IAV replication. However, in mouse models of IAV the effectiveness of 3pRNA reported to date differs markedly between studies. Myxoma resistance (Mx)1 is an ISG protein which mediates potent anti-IAV activity, however most inbred mouse strains do not express a functional Mx1. Herein, we utilised C57BL/6 mice that do (B6.A2G-Mx1) and do not (B6-WT) express functional Mx1 to assess the ability of prophylactic 3pRNA treatment to induce ISGs and to protect against subsequent IAV infection. In vitro, 3pRNA treatment of primary lung cells from B6-WT and B6.A2G-Mx1 mice resulted in ISG induction however inhibition of IAV infection was more potent in cells from B6.A2G-Mx1 mice. In vivo, a single intravenous injection of 3pRNA resulted in ISG induction in lungs of both B6-WT and B6.A2G-Mx1 mice, however potent and long-lasting protection against subsequent IAV challenge was only observed in B6.A2G-Mx1 mice. Thus, despite broad ISG induction, expression of a functional Mx1 is critical for potent and long-lasting RIG-I agonist-mediated protection in the mouse model of IAV infection.

Deficiency in coatomer complex I causes aberrant activation of STING signalling.

Coatomer complex I (COPI) mediates retrograde vesicular trafficking from Golgi to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency in subunit alpha causes COPA syndrome and is associated with type I IFN signalling, although the upstream innate immune sensor involved was unknown. Using in vitro models we find aberrant activation of the STING pathway due to deficient retrograde but probably not intra-Golgi transport. Further we find the upstream cytosolic DNA sensor cGAS as essentially required to drive type I IFN signalling. Genetic deletion of COPI subunits COPG1 or COPD similarly induces type I IFN activation in vitro, which suggests that inflammatory diseases associated with mutations in other COPI subunit genes may exist. Finally, we demonstrate that inflammation in COPA syndrome patient peripheral blood mononuclear cells and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS/STING pathway as a promising therapeutic approach.

Recessive NLRC4-Autoinflammatory Disease Reveals an Ulcerative Colitis Locus.

PURPOSE: NLRC4-associated autoinflammatory disease (NLRC4-AID) is an autosomal dominant condition presenting with a range of clinical manifestations which can include macrophage activation syndrome (MAS) and severe enterocolitis. We now report the first homozygous mutation in NLRC4 (c.478G > A, p.A160T) causing autoinflammatory disease with immune dysregulation and find that heterozygous carriers in the general population are at increased risk of developing ulcerative colitis. METHODS: Circulating immune cells and inflammatory markers were profiled and historical clinical data interrogated. DNA was extracted and sequenced using standard procedures. Inflammasome activation assays for ASC speck formation, pyroptosis, and IL-1ß/IL-18 secretion confirmed pathogenicity of the mutation in vitro. Genome-wide association of NLRC4 (A160T) with ulcerative colitis was examined using data from the IBD exomes portal. RESULTS: A 60-year-old Brazilian female patient was evaluated for recurrent episodes of systemic inflammation from six months of age. Episodes were characterized by recurrent low-grade fever, chills, oral ulceration, uveitis, arthralgia, and abdominal pain, followed by diarrhea with mucus and variable skin rash. High doses of corticosteroids were somewhat effective in controlling disease and anti-IL-1ß therapy partially controlled symptoms. While on treatment, serum IL-1ß and IL-18 levels remained elevated. Genetic investigations identified a homozygous mutation in NLRC4 (A160T), inherited in a recessive fashion. Increased ASC speck formation and IL-1ß/IL-18 secretion confirmed pathogenicity when NLRC4 (A160T) was analyzed in human cell lines. This allele is significantly enriched in patients with ulcerative colitis: OR 2.546 (95% 1.778-3.644), P = 0.01305. CONCLUSION: NLRC4 (A160T) can either cause recessively inherited autoinflammation and immune dysregulation, or function as a heterozygous risk factor for the development of ulcerative colitis.

ADAR1 edits the SenZ and SenZ-ability of RNA.

Some RNAs can assume a Z conformation, an unusual, left-handed turn. In this issue of Immunity, three studies report that mutations in the Zα-RNA binding domain of the adenosine deaminase ADAR1 are sufficient to induce autoinflammatory disease in mice, which models human Aicardí-Goutières syndrome, highlighting the important role of Z-RNA editing in limiting innate immune recognition of endogenous RNA.

MAPK-pathway inhibition mediates inflammatory reprogramming and sensitizes tumors to targeted activation of innate immunity sensor RIG-I.

Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8+ T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients.

Malaria parasites both repress host CXCL10 and use it as a cue for growth acceleration.

Pathogens are thought to use host molecular cues to control when to initiate life-cycle transitions, but these signals are mostly unknown, particularly for the parasitic disease malaria caused by Plasmodium falciparum. The chemokine CXCL10 is present at high levels in fatal cases of cerebral malaria patients, but is reduced in patients who survive and do not have complications. Here we show a Pf 'decision-sensing-system' controlled by CXCL10 concentration. High CXCL10 expression prompts P. falciparum to initiate a survival strategy via growth acceleration. Remarkably, P. falciparum inhibits CXCL10 synthesis in monocytes by disrupting the association of host ribosomes with CXCL10 transcripts. The underlying inhibition cascade involves RNA cargo delivery into monocytes that triggers RIG-I, which leads to HUR1 binding to an AU-rich domain of the CXCL10 3'UTR. These data indicate that when the parasite can no longer keep CXCL10 at low levels, it can exploit the chemokine as a cue to shift tactics and escape.

An epigenetic GPI anchor defect impairs TLR4 signaling in the B cell transdifferentiation model for primary human monocytes BLaER1.

Antigen-presenting myeloid cells like monocytes detect invading pathogens via pattern recognition receptors (PRRs) and initiate adaptive and innate immune responses. As analysis of PRR signaling in primary human monocytes is hampered by their restricted expandability, human monocyte models like THP-1 cells are commonly used for loss-of-function studies, such as with CRISPR-Cas9 editing. A recently developed transdifferentiation cell culture system, BLaER1, enables lineage conversion from malignant B cells to monocytes and was found superior to THP-1 in mimicking PRR signaling, thus being the first model allowing TLR4 and inflammasome pathway analysis. Here, we identified an important caveat when investigating TLR4-driven signaling in BLaER1 cells. We show that this model contains glycosylphosphatidylinositol (GPI) anchor-deficient cells, which lack CD14 surface expression when differentiated to monocytes, resulting in diminished LPS/TLR4 but not TLR7/TLR8 responsiveness. This GPI anchor defect is caused by epigenetic silencing of PIGH, leading to a random distribution of intact and PIGH-deficient clones after single-cell cloning. Overexpressing PIGH restored GPI-anchored protein (including CD14) expression and LPS responsiveness. When studying CD14- or other GPI-anchored protein-dependent pathways, researchers should consider this anomaly and ensure equal GPI-anchored protein expression when comparing cells that have undergone single-cell cloning, e. g. after CRISPR-Cas9 editing.