TLR8 Is a Sensor of RNase T2 Degradation Products.

TLR8 is among the highest-expressed pattern-recognition receptors in the human myeloid compartment, yet its mode of action is poorly understood. TLR8 engages two distinct ligand binding sites to sense RNA degradation products, although it remains unclear how these ligands are formed in cellulo in the context of complex RNA molecule sensing. Here, we identified the lysosomal endoribonuclease RNase T2 as a non-redundant upstream component of TLR8-dependent RNA recognition. RNase T2 activity is required for rendering complex single-stranded, exogenous RNA molecules detectable for TLR8. This is due to RNase T2's preferential cleavage of single-stranded RNA molecules between purine and uridine residues, which critically contributes to the supply of catabolic uridine and the generation of purine-2',3'-cyclophosphate-terminated oligoribonucleotides. Thus-generated molecules constitute agonistic ligands for the first and second binding pocket of TLR8. Together, these results establish the identity and origin of the RNA-derived molecular pattern sensed by TLR8.

Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses.

CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9-guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.

STING agonism turns human T cells into interferon-producing cells but impedes their functionality.

The cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) axis is the predominant DNA sensing system in cells of the innate immune system. However, human T cells also express high levels of STING, while its role and physiological trigger remain largely unknown. Here, we show that the cGAS-STING pathway is indeed functional in human primary T cells. In the presence of a TCR-engaging signal, both cGAS and STING activation switches T cells into type I interferon-producing cells. However, T cell function is severely compromised following STING activation, as evidenced by increased cell death, decreased proliferation, and impaired metabolism. Interestingly, these different phenotypes bifurcate at the level of STING. While antiviral immunity and cell death require the transcription factor interferon regulatory factor 3 (IRF3), decreased proliferation is mediated by STING independently of IRF3. In summary, we demonstrate that human T cells possess a functional cGAS-STING signaling pathway that can contribute to antiviral immunity. However, regardless of its potential antiviral role, the activation of the cGAS-STING pathway negatively affects T cell function at multiple levels. Taken together, these results could help inform the future development of cGAS-STING-targeted immunotherapies.

CARD8 inflammasome activation triggers pyroptosis in human T cells.

Inflammasomes execute a unique type of cell death known as pyroptosis. Mostly characterized in myeloid cells, caspase-1 activation downstream of an inflammasome sensor results in the cleavage and activation of gasdermin D (GSDMD), which then forms a lytic pore in the plasma membrane. Recently, CARD8 was identified as a novel inflammasome sensor that triggers pyroptosis in myeloid leukemia cells upon inhibition of dipeptidyl-peptidases (DPP). Here, we show that blocking DPPs using Val-boroPro triggers a lytic form of cell death in primary human CD4 and CD8 T cells, while other prototypical inflammasome stimuli were not active. This cell death displays morphological and biochemical hallmarks of pyroptosis. By genetically dissecting candidate components in primary T cells, we identify this response to be dependent on the CARD8-caspase-1-GSDMD axis. Moreover, DPP9 constitutes the relevant DPP restraining CARD8 activation. Interestingly, this CARD8-induced pyroptosis pathway can only be engaged in resting, but not in activated T cells. Altogether, these results broaden the relevance of inflammasome signaling and associated pyroptotic cell death to T cells, central players of the adaptive immune system.

Irgm2 and Gate-16 put a break on caspase-11 activation.

In infections caused by gram-negative bacteria, the bacterial cell wall component lipopolysaccharide (LPS) acts as a potent pathogen-associated molecular pattern (PAMP) that triggers the innate immune system. This is accomplished by two pattern recognition receptor systems. Toll-like receptor 4 (TLR4) senses extracellular LPS and induces a broad pro-inflammatory transcriptional program and also antiviral interferons. A complementary system detects intracellular LPS. As such, upon its release into the cytoplasm, LPS can directly engage the protease caspase-4 (caspase-11 in the murine system) and thereby trigger a pro-inflammatory cell death program known as pyroptosis (Rathinam et al, 2019). This is mediated by active caspase-4 cleaving its substrate gasdermin D (GSDMD). The thereby released N-terminal fragment of GSDMD inserts into the cell membrane and forms a cytotoxic pore. As a consequence, the cell ruptures and releases its pro-inflammatory content. In addition, the GSDMD pore results in potassium efflux that can activate the NLRP3 inflammasome. NLRP3 in turn activates caspase-1, which matures pro-IL-1ß and pro-IL-18, further perpetuating the inflammatory nature of this cell death. Given its unconventional mode of NLRP3 activation, this pathway has been coined the non-canonical inflammasome.

The patients' perspective on the burden of idiopathic intracranial hypertension.

BACKGROUND: Idiopathic intracranial hypertension (IIH) is characterized by increased intracranial pressure without evidence of a tumor or any other underlying cause. Headache and visual disturbances are frequent complaints of IIH patients, but little is known about other symptoms. In this study, we evaluated the patients' perspective on the burden of IIH. METHODS: For this cross-sectional study, we developed an online survey for patients with IIH containing standardized evaluations of headache (HIT-6), sleep (PROMIS Sleep Disturbance Scale) and depression (MDI) in relation to BMI, lumbar puncture opening pressure (LP OP) and treatment. RESULTS: Between December 2019 and February 2020, 306 patients completed the survey. 285 (93 %) were female, mean age was 36.6 years (± 10.8), mean BMI 34.2 (± 7.3) and mean LP OP at diagnosis was 37.8 cmH2O (± 9.5). 219 (72 %) of the participants were obese (BMI ≥ 30); 251 (82 %) reported severe impacting headaches, 140 (46 %) were suffering from sleep disturbances and 169 (56 %) from depression. Higher MDI scores correlated with higher BMI and increased sleep disturbances. Patients with a normalized LP opening pressure reported less headaches, less sleep disturbances and less depression than those with a constantly elevated opening pressure. CONCLUSIONS: In addition to headaches and visual disturbances, sleep disturbances and depression are frequent symptoms in IIH and contribute to the patients' burden. Structured questionnaires can help to identify IIH patients' needs and can lead to personalized and better treatment.

Live Cell Imaging of T Cell Pyroptosis.

Pyroptosis is a lytic, pro-inflammatory cell death program that is tightly regulated by inflammasomes in most cases. Inflammasome-dependent pyroptosis is best characterized in myeloid cells, but there is growing evidence that this cell death program also functions in human T cells. Several studies have suggested a role for inflammasome components in T-cell biology but often do not unambiguously clarify whether this means that T cells progress to pyroptosis. Pyroptosis has distinct morphological features, such as early loss of membrane integrity and ballooning, that allow it to be distinguished from apoptosis in a microscopic experiment. However, the most stringent definition of inflammasome-dependent pyroptosis is that it is genetically dependent on a pro-inflammatory caspase (caspase-1 or caspase-4) and the pore-forming protein gasdermin D (GSDMD). Therefore, using live cell imaging of T cells in combination with a genetic loss-of-function setup is the most reliable tool for us to unequivocally demonstrate that a T cell undergoes pyroptosis. Parallel live cell imaging of T cells and macrophages is limited due to the fact that T cells do not adhere while macrophages do. This can be overcome by using so-called micro-inserts that hold the cells in a limited area that can be monitored by microscopic field of view. Here we describe in detail how live cell imaging of human T cells and monocyte-derived macrophages undergoing pyroptosis can be performed.

Inflammasomes in T cells.

The concept of non-self recognition through germ-line encoded pattern recognition receptors (PRRs) has been well-established for professional innate immune cells. However, there is growing evidence that also T cells employ PRRs and associated effector functions in response to certain non-self or damage signals. Inflammasomes constitute a special subgroup of PRRs that is hardwired to a signaling cascade that culminates in the activation of caspase-1. Active caspase-1 processes pro-inflammatory cytokines of the IL-1 family and also triggers a lytic programmed cell death pathway known as pyroptosis. An increasing body of literature suggests that inflammasomes are also functional in T cells. On the one hand, conventional inflammasome signaling cascades have been described that operate similarly to pathways characterized in innate immune cells. On the other hand, unconventional functions have been suggested, in which certain inflammasome components play a role in unrelated processes, such as cell fate decisions and functions of T helper cells. In this review, we discuss our current knowledge on inflammasome functions in T cells and the biological implications of these findings for health and disease.

Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection.

The antiviral immune response to SARS-CoV-2 infection can limit viral spread and prevent development of pneumonic COVID-19. However, the protective immunological response associated with successful viral containment in the upper airways remains unclear. Here, we combine a multi-omics approach with longitudinal sampling to reveal temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients and associate specific immune trajectories with upper airway viral containment. We see a distinct systemic rather than local immune state associated with viral containment, characterized by interferon stimulated gene (ISG) upregulation across circulating immune cell subsets in non-pneumonic SARS-CoV2 infection. We report reduced cytotoxic potential of Natural Killer (NK) and T cells, and an immune-modulatory monocyte phenotype associated with protective immunity in COVID-19. Together, we show protective immune trajectories in SARS-CoV2 infection, which have important implications for patient prognosis and the development of immunomodulatory therapies.

Defective Interfering Genomes and the Full-Length Viral Genome Trigger RIG-I After Infection With Vesicular Stomatitis Virus in a Replication Dependent Manner.

Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/ß. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics.

Esophageal Blood Flow May Not Be Directly Influenced by Anastomotic Tension: An Exploratory Laser Doppler Study in Swine.

BACKGROUND: Anastomotic tension has been linked to leakage and stenosis in esophageal surgery in both adults and children. We aimed to determine the effects of esophageal topography, operative technique, and anastomotic tension on esophageal blood flow. MATERIALS AND METHODS: We divided and reanastomosed the esophagi at the carinal level with increasing levels of anastomotic tension in piglets (n = 10) and sham controls (n = 4). We examined esophageal blood flow pre- and postoperatively using laser Doppler flowmetry at the anastomosis and two predetermined proximal and distal points. Blood flow in relation to distance from the anastomosis was examined by multivariate linear regression. RESULTS: Thoracotomy alone did not influence perfusion at the carinal level in shams (Δ = 3.13 laser Doppler units, 95% confidence interval: -3.4 to 9.7, p = 0.2686). We constructed a (F[5,134] = 6.34, p < 0.0001) multinomial regression model based on distance to the anastomotic site and pre-/postoperative measurements (adjusted R 2 = 0.1624). Tissue blood flow was higher distant to the carina, but lower postoperatively and not influenced by the tension resulting from the extent of resection (F[1, 8] = 1.134, p = 0.318). CONCLUSION: Esophageal blood flow is higher at greater distances to the carinal level and hampered by esophageal division and reanastomosis. The extent of resection has less influence than previously assumed. Therefore, leakage and stenosis after esophageal anastomosis may not solely be caused by insufficient anastomotic blood flow.

Mitochondrial dsRNA: A New DAMP for MDA5.

The role of mitochondria as a signaling platform downstream of the RNA sensors RIG-I and MDA5 is well defined. Now, a recent study in Nature by Dhir et al. (2018) identifies mitochondrial dsRNA as an immunogenic ligand, adding another intriguing aspect to the role of mitochondria in innate immunity.

Isolation of RIG-I-associated RNAs from virus-infected cells.

When a novel innate pattern recognition receptor (PRR) is identified, a question comes up immediately: Which molecular pattern(s) can it recognize? One approach that can be taken to answer this question for nucleic acid-binding receptors is the detailed analysis of synthetic ligands (DNA, RNA, or hybrids) to narrow in on the minimal patterns that activate a given receptor. However, this may not always lead to a satisfying answer. A complementary albeit technically more demanding way to tackle this question is to examine which nucleic acids are actually bound by the receptor in a setting of cellular infection. Here, we describe a basic protocol to isolate RNAs bound to the RNA receptors of the RIG-I-like helicase family from virus-infected cells via immunoprecipitation (IP). The isolated RNA can then be used to analyze its origin, characteristics, and immunostimulatory properties with a variety of methods.