CXCR5(+) follicular cytotoxic T cells control viral infection in B cell follicles.

During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell-derived malignancies.

SARS-CoV-2 mouse adaptation selects virulence mutations that cause TNF-driven age-dependent severe disease with human correlates.

The diversity of COVID-19 disease in otherwise healthy people, from seemingly asymptomatic infection to severe life-threatening disease, is not clearly understood. We passaged a naturally occurring near-ancestral SARS-CoV-2 variant, capable of infecting wild-type mice, and identified viral genomic mutations coinciding with the acquisition of severe disease in young adult mice and lethality in aged animals. Transcriptomic analysis of lung tissues from mice with severe disease elucidated a host antiviral response dominated mainly by interferon and IL-6 pathway activation in young mice, while in aged animals, a fatal outcome was dominated by TNF and TGF-ß signaling. Congruent with our pathway analysis, we showed that young TNF-deficient mice had mild disease compared to controls and aged TNF-deficient animals were more likely to survive infection. Emerging clinical correlates of disease are consistent with our preclinical studies, and our model may provide value in defining aberrant host responses that are causative of severe COVID-19.

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.

Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice.

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

Epigenetic Silencing of RIPK3 in Hepatocytes Prevents MLKL-mediated Necroptosis From Contributing to Liver Pathologies.

BACKGROUND & AIMS: Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/ nonalcoholic steatohepatitis (NASH); however, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology. METHODS: Several animal models of human liver pathology, including diet-induced steatohepatitis in male mice and diverse infections in both male and female mice, were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. RESULTS: Identical infection kinetics and pathologic outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared with control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed a high-fat diet to induce NASH. Under all conditions tested, we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis. CONCLUSIONS: We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathologic conditions tested in this study.

Mpeg1 is not essential for antibacterial or antiviral immunity, but is implicated in antigen presentation.

To control infections phagocytes can directly kill invading microbes. Macrophage-expressed gene 1 (Mpeg1), a pore-forming protein sometimes known as perforin-2, is reported to be essential for bacterial killing following phagocytosis. Mice homozygous for the mutant allele Mpeg1tm1Pod succumb to bacterial infection and exhibit deficiencies in bacterial killing in vitro. Here we describe a new Mpeg mutant allele Mpeg1tm1.1Pib on the C57BL/6J background. Mice homozygous for the new allele are not abnormally susceptible to bacterial or viral infection, and irrespective of genetic background show no perturbation in bacterial killing in vitro. Potential reasons for these conflicting findings are discussed. In further work, we show that cytokine responses to inflammatory mediators, as well as antibody generation, are also normal in Mpeg1tm1.1Pib/tm1.1Pib mice. We also show that Mpeg1 is localized to a CD68-positive endolysosomal compartment, and that it exists predominantly as a processed, two-chain disulfide-linked molecule. It is abundant in conventional dendritic cells 1, and mice lacking Mpeg1 do not present the model antigen ovalbumin efficiently. We conclude that Mpeg1 is not essential for innate antibacterial protection or antiviral immunity, but may play a focused role early in the adaptive immune response.

The role of MKK4 in T-cell development and immunity to viral infections.

The stress-activated protein kinases (SAPKs)/c-Jun-N-terminal-kinases (JNK) are members of the mitogen-activated protein kinase family. These kinases are responsible for transducing cellular signals through a phosphorylation-dependent signaling cascade. JNK activation in immune cells can lead to a range of critical cellular responses that include proliferation, differentiation and apoptosis. MKK4 is a SAPK that can activate both JNK1 and JNK2; however, its role in T-cell development and function has been controversial. Additionally, loss of either JNK1 or JNK2 has opposing effects in the generation of T-cell immunity to viral infection and cancer. We used mice with a conditional loss of MKK4 in T cells to investigate the in vivo role of MKK4 in T-cell development and function during lymphocytic choriomeningitis virus (LCMV) infection. We found no physiologically relevant differences in T-cell responses or immunity to either acute or chronic LCMV in the absence of MKK4.

Pseudotumor presentation of CMV disease: Diagnostic dilemma and association with immunomodulating therapy.

Cytomegalovirus (CMV) is a significant cause of morbidity and mortality in the immunocompromised host. Atypical presentations which include pseudotumors or "cancer mimics" have been described. The etiology of these lesions remains unclear. The authors describe two previously unpublished cases that have arisen in the context of newer immunomodulating therapy and review the existing non-HIV-associated CMV pseudotumors described in the literature.

Granzyme K-deficient mice show no evidence of impaired antiviral immunity.

The biological role of granzyme K, a serine protease of cytotoxic T lymphocytes (CTL), is controversial. It has been reported to induce perforin-mediated cell death in vitro, but is also reported to be non-cytotoxic and to operate in inflammatory processes. To elucidate the biological role of this protease, we have deleted the granzyme K gene in mice (mutant allele: Gzmktm1.1Pib; MGI:5636646). Gzmk -/- mice are healthy, anatomically normal, fecund and show normal hematopoietic development. Gzmk -/- mice readily recover from lymphocytic choriomeningitis virus and mouse pox Ectromelia virus infection. Ex vivo, virus-specific granzyme K-deficient CTL are indistinguishable from those of wild-type mice in apoptosis induction of target cells. These data suggest that granzyme K does not play an essential role in viral immunity or cytotoxicity. Our granzyme K knockout line completes the collection of mouse models for the human granzymes, and will further our understanding of their biological roles and relationships.

Nucleotide oligomerization domain 1 enhances IFN-γ signaling in gastric epithelial cells during Helicobacter pylori infection and exacerbates disease severity.

Virulent Helicobacter pylori strains that specifically activate signaling in epithelial cells via the innate immune molecule, nucleotide oligomerization domain 1 (NOD1), are more frequently associated with IFN-γ-dependent inflammation and with severe clinical outcomes (i.e., gastric cancer and peptic ulceration). In cell culture models, we showed that H. pylori activation of the NOD1 pathway caused enhanced proinflammatory signaling in epithelial cells in response to IFN-γ stimulation through the direct effects of H. pylori on two components of the IFN-γ signaling pathway, STAT1 and IFN regulatory factor 1 (IRF1). Specifically, H. pylori activation of the NOD1 pathway was shown to increase the levels of STAT1-Tyr(701)/Ser(727) phosphorylation and IRF1 expression/synthesis in cells, resulting in enhanced production of the NOD1- and IFN-γ-regulated chemokines, IL-8- and IFN-γ-induced protein 10, respectively. Consistent with the notion that heightened proinflammatory signaling in epithelial cells may have an impact on disease severity, we observed significantly increased expression levels of NOD1, CXCL8, IRF1, and CXCL10 in human gastric biopsies displaying severe gastritis, when compared with those without gastritis (p < 0.05, p < 0.001, p < 0.01, and p < 0.05, respectively). Interestingly, NOD1, CXCL8, and IRF1 expression levels were also significantly upregulated in gastric tumor tissues, when compared with paired nontumor samples (p < 0.0001, p < 0.05, and p < 0.05, respectively). Thus, we propose that cross-talk between NOD1 and IFN-γ signaling pathways contribute to H. pylori-induced inflammatory responses, potentially revealing a novel mechanism whereby virulent H. pylori strains promote more severe disease.

Necroptosis does not drive disease pathogenesis in a mouse infective model of SARS-CoV-2 in vivo.

Necroptosis, a type of lytic cell death executed by the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) has been implicated in the detrimental inflammation caused by SARS-CoV-2 infection. We minimally and extensively passaged a single clinical SARS-CoV-2 isolate to create models of mild and severe disease in mice allowing us to dissect the role of necroptosis in SARS-CoV-2 disease pathogenesis. We infected wild-type and MLKL-deficient mice and found no significant differences in viral loads or lung pathology. In our model of severe COVID-19, MLKL-deficiency did not alter the host response, ameliorate weight loss, diminish systemic pro-inflammatory cytokines levels, or prevent lethality in aged animals. Our in vivo models indicate that necroptosis is dispensable in the pathogenesis of mild and severe COVID-19.

A necroptosis-independent function of RIPK3 promotes immune dysfunction and prevents control of chronic LCMV infection.

Necroptosis is a lytic and inflammatory form of cell death that is highly constrained to mitigate detrimental collateral tissue damage and impaired immunity. These constraints make it difficult to define the relevance of necroptosis in diseases such as chronic and persistent viral infections and within individual organ systems. The role of necroptotic signalling is further complicated because proteins essential to this pathway, such as receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), have been implicated in roles outside of necroptotic signalling. We sought to address this issue by individually defining the role of RIPK3 and MLKL in chronic lymphocytic choriomeningitis virus (LCMV) infection. We investigated if necroptosis contributes to the death of LCMV-specific CD8+ T cells or virally infected target cells during infection. We provide evidence showing that necroptosis was redundant in the pathogenesis of acute forms of LCMV (Armstrong strain) and the early stages of chronic (Docile strain) LCMV infection in vivo. The number of immune cells, their specificity and reactivity towards viral antigens and viral loads are not altered in the absence of either MLKL or RIPK3 during acute and during the early stages of chronic LCMV infection. However, we identified that RIPK3 promotes immune dysfunction and prevents control of infection at later stages of chronic LCMV disease. This was not phenocopied by the loss of MLKL indicating that the phenotype was driven by a necroptosis-independent function of RIPK3. We provide evidence that RIPK3 signaling evoked a dysregulated type 1 interferone response which we linked to an impaired antiviral immune response and abrogated clearance of chronic LCMV infection.

Venetoclax, alone and in combination with the BH3 mimetic S63845, depletes HIV-1 latently infected cells and delays rebound in humanized mice.

HIV-1 persists indefinitely in people living with HIV (PLWH) on antiretroviral therapy (ART). If ART is stopped, the virus rapidly rebounds from long-lived latently infected cells. Using a humanized mouse model of HIV-1 infection and CD4+ T cells from PLWH on ART, we investigate whether antagonizing host pro-survival proteins can prime latent cells to die and facilitate HIV-1 clearance. Venetoclax, a pro-apoptotic inhibitor of Bcl-2, depletes total and intact HIV-1 DNA in CD4+ T cells from PLWH ex vivo. This venetoclax-sensitive population is enriched for cells with transcriptionally higher levels of pro-apoptotic BH3-only proteins. Furthermore, venetoclax delays viral rebound in a mouse model of persistent HIV-1 infection, and the combination of venetoclax with the Mcl-1 inhibitor S63845 achieves a longer delay in rebound compared with either intervention alone. Thus, selective inhibition of pro-survival proteins can induce death of HIV-1-infected cells that persist on ART, extending time to viral rebound.

Endothelial Caspase-8 prevents fatal necroptotic hemorrhage caused by commensal bacteria.

Caspase-8 transduces signals from death receptor ligands, such as tumor necrosis factor, to drive potent responses including inflammation, cell proliferation or cell death. This is a developmentally essential function because in utero deletion of endothelial Caspase-8 causes systemic circulatory collapse during embryogenesis. Whether endothelial Caspase-8 is also required for cardiovascular patency during adulthood was unknown. To address this question, we used an inducible Cre recombinase system to delete endothelial Casp8 in 6-week-old conditionally gene-targeted mice. Extensive whole body vascular gene targeting was confirmed, yet the dominant phenotype was fatal hemorrhagic lesions exclusively within the small intestine. The emergence of these intestinal lesions was not a maladaptive immune response to endothelial Caspase-8-deficiency, but instead relied upon aberrant Toll-like receptor sensing of microbial commensals and tumor necrosis factor receptor signaling. This lethal phenotype was prevented in compound mutant mice that lacked the necroptotic cell death effector, MLKL. Thus, distinct from its systemic role during embryogenesis, our data show that dysregulated microbial- and death receptor-signaling uniquely culminate in the adult mouse small intestine to unleash MLKL-dependent necroptotic hemorrhage after loss of endothelial Caspase-8. These data support a critical role for Caspase-8 in preserving gut vascular integrity in the face of microbial commensals.

Caspase-8 has dual roles in regulatory T cell homeostasis balancing immunity to infection and collateral inflammatory damage.

Targeting the potent immunosuppressive properties of FOXP3+ regulatory T cells (Tregs) has substantial therapeutic potential for treating autoimmune and inflammatory diseases. Yet, the molecular mechanisms controlling Treg homeostasis, particularly during inflammation, remain unclear. We report that caspase-8 is a central regulator of Treg homeostasis in a context-specific manner that is decisive during immune responses. In mouse genetic models, targeting caspase-8 in Tregs led to accumulation of effector Tregs resistant to apoptotic cell death. Conversely, inflammation induced the MLKL-dependent necroptosis of caspase-8-deficient lymphoid and tissue Tregs, which enhanced immunity to a variety of chronic infections to promote clearance of viral or parasitic pathogens. However, improved immunity came at the risk of lethal inflammation in overwhelming infections. Caspase-8 inhibition using a clinical-stage compound revealed that human Tregs have heightened sensitivity to necroptosis compared with conventional T cells. These findings reveal a fundamental mechanism in Tregs that could be targeted to manipulate the balance between immune tolerance versus response for therapeutic benefit.

Helicobacter pylori induces MAPK phosphorylation and AP-1 activation via a NOD1-dependent mechanism.

Helicobacter pylori rapidly activates MAPKs and transcription factors, NF-kappaB and AP-1, in gastric epithelial cells following host attachment. Activation of these signal transducers is largely dependent on the cag pathogenicity island (cagPAI)-encoded Type IV Secretion System. H. pylori was shown to translocate peptidoglycan through the Type IV Secretion System, which is recognized by the pathogen recognition molecule, NOD1, thus resulting in NF-kappaB activation. The mechanisms of H. pylori-induced MAPK and AP-1 activation, however, are less well defined and therefore, we assessed the contribution of NOD1 to their activation. For this, we used gastric epithelial cell lines, stably expressing siRNA to either NOD1 or a control gene. In siNOD1-expressing cells stimulated with cagPAI(+) H. pylori, we observed significant reductions in p38 and ERK phosphorylation (p < 0.05), whereas the levels of Jnk phosphorylation remained unchanged. Consistent with a previous report, however, we were able to demonstrate NOD1-dependent Jnk phosphorylation by the invasive pathogen Shigella flexneri, highlighting pathogen-specific host responses to infection. We also show that NOD1 was essential for H. pylori induction of not only NF-kappaB, but also AP-1 activation, implying that NOD1 induces robust proinflammatory responses, in an attempt to rapidly control infection. Pharmacological inhibition of p38 and ERK activity significantly reduced IL-8 production in response to H. pylori, further emphasizing the importance of MAPKs in innate immune responses to the pathogen. Thus, for the first time we have shown the important role for NOD1 in MAPK and AP-1 activation in response to cagPAI(+) H. pylori.

Landscape of human antibody recognition of the SARS-CoV-2 receptor binding domain.

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).

Shiga toxin-producing Escherichia coli strains negative for locus of enterocyte effacement.

Most Shiga toxin-producing Escherichia coli (STEC) infections that are associated with severe sequelae such as hemolytic uremic syndrome (HUS) are caused by attaching and effacing pathogens that carry the locus of enterocyte effacement (LEE). However, a proportion of STEC isolates that do not carry LEE have been associated with HUS. To clarify the emergence of LEE-negative STEC, we compared the genetic composition of the virulence plasmids pO113 and pO157 from LEE-negative and LEE-positive STEC, respectively. The complete nucleotide sequence of pO113 showed that several plasmid genes were shared by STEC O157:H7. In addition, allelic profiling of the ehxA gene demonstrated that pO113 belongs to a different evolutionary lineage than pO157 and that the virulence plasmids of LEE-negative STEC strains were highly related. In contrast, multilocus sequence typing of 17 LEE-negative STEC isolates showed several clonal groups, suggesting that pathogenic LEE-negative STEC has emerged several times throughout its evolution.

Cell Traversal Activity Is Important for Plasmodium falciparum Liver Infection in Humanized Mice.

Malaria sporozoites are deposited into the skin by mosquitoes and infect hepatocytes. The molecular basis of how Plasmodium falciparum sporozoites migrate through host cells is poorly understood, and direct evidence of its importance in vivo is lacking. Here, we generated traversal-deficient sporozoites by genetic disruption of sporozoite microneme protein essential for cell traversal (PfSPECT) or perforin-like protein 1 (PfPLP1). Loss of either gene did not affect P. falciparum growth in erythrocytes, in contrast with a previous report that PfPLP1 is essential for merozoite egress. However, although traversal-deficient sporozoites could invade hepatocytes in vitro, they could not establish normal liver infection in humanized mice. This is in contrast with NF54 sporozoites, which infected the humanized mice and developed into exoerythrocytic forms. This study demonstrates that SPECT and perforin-like protein 1 (PLP1) are critical for transcellular migration by P. falciparum sporozoites and demonstrates the importance of cell traversal for liver infection by this human pathogen.