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Repeated intravenous (IV) administration of radiation-attenuated sporozoite (RAS) vaccines induces Plasmodium-specific CD8+ liver-resident memory T (Trm) cells in mice and achieves sterile protection against challenge. Our heterologous “prime-and-trap” vaccine strategy was previously shown to simplify and improve upon RAS vaccination. Prime-and-trap vaccination combines epidermal priming by DNA-encoded circumsporozoite protein (CSP) antigen followed by a single IV dose of freshly dissected RAS (fresh-RAS) to direct and trap activated and expanding CD8+ T cells in the liver. Prime-and-trap vaccination protects mice against wild-type sporozoite (spz) challenge. Assessment of prime-and-trap vaccines in nonhuman primate (NHP) models and/or humans would be greatly enabled if fresh-RAS could be replaced by cryopreserved RAS (cryo-RAS). Here, we investigated if fresh-RAS could be replaced with cryo-RAS for prime-and-trap vaccination in BALB/cj mice. Despite a reduction in spz vaccine liver burden following cryo-RAS administration compared with fresh-RAS, cryo-RAS induced a similar level of Plasmodium yoelii (Py) CSP-specific CD8+ liver Trm cells and completely protected mice against Py spz challenge 112 days after vaccination. Additionally, when the glycolipid adjuvant 7DW8-5 was co-administered with cryo-RAS, 7DW8-5 permitted the dose of cryo-RAS to be reduced four-fold while still achieving high rates of sterile protection. In summary, cryo-RAS with and without 7DW8-5 were compatible with prime-and-trap malaria vaccination in a mouse model, which may accelerate the pathway for this vaccine strategy to move to NHPs and humans.

The tight junction protein claudin-2 is upregulated in disease. Although many studies have linked intestinal barrier loss to local and systemic disease, these have relied on macromolecular probes. In vitro analyses show, however, that these probes cannot be accommodated by size- and charge-selective claudin-2 channels. We sought to define the impact of claudin-2 channels on disease. Transgenic claudin-2 overexpression or IL-13-induced claudin-2 upregulation increased intestinal small cation permeability in vivo. IL-13 did not, however, affect permeability in claudin-2-knockout mice. Claudin-2 is therefore necessary and sufficient to effect size- and charge-selective permeability increases in vivo. In chronic disease, T cell transfer colitis severity was augmented or diminished in claudin-2-transgenic or -knockout mice, respectively. We translated the in vitro observation that casein kinase-2 (CK2) inhibition blocks claudin-2 channel function to prevent acute, IL-13-induced, claudin-2-mediated permeability increases in vivo. In chronic immune-mediated colitis, CK2 inhibition attenuated progression in claudin-2-sufficient, but not claudin-2-knockout, mice, i.e., the effect was claudin-2 dependent. Paracellular flux mediated by claudin-2 channels can therefore promote immune-mediated colitis progression. Although the mechanisms by which claudin-2 channels intensify disease remain to be defined, these data suggest that claudin-2 may be an accessible target in immune-mediated disorders, including inflammatory bowel disease.

Regulated cell death is essential for the maintenance of cellular and tissue homeostasis. In the hematopoietic system, genetic defects in apoptotic cell death generally produce the accumulation of immune cells, inflammation and autoimmunity. In contrast, we found that genetic deletion of caspases of the mitochondrial apoptosis pathway reduces natural killer (NK) cell numbers and makes NK cells functionally defective in vivo and in vitro. Caspase deficiency results in constitutive activation of a type I interferon (IFN) response, due to leakage of mitochondrial DNA and activation of the cGAS/STING pathway. The NK cell defect in caspase-deficient mice is independent of the type I IFN response, but the phenotype is partially rescued by cGAS or STING deficiency. Finally, caspase deficiency alters NK cells in a cell-extrinsic manner. Type I IFNs and NK cells are two essential effectors of antiviral immunity, and our results demonstrate that they are both regulated in a caspase-dependent manner. Beyond caspase-deficient animals, our observations may have implications in infections that trigger mitochondrial stress and caspase-dependent cell death.

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs. M. G.-L. was a postdoctoral fellow supported by the Plan Estratégico de Investigación e Innovación en Salud (PERIS, SLT002/16/00179) of the Generalitat de Catalunya, Spain. M. D.-V. was a predoctoral fellow supported by Secretaria d’Universitats i Recerca del Departament d’Economia i Creixement, Generalitat de Catalunya (2017 FI_B2_00029). I. A.-H. is a predoctoral fellow supported by the Ministerio de Economia y Competitividad (FPI BES-2017081657). C. S. was funded by the German Research Foundation (DFG; fellowship SCHA2047/1-1). We acknowledge support from the National Institute of Health R21 program (1R21AI135680-01). The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech), and it is supported by “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2017SGR595) and acknowledges support of the Spanish Ministry of Science and Innovation to the EMBL partnership. We also acknowledge support from the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S) and support from the Generalitat de Catalunya through the CERCA Program. This research is part of the ISGlobal’s Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundación Ramón Areces. Work in the laboratory of Carmen Fernandez-Becerra and Hernando A. del Portillo is funded by the Ministerio Español de Economía y Competitividad (SAF2016 80655-R) and by and by the Ministerio de Ciencia e Innovación (PID2019-111795RB-I00). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Tissue-resident memory CD8+ T (Trm) cells in the liver are critical for long-term protection against pre-erythrocytic Plasmodium infection. Such protection can usually be induced with three to five doses of i.v. administered radiation-attenuated sporozoites (RAS). To simplify and accelerate vaccination, we tested a DNA vaccine designed to induce potent T cell responses against the SYVPSAEQI epitope of Plasmodium yoelii circumsporozoite protein. In a heterologous “prime-and-trap” regimen, priming using gene gun–administered DNA and boosting with one dose of RAS attracted expanding Ag-specific CD8+ T cell populations to the liver, where they became Trm cells. Vaccinated in this manner, BALB/c mice were completely protected against challenge, an outcome not reliably achieved following one dose of RAS or following DNA-only vaccination. This study demonstrates that the combination of CD8+ T cell priming by DNA and boosting with liver-homing RAS enhances formation of a completely protective liver Trm cell response and suggests novel approaches for enhancing T cell–based pre-erythrocytic malaria vaccines.

Key Points Next-generation humanized mice differentially support human HSPC maintenance and myelopoiesis. MISTRG mice support long-term human HSPC maintenance demonstrated by quaternary transplantation and development of human tissue macrophages.

Tissue-resident memory CD8

The sensing of viral nucleic acids within the cytosol is essential for the induction of innate immune responses following infection. However, this sensing occurs within cells that have already been infected. The death of infected cells can be beneficial to the host by eliminating the virus’s replicative niche and facilitating the release of inflammatory mediators. In this study, we show that sensing of intracellular DNA or RNA by cGAS-STING or RIG-I-MAVS, respectively, leads to activation of RIPK3 and necroptosis in bone marrow–derived macrophages. Notably, this requires signaling through both type I IFN and TNF receptors, revealing synergy between these pathways to induce cell death. Furthermore, we show that hyperactivation of STING in mice leads to a shock-like phenotype, the mortality of which requires activation of the necroptotic pathway and IFN and TNF cosignaling, demonstrating that necroptosis is one outcome of STING signaling in vivo.

Malaria eradication necessitates new tools to fight the evolving and complex Plasmodium pathogens. These tools include prophylactic drugs that eliminate Plasmodium liver stages and consequently prevent clinical disease, decrease transmission, and reduce the propensity for resistance development. Currently, the identification of these drugs relies on in vitro P. falciparum liver stage assays or in vivo causal prophylaxis assays using rodent malaria parasites; there is no method to directly test in vivo liver stage activity of candidate antimalarials against the human malaria-causing parasite P. falciparum. Here, we use a liver-chimeric humanized mouse (FRG huHep) to demonstrate in vivo P. falciparum liver stage development and describe the efficacy of clinically used and candidate antimalarials with prophylactic activity. We show that daily administration of atovaquone-proguanil (ATQ-PG; ATQ, 30 mg/kg, and PG, 10 mg/kg) protects 5 of 5 mice from liver stage infection, consistent with the use in humans as a causal prophylactic drug. Single-dose primaquine (60 mg/kg) has similar activity to that observed in humans, demonstrating the activity of this drug (and its active metabolites) in FRG huHep mice. We also show that DSM265, a selective Plasmodial dihydroorotate dehydrogenase inhibitor with causal prophylactic activity in humans, reduces liver stage burden in FRG huHep mice. Finally, we measured liver stage-to-blood stage transition of the parasite, the ultimate readout of prophylactic activity and measurement of infective capacity of parasites in the liver, to show that ATQ-PG reduces blood stage patency to below the limit of quantitation by quantitative PCR (qPCR). The FRG huHep model, thus, provides a platform for preclinical evaluation of drug candidates for liver stage causal prophylactic activity, pharmacokinetic/pharmacodynamics studies, and biological studies to investigate the mechanism of action of liver stage active antimalarials.

The conserved eukaryotic Pan2–Pan3 deadenylation complex shortens cytoplasmic mRNA 3′ polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C-terminal domain and via an N-terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N-terminal WD40 domain to the C-terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high-affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2–Pan3 complex.

Necroptosis is a form of programmed cell death defined by activation of the kinase receptor interacting protein kinase 3 and its downstream effector, the pseudokinase mixed lineage kinase domain-like (MLKL). Activated MLKL translocates to the cell membrane and disrupts it, leading to loss of cellular ion homeostasis. In this study, we use a system in which this event can be specifically triggered by a small-molecule ligand to show that MLKL activation is sufficient to induce the processing and release of bioactive IL-1β. MLKL activation triggers potassium efflux and assembly of the NLRP3 inflammasome, which is required for the processing and activity of IL-1β released during necroptosis. Notably, MLKL activation also causes cell membrane disruption, which allows efficient release of IL-1β independently of the recently described pyroptotic effector gasdermin-D. Taken together, our findings indicate that MLKL is an endogenous activator of the NLRP3 inflammasome, and that MLKL activation provides a mechanism for concurrent processing and release of IL-1β independently of gasdermin-D.

Summary Receptor-interacting protein kinase-3 (RIPK3) is an activator of necroptotic cell death, but recent work has implicated additional roles for RIPK3 in inflammatory signaling independent of cell death. However, while necroptosis has been shown to contribute to antiviral immunity, death-independent roles for RIPK3 in host defense have not been demonstrated. Using a mouse model of West Nile virus (WNV) encephalitis, we show that RIPK3 restricts WNV pathogenesis independently of cell death. Ripk3 −/− mice exhibited enhanced mortality compared to wild-type (WT) controls, while mice lacking the necroptotic effector MLKL, or both MLKL and caspase-8, were unaffected. The enhanced susceptibility of Ripk3 −/− mice arose from suppressed neuronal chemokine expression and decreased central nervous system (CNS) recruitment of T lymphocytes and inflammatory myeloid cells, while peripheral immunity remained intact. These data identify pleiotropic functions for RIPK3 in the restriction of viral pathogenesis and implicate RIPK3 as a key coordinator of immune responses within the CNS.

Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8 DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense., Author Summary TLR signaling induces expression of key inflammatory cytokines and pro-survival factors that facilitate control of microbial infection. TLR signaling can also engage cell death pathways through activation of enzymes known as caspases. Caspase-8 activates apoptosis in response to infection by pathogens that interfere with NF-κB signaling, including Yersinia, but has also recently been linked to control of inflammatory gene expression. Pathogenic Yersinia can cause severe disease ranging from gastroenteritis to plague. While caspase-8 mediates cell death in response to Yersinia infection as well as other signals, its precise role in gene expression and host defense during in vivo infection is unknown. Here, we show that caspase-8 activity promotes cell-intrinsic cytokine expression, independent of its role in cell death in response to Yersinia infection. Our studies further demonstrate that caspase-8 enzymatic activity plays a previously undescribed role in ensuring optimal TLR-induced gene expression by innate cells during bacterial infection. This work sheds new light on mechanisms that regulate essential innate anti-bacterial immune defense.

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis., Author Summary DNA polymerase V from the bacterium Escherichia coli is one of a class of DNA polymerases that replicate DNA inaccurately. They thus generate mutations at elevated levels. Whereas this might seem incongruous with the goal of accurate transmission of genetic information from one generation to the next, it is actually part of a specialized bacterial DNA repair process that comes into play principally when cells are severely stressed by high loads of DNA damage. Polymerase V is normally inactive. The transfer of one subunit from the bacterial recombinase RecA, and the addition of ATP, leads to the formation of the active pol V Mutasome (Mut) (UmuD′2C-RecA-ATP). The current study delves deeper into this process, initiating the task of mapping out the molecular details of the interaction between RecA and UmuD′2C. One surface region on the RecA protein required for this activation is defined, the step in the activation process that is affected by this surface is identified, and a direct interaction (or at least very close proximity) between this surface and particular amino acid residues in the UmuC protein is demonstrated. A new RecA variant protein is generated that provides improved separation of function, in that the activation of pol V is abolished while minimally affecting other RecA functions. The study also provides a molecular consummation for a series of incisive genetic studies carried out nearly two decades ago.

The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.

P1 bacteriophage recombination enhancement function (Ref) protein is an HNH class and RecA-dependent endonuclease. RecA-bound oligonucleotides homologous to a specific region on circular dsDNA will catalyze RecA strand-invasion, creating a displacement loop (D-loop). Upon Ref addition, double-stranded breaks (DSBs) are created within the D-loop. Thus, the Ref protein, in concert with RecA, is a programmable endonuclease. The closely related φW39 Ref protein exhibits increased RecA-dependent endonuclease activity compared to P1 Ref. P1 Ref protein missing the disordered 76 N-terminus residues is incapable of DNA binding and exhibits decreased nuclease activity, which indicates the importance of the N-terminal region for these functions. Characterization of N-terminal truncations based on charge distribution for φW39 Refs ΔN21, ΔN47, ΔN59, ΔN66, and ΔN74 revealed modulated activity for DSB formation and DNA binding. In addition to these in vitro studies, cells transformed with a plasmid containing P1 Ref an...

Activation of the NLRP3 inflammasome results in caspase-1 dependent processing and secretion of IL-1β. Inflammasome activation and IL-1β maturation is thought to require two signals: Signal I leads to transcriptional up-regulation of inflammasome components and pro-IL-1β. Various agents such as ATP and nigericin can function as signal II to activate the NLRP3 inflammasome through a mechanism that is thought to involve disrupted ion homeostasis. This in turn leads to IL-1β processing and release, as well as pyroptotic cell death. Notably, the execution of another cell death program, necroptosis, also involves cellular membrane disruption and ion flux, through the action of the protein MLKL. Here, we demonstrate that the execution of necroptosis by MLKL can lead to NLRP3 inflammasome activation and the secretion of bioactive IL-1β. We further show that IL-1β processing is independent from the cell death function of MLKL. This mechanism represents a novel inflammatory signal emanating from necroptotic cells, as well as an unexpected mechanistic cross-talk between necroptosis and inflammasome activation.

Necroptosis is a form of programmed cell death and is distinct from apoptosis in its mechanism and morphology; apoptosis is thought to be a non-inflammatory form of cell death whereas necroptosis is thought to be inflammatory. Necroptosis occurs in response to viral infection and is important for control of DNA and RNA viruses in vivo. While much of the necroptotic pathway has been characterized in vitro, the effects of necroptosis are poorly understood in vivo. We recently described a system by which necroptosis can be triggered using a version of the pro-necroptotic kinase RIPK3 fused to a ligand binding domain. Here, we characterize a novel transgenic model in which this inducible form of RIPK3 is expressed in vivo, allowing induction of necroptosis in primary tissues. We find that widespread activation of necroptosis in vivo using this model triggers sepsis-like systemic inflammation.