Immune response to intravenous immunoglobulin in patients with Kawasaki disease and MIS-C.

BACKGROUNDMultisystem inflammatory syndrome in children (MIS-C) is a rare but potentially severe illness that follows exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Kawasaki disease (KD) shares several clinical features with MIS-C, which prompted the use of intravenous immunoglobulin (IVIG), a mainstay therapy for KD. Both diseases share a robust activation of the innate immune system, including the IL-1 signaling pathway, and IL-1 blockade has been used for the treatment of both MIS-C and KD. The mechanism of action of IVIG in these 2 diseases and the cellular source of IL-1ß have not been defined.METHODSThe effects of IVIG on peripheral blood leukocyte populations from patients with MIS-C and KD were examined using flow cytometry and mass cytometry (CyTOF) and live-cell imaging.RESULTSCirculating neutrophils were highly activated in patients with KD and MIS-C and were a major source of IL-1ß. Following IVIG treatment, activated IL-1ß+ neutrophils were reduced in the circulation. In vitro, IVIG was a potent activator of neutrophil cell death via PI3K and NADPH oxidase, but independently of caspase activation.CONCLUSIONSActivated neutrophils expressing IL-1ß can be targeted by IVIG, supporting its use in both KD and MIS-C to ameliorate inflammation.FUNDINGPatient Centered Outcomes Research Institute; NIH; American Asthma Foundation; American Heart Association; Novo Nordisk Foundation; NIGMS; American Academy of Allergy, Asthma and Immunology Foundation.

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection.

We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.

ß-glucan-dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment.

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. ß-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.

Single-cell cloning of human T-cell lines reveals clonal variation in cell death responses to chemotherapeutics.

Genetic modification of human leukemic cell lines using CRISPR-Cas9 has become a staple of gene-function studies. Single-cell cloning of modified cells is frequently used to facilitate studies of gene function. Inherent in this approach is an assumption that the genetic drift, amplified in some cell lines by mutations in DNA replication and repair machinery, as well as non-genetic factors will not introduce significant levels of experimental cellular heterogeneity in clones derived from parental populations. In this study, we characterize the variation in cell death of fifty clonal cell lines generated from human Jurkat and MOLT-4 T-cells edited by CRISPR-Cas9. We demonstrate a wide distribution of sensitivity to chemotherapeutics between non-edited clonal human leukemia T-cell lines, and also following CRISPR-Cas9 editing at the NLRP1 locus, or following transfection with non-targeting sgRNA controls. The cell death sensitivity profile of clonal cell lines was consistent across experiments and failed to revert to the non-clonal parental phenotype. Whole genome sequencing of two clonal cell lines edited by CRISPR-Cas9 revealed unique and shared genetic variants, which had minimal read support in the non-clonal parental population and were not suspected CRISPR-Cas9 off-target effects. These variants included genes related to cell death and drug metabolism. The variation in cell death phenotype of clonal populations of human T-cell lines may be a consequence of T-cell line genetic instability, and to a lesser extent clonal heterogeneity in the parental population or CRISPR-Cas9 off-target effects not predicted by current models. This work highlights the importance of genetic variation between clonal T-cell lines in the design, conduct, and analysis of experiments to investigate gene function after single-cell cloning.

Interconversion between Tumorigenic and Differentiated States in Acute Myeloid Leukemia.

Tumors are composed of phenotypically heterogeneous cancer cells that often resemble various differentiation states of their lineage of origin. Within this hierarchy, it is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategies that specifically eradicate CSCs or induce their differentiation. The clinical success of these approaches depends on CSC differentiation being unidirectional rather than reversible, yet this question remains unresolved even in prototypically hierarchical malignancies, such as acute myeloid leukemia (AML). Here, we show in murine and human models of AML that, upon perturbation of endogenous expression of the lineage-determining transcription factor PU.1 or withdrawal of established differentiation therapies, some mature leukemia cells can de-differentiate and reacquire clonogenic and leukemogenic properties. Our results reveal plasticity of CSC maturation in AML, highlighting the need to therapeutically eradicate cancer cells across a range of differentiation states.

Selective FcγR Co-engagement on APCs Modulates the Activity of Therapeutic Antibodies Targeting T Cell Antigens.

The co-engagement of fragment crystallizable (Fc) gamma receptors (FcγRs) with the Fc region of recombinant immunoglobulin monoclonal antibodies (mAbs) and its contribution to therapeutic activity has been extensively studied. For example, Fc-FcγR interactions have been shown to be important for mAb-directed effector cell activities, as well as mAb-dependent forward signaling into target cells via receptor clustering. Here we identify a function of mAbs targeting T cell-expressed antigens that involves FcγR co-engagement on antigen-presenting cells (APCs). In the case of mAbs targeting CTLA-4 and TIGIT, the interaction with FcγR on APCs enhanced antigen-specific T cell responses and tumoricidal activity. This mechanism extended to an anti-CD45RB mAb, which led to FcγR-dependent regulatory T cell expansion in mice.

Cutting Edge: Blockade of Inhibitor of Apoptosis Proteins Sensitizes Neutrophils to TNF- but Not Lipopolysaccharide-Mediated Cell Death and IL-1ß Secretion.

The mammalian inhibitor of apoptosis proteins (IAPs) are key regulators of cell death and inflammation. A major function of IAPs is to block the formation of a cell death-inducing complex, termed the ripoptosome, which can trigger caspase-8-dependent apoptosis or caspase-independent necroptosis. Recent studies report that upon TLR4 or TNF receptor 1 (TNFR1) signaling in macrophages, the ripoptosome can also induce NLRP3 inflammasome formation and IL-1ß maturation. Whether neutrophils have the capacity to assemble a ripoptosome to induce cell death and inflammasome activation during TLR4 and TNFR1 signaling is unclear. In this study, we demonstrate that murine neutrophils can signal via TNFR1-driven ripoptosome assembly to induce both cell death and IL-1ß maturation. However, unlike macrophages, neutrophils suppress TLR4-dependent cell death and NLRP3 inflammasome activation during IAP inhibition via deficiencies in the CD14/TRIF arm of TLR4 signaling.

Defining a therapeutic window for kinase inhibitors in leukemia to avoid neutropenia.

Neutropenia represents one of the major dose-limiting toxicities of many current cancer therapies. To circumvent the off-target effects of cytotoxic chemotherapeutics, kinase inhibitors are increasingly being used as an adjunct therapy to target leukemia. In this study, we conducted a screen of leukemic cell lines in parallel with primary neutrophils to identify kinase inhibitors with the capacity to induce apoptosis of myeloid and lymphoid cell lines whilst sparing primary mouse and human neutrophils. We have utilized a high-throughput live cell imaging platform to demonstrate that cytotoxic drugs have limited effects on neutrophil viability but are toxic to hematopoietic progenitor cells, with the exception of the topoisomerase I inhibitor SN-38. The parallel screening of kinase inhibitors revealed that mouse and human neutrophil viability is dependent on cyclin-dependent kinase (CDK) activity but surprisingly only partially dependent on PI3 kinase and JAK/STAT signaling, revealing dominant pathways contributing to neutrophil viability. Mcl-1 haploinsufficiency sensitized neutrophils to CDK inhibition, demonstrating that Mcl-1 is a direct target for CDK inhibitors. This study reveals a therapeutic window for the kinase inhibitors BEZ235, BMS-3, AZD7762, and (R)-BI-2536 to induce apoptosis of leukemia cell lines whilst maintaining immunocompetence and hemostasis.

Phosphatidylserine externalization, "necroptotic bodies" release, and phagocytosis during necroptosis.

Necroptosis is a regulated, nonapoptotic form of cell death initiated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins. It is considered to be a form of regulated necrosis, and, by lacking the "find me" and "eat me" signals that are a feature of apoptosis, necroptosis is considered to be inflammatory. One such "eat me" signal observed during apoptosis is the exposure of phosphatidylserine (PS) on the outer plasma membrane. Here, we demonstrate that necroptotic cells also expose PS after phosphorylated mixed lineage kinase-like (pMLKL) translocation to the membrane. Necroptotic cells that expose PS release extracellular vesicles containing proteins and pMLKL to their surroundings. Furthermore, inhibition of pMLKL after PS exposure can reverse the process of necroptosis and restore cell viability. Finally, externalization of PS by necroptotic cells drives recognition and phagocytosis, and this may limit the inflammatory response to this nonapoptotic form of cell death. The exposure of PS to the outer membrane and to extracellular vesicles is therefore a feature of necroptotic cell death and may serve to provide an immunologically-silent window by generating specific "find me" and "eat me" signals.

A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization.

The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as "lipid rafts." Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01). HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05), and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05). Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.

A motive for killing: effector functions of regulated lytic cell death.

Immunological responses activated by pathogen recognition come in many guises. The proliferation, differentiation and recruitment of immune cells, and the production of inflammatory cytokines and chemokines are central to lifelong immunity. Cell death serves as a key function in the resolution of innate and adaptive immune responses. It also coordinates cell-intrinsic effector functions to restrict infection. Necrosis was formally considered a passive form of cell death or a consequence of pathogen virulence factor expression, and necrotic tissue is frequently associated with infection. However, there is now emerging evidence that points to a role for regulated forms of necrosis, such as pyroptosis and necroptosis, driving inflammation and shaping the immune response.

IL-18 Production from the NLRP1 Inflammasome Prevents Obesity and Metabolic Syndrome.

Interleukin-18 (IL-18) is activated by Caspase-1 in inflammasome complexes and has anti-obesity effects; however, it is not known which inflammasome regulates this process. We found that mice lacking the NLRP1 inflammasome phenocopy mice lacking IL-18, with spontaneous obesity due to intrinsic lipid accumulation. This is exacerbated when the mice are fed a high-fat diet (HFD) or a high-protein diet, but not when mice are fed a HFD with low energy density (high fiber). Furthermore, mice with an activating mutation in NLRP1, and hence increased IL-18, have decreased adiposity and are resistant to diet-induced metabolic dysfunction. Feeding these mice a HFD further increased plasma IL-18 concentrations and strikingly resulted in loss of adipose tissue mass and fatal cachexia, which could be prevented by genetic deletion of IL-18. Thus, NLRP1 is an innate immune sensor that functions in the context of metabolic stress to produce IL-18, preventing obesity and metabolic syndrome.

Fight or flight: regulation of emergency hematopoiesis by pyroptosis and necroptosis.

PURPOSE OF REVIEW: A feature of the innate immune response that is conserved across kingdoms is the induction of cell death. In this review, we discuss the direct and indirect effects of increased inflammatory cell death, including pyroptosis - a caspase-1-dependent cell death - and necroptosis - a receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein-dependent, caspase-independent cell death - on emergency hematopoiesis. RECENT FINDINGS: Activation of nonapoptotic cell death pathways during infection can trigger release of cytokines and/or damage-associated molecular patterns such as interleukin (IL)-1α, IL-1ß, IL-18, IL-33, high-mobility group protein B1, and mitochondrial DNA to promote emergency hematopoiesis. During systemic infection, pyroptosis and necroptosis can directly kill hematopoietic stem and progenitor cells, which results in impaired hematopoiesis, cytopenia, and immunosuppression. Although originally described as discrete entities, there now appear to be more intimate connections between the nonapoptotic and death receptor signaling pathways. SUMMARY: The choice to undergo pyroptotic and necroptotic cell death constitutes a rapid response system serving to eliminate infected cells, including hematopoietic stem and progenitor cells. This system has the potential to be detrimental to emergency hematopoiesis during severe infection. We discuss the potential of pharmacological intervention for the pyroptosis and necroptosis pathways that may be beneficial during periods of infection and emergency hematopoiesis.

Aberrant actin depolymerization triggers the pyrin inflammasome and autoinflammatory disease that is dependent on IL-18, not IL-1ß.

Gain-of-function mutations that activate the innate immune system can cause systemic autoinflammatory diseases associated with increased IL-1ß production. This cytokine is activated identically to IL-18 by an intracellular protein complex known as the inflammasome; however, IL-18 has not yet been specifically implicated in the pathogenesis of hereditary autoinflammatory disorders. We have now identified an autoinflammatory disease in mice driven by IL-18, but not IL-1ß, resulting from an inactivating mutation of the actin-depolymerizing cofactor Wdr1. This perturbation of actin polymerization leads to systemic autoinflammation that is reduced when IL-18 is deleted but not when IL-1 signaling is removed. Remarkably, inflammasome activation in mature macrophages is unaltered, but IL-18 production from monocytes is greatly exaggerated, and depletion of monocytes in vivo prevents the disease. Small-molecule inhibition of actin polymerization can remove potential danger signals from the system and prevents monocyte IL-18 production. Finally, we show that the inflammasome sensor of actin dynamics in this system requires caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain, and the innate immune receptor pyrin. Previously, perturbation of actin polymerization by pathogens was shown to activate the pyrin inflammasome, so our data now extend this guard hypothesis to host-regulated actin-dependent processes and autoinflammatory disease.

RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL.

RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3-caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1ß inflammatory responses independent of MLKL and necroptotic cell death.

Fas regulates neutrophil lifespan during viral and bacterial infection.

The regulation of neutrophil lifespan is critical for a circumscribed immune response. Neutrophils are sensitive to Fas/CD95 death receptor signaling in vitro, but it is unknown if Fas regulates neutrophil lifespan in vivo. We hypothesized that FasL-expressing CD8(+) T cells, which kill antigen-stimulated T cells during chronic viral infection, can also induce neutrophil death in tissues during infection. With the use of LysM-Cre Fas(fl/fl) mice, which lack Fas expression in macrophages and neutrophils, we show that Fas regulates neutrophil lifespan during lymphocytic choriomeningitis virus (LCMV) infection in the lung, peripheral blood, and spleen. Fas also contributed to the regulation of neutrophil numbers in the colon of Citrobacter rodentium-infected mice. To examine the effects of infection on Fas activation in neutrophils, we primed neutrophils with TLR ligands or IL-18, resulting in ablation of Fas death receptor signaling. These data provide the first in vivo genetic evidence that neutrophil lifespan is controlled by death receptor signaling and provide a mechanism to account for neutrophil resistance to Fas stimulation during infection.

RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.