Discovery of a Novel Missense Variant in NLRP3 Causing Atypical Cryopyrin-Associated Periodic Syndromes With Hearing Loss as the Primary Presentation, Responsive to Anti-Interleukin-1 Therapy.

OBJECTIVE: Cryopyrin-associated periodic syndromes (CAPS), also known as NLRP3-associated autoinflammatory diseases, are a spectrum of rare autoinflammatory diseases caused by gain-of-function variants in the NLRP3 gene, resulting in inflammasome hyperactivation and dysregulated release of interleukin-1ß (IL-1ß). Many patients with CAPS develop progressive sensorineural hearing loss (SNHL) because of cochlear autoinflammation, which may be the sole manifestation in rare cases. This study was undertaken to establish the suspected diagnosis of CAPS in a family presenting with autosomal-dominant progressive/acute SNHL and a novel missense variant in the NLRP3 gene of unknown significance (NM_001079821.3:c.1784G>A p.Ser595Asn). METHODS: We conducted an ex vivo functional assessment of the NLRP3 inflammasome in heterozygous individuals (n = 10) and healthy family members (n = 5). RESULTS: The assay revealed hyperactivation of the inflammasome among heterozygous individuals, supporting the hypothesis that this missense variant is a pathogenic gain-of-function variant. Administration of IL-1 receptor antagonist resulted in a substantial clinical improvement among pediatric patients, who exhibited near resolution of hearing impairment within 1 to 3 months of treatment. CONCLUSION: Our findings highlight the crucial role of early diagnosis and treatment with an anti-IL-1 agent in reversing cochlear damage. Furthermore, our results suggest that high- and ultrahigh-frequency ranges need to be included in the auditory assessment to enable early detection of subclinical SNHL. Finally, incorporating functional inflammasome assessment as part of the clinical evaluation could establish the diagnosis in inconclusive cases.

NET formation is a default epigenetic program controlled by PAD4 in apoptotic neutrophils.

Neutrophil extracellular traps (NETs) not only counteract bacterial and fungal pathogens but can also promote thrombosis, autoimmunity, and sterile inflammation. The presence of citrullinated histones, generated by the peptidylarginine deiminase 4 (PAD4), is synonymous with NETosis and is considered independent of apoptosis. Mitochondrial- and death receptor-mediated apoptosis promote gasdermin E (GSDME)-dependent calcium mobilization and membrane permeabilization leading to histone H3 citrullination (H3Cit), nuclear DNA extrusion, and cytoplast formation. H3Cit is concentrated at the promoter in bone marrow neutrophils and redistributes in a coordinated process from promoter to intergenic and intronic regions during apoptosis. Loss of GSDME prevents nuclear and plasma membrane disruption of apoptotic neutrophils but prolongs early apoptosis-induced cellular changes to the chromatin and cytoplasmic granules. Apoptotic signaling engages PAD4 in neutrophils, establishing a cellular state that is primed for NETosis, but that occurs only upon membrane disruption by GSDME, thereby redefining the end of life for neutrophils.

A Vibrio T6SS-Mediated Lethality in an Aquatic Animal Model.

Bacteria belonging to the genus Vibrio include many known and emerging pathogens. Horizontal gene transfer of pathogenicity islands is a major contributor to the emergence of new pathogenic Vibrio strains. Here, we use the brine shrimp Artemia salina as a model and show that the marine bacterium Vibrio proteolyticus uses a horizontally shared type VI secretion system, T6SS3, to intoxicate a eukaryotic host. Two T6SS3 effectors, which were previously shown to induce inflammasome-mediated pyroptotic cell death in mammalian phagocytic cells, contribute to this toxicity. Furthermore, we find a novel T6SS3 effector that also contributes to the lethality mediated by this system against Artemia salina. Therefore, our results reveal a T6SS that is shared among diverse vibrios and mediates host lethality, indicating that it can lead to the emergence of new pathogenic strains. IMPORTANCE The rise in sea surface temperature has been linked to the spread of bacteria belonging to the genus Vibrio and the human illnesses associated with them. Since vibrios often share virulence traits horizontally, a better understanding of their virulence potential and determinants can prepare us for new emerging pathogens. In this work, we showed that a toxin delivery system found in various vibrios mediates lethality in an aquatic animal. Taken together with previous reports showing that the same system induces inflammasome-mediated cell death in mammalian phagocytic cells, our findings suggest that this delivery system and its associated toxins may contribute to the emergence of pathogenic strains.

Adaptive immune response to BNT162b2 mRNA vaccine in immunocompromised adolescent patients.

Protective immunity against COVID-19 is orchestrated by an intricate network of innate and adaptive anti-viral immune responses. Several vaccines have been rapidly developed to combat the destructive effects of COVID-19, which initiate an immunological cascade that results in the generation of neutralizing antibodies and effector T cells towards the SARS-CoV-2 spike protein. Developing optimal vaccine-induced anti-SARS- CoV-2 protective immunity depends on a fully competent immune response. Some evidence was gathered on the effects of vaccination outcomes in immunocompromised adult individuals. Nonetheless, protective immunity elicited by the Pfizer Biontech BNT162b2 vaccine in immunocompromised adolescents received less attention and was mainly focused on the antibody response and their neutralization potential. The overall immune response, including T-cell activities, was largely understudied. In this study, we characterized the immune response of vaccinated immunocompromised adolescents. We found that immunocompromised adolescents, which may fail to elicit a humoral response and develop antibodies, may still develop cellular T-cell immunity towards SARS-CoV-2 infections. Furthermore, most immunocompromised adolescents due to genetic disorders or drugs (Kidney and liver transplantation) still develop either humoral, cellular or both arms of immunity towards SARS-CoV-2 infections. We also demonstrate that most patients could mount a cellular or humoral response even after six months post 2nd vaccination. The findings that adolescents immunocompromised patients respond to some extent to vaccination are promising. Finally, they question the necessity for additional vaccination boosting regimens for this population who are not at high risk for severe disease, without further testing of their post-vaccination immune status.

Differential regulation of Type 1 and Type 2 mouse eosinophil activation by apoptotic cells.

Eosinophils are multifunctional, evolutionary conserved leukocytes that are involved in a plethora of responses ranging from regulation of tissue homeostasis, host defense and cancer. Although eosinophils have been studied mostly in the context of Type 2 inflammatory responses, it is now evident that they participate in Type 1 inflammatory responses and can respond to Type 1 cytokines such as IFN-γ. Notably, both Type 1- and Type 2 inflammatory environments are characterized by tissue damage and cell death. Collectively, this raises the possibility that eosinophils can interact with apoptotic cells, which can alter eosinophil activation in the inflammatory milieu. Herein, we demonstrate that eosinophils can bind and engulf apoptotic cells. We further show that exposure of eosinophils to apoptotic cells induces marked transcriptional changes in eosinophils, which polarize eosinophils towards an anti-inflammatory phenotype that is associated with wound healing and cell migration. Using an unbiased RNA sequencing approach, we demonstrate that apoptotic cells suppress the inflammatory responses of eosinophils that were activated with IFN-γ + E. coli (e.g., Type 1 eosinophils) and augment IL-4-induced eosinophil activation (e.g., Type 2 eosinophils). These data contribute to the growing understanding regarding the heterogeneity of eosinophil activation patterns and highlight apoptotic cells as potential regulators of eosinophil polarization.

Post-phagocytosis activation of NLRP3 inflammasome by two novel T6SS effectors.

The type VI secretion system (T6SS) is used by bacteria to deliver toxic effectors directly into target cells. Most T6SSs mediate antibacterial activities, whereas the potential anti-eukaryotic role of T6SS remains understudied. Here, we found a Vibrio T6SS that delivers two novel effectors into mammalian host immune cells. We showed that these effectors induce a pyroptotic cell death in a phagocytosis-dependent manner; we identified the NLRP3 inflammasome as being the underlying mechanism leading to the T6SS-induced pyroptosis. Moreover, we identified a compensatory T6SS-induced pathway that is activated upon inhibition of the canonical pyroptosis pathway. Genetic analyses revealed possible horizontal spread of this T6SS and its anti-eukaryotic effectors into emerging pathogens in the marine environment. Our findings reveal novel T6SS effectors that activate the host inflammasome and possibly contribute to virulence and to the emergence of bacterial pathogens.

Proteomic analysis of necroptotic extracellular vesicles.

Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.

Walking down the memory lane with SARS-CoV-2 B cells.

The B-cell response to COVID-19 vaccines in convalescent individuals.

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.

Metastasis-Entrained Eosinophils Enhance Lymphocyte-Mediated Antitumor Immunity.

The recognition of the immune system as a key component of the tumor microenvironment (TME) led to promising therapeutics. Because such therapies benefit only subsets of patients, understanding the activities of immune cells in the TME is required. Eosinophils are an integral part of the TME especially in mucosal tumors. Nonetheless, their role in the TME and the environmental cues that direct their activities are largely unknown. We report that breast cancer lung metastases are characterized by resident and recruited eosinophils. Eosinophil recruitment to the metastatic sites in the lung was regulated by G protein-coupled receptor signaling but independent of CCR3. Functionally, eosinophils promoted lymphocyte-mediated antitumor immunity. Transcriptome and proteomic analyses identified the TME rather than intrinsic differences between eosinophil subsets as a key instructing factor directing antitumorigenic eosinophil activities. Specifically, TNFα/IFNγ-activated eosinophils facilitated CD4+ and CD8+ T-cell infiltration and promoted antitumor immunity. Collectively, we identify a mechanism by which the TME trains eosinophils to adopt antitumorigenic properties, which may lead to the development of eosinophil-targeted therapeutics. SIGNIFICANCE: These findings demonstrate antitumor activities of eosinophils in the metastatic tumor microenvironment, suggesting that harnessing eosinophil activity may be a viable clinical strategy in patients with cancer.

BNT162b2 vaccination effectively prevents the rapid rise of SARS-CoV-2 variant B.1.1.7 in high-risk populations in Israel.

Since the emergence of the SARS-CoV-2 pandemic, various genetic variants have been described. The B.1.1.7 variant, which emerged in England during December 2020, is associated with increased infectivity. Therefore, its pattern of spread is of great importance. The Israeli government established three national programs: massive RT-PCR testing, focused surveillance in nursing homes, and robust prioritized vaccination with BNT162b2. To define the impact of the aforementioned programs, we analyze data from ∼300,000 RT-PCR samples collected from December 6, 2020, to February 10, 2021. We reveal that the B.1.1.7 is 45% (95% confidence interval [CI]: 20%-60%) more transmissible than the wild-type strain and has become the dominant strain in Israel within 3.5 weeks. Despite the rapid increase in viral spread, focused RT-PCR testing and prioritized vaccination programs are capable of preventing the spread of the B.1.1.7 variant in the elderly. Therefore, proactive surveillance programs, combined with prioritized vaccination, are achievable and can reduce severe illness and subsequent death.

Rapid seroconversion and persistent functional IgG antibodies in severe COVID-19 patients correlates with an IL-12p70 and IL-33 signature.

Despite ongoing efforts to characterize the host response toward SARS-CoV-2, a major gap in our knowledge still exists regarding the magnitude and duration of the humoral response. Analysis of the antibody response in mild versus moderate/severe patients, using our new developed quantitative electrochemiluminescent assay for detecting IgM/IgA/IgG antibodies toward SARS-CoV-2 antigens, revealed a rapid onset of IgG/IgA antibodies, specifically in moderate/severe patients. IgM antibodies against the viral receptor binding domain, but not against nucleocapsid protein, were detected at early stages of the disease. Furthermore, we observed a marked reduction in IgM/IgA antibodies over-time. Adapting our assay for ACE2 binding-competition, demonstrated that the presence of potentially neutralizing antibodies is corelated with IgG/IgA. Finally, analysis of the cytokine profile in COVID-19 patients revealed unique correlation of an IL-12p70/IL33 and IgG seroconversion, which correlated with disease severity. In summary, our comprehensive analysis has major implications on the understanding and monitoring of SARS-CoV-2 infections.

NLRP1 variant M1184V decreases inflammasome activation in the context of DPP9 inhibition and asthma severity.

BACKGROUND: NLRP1 is an innate immune sensor that can form cytoplasmic inflammasome complexes. Polymorphisms in NLRP1 are linked to asthma; however, there is currently no functional or mechanistic explanation for this. OBJECTIVE: We sought to clarify the role of NLRP1 in asthma pathogenesis. METHODS: Results from the GALA II cohort study were used to identify a link between NLRP1 and asthma in Mexican Americans. In vitro and in vivo models for NLRP1 activation were applied to investigate the role of this inflammasome in asthma at the molecular level. RESULTS: We document the association of an NLRP1 haplotype with asthma for which the single nucleotide polymorphism rs11651270 (M1184V) individually is the most significant. Surprisingly, M1184V increases NLRP1 activation in the context of N-terminal destabilization, but decreases NLRP1 activation on dipeptidyl peptidase 9 inhibition. In vitro studies demonstrate that M1184V increases binding to dipeptidyl peptidase 9, which can account for its inhibitory role in this context. In addition, in vivo data from a mouse model of airway inflammation reveal a protective role for NLRP1 inflammasome activation reducing eosinophilia in this setting. CONCLUSIONS: Linking our in vitro and in vivo results, we found that the NLRP1 variant M1184V reduces inflammasome activation in the context of dipeptidyl peptidase 9 inhibition and could thereby increase asthma severity. Our studies may have implications for the treatment of asthma in patients carrying this variant of NLRP1.

Necroptotic extracellular vesicles - present and future.

Necroptosis is a receptor-interacting protein kinase 3 (RIPK3)-/mixed lineage kinase domain-like(MLKL)-dependent and caspase-independent form of cell death that promotes inflammation. We, and others, have recently shown that necroptotic cells release extracellular vesicles (EVs). This finding has highlighted that the impact of necroptosis extends well beyond its cell death function. In this review, we summarize the general characteristics, biogenesis, and function of EVs, as well as their role in cancer. In addition, we outline our current knowledge regarding necroptotic EVs, including their recently discovered and analyzed proteome. We examine the accumulating evidence for a role for necroptosis in anti-tumor immunity. Finally, we suggest that necroptotic EVs play an important role in the necroptosis-induced immune response and may, therefore, be exploited in cancer immunotherapy applications.

Potential Antigenic Cross-reactivity Between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Dengue Viruses.

BACKGROUND: Coronavirus disease 2019 (COVID-19) and dengue fever are difficult to distinguish given shared clinical and laboratory features. Failing to consider COVID-19 due to false-positive dengue serology can have serious implications. We aimed to assess this possible cross-reactivity. METHODS: We analyzed clinical data and serum samples from 55 individuals with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To assess dengue serology status, we used dengue-specific antibodies by means of lateral-flow rapid test, as well as enzyme-linked immunosorbent assay (ELISA). Additionally, we tested SARS-CoV-2 serology status in patients with dengue and performed in-silico protein structural analysis to identify epitope similarities. RESULTS: Using the dengue lateral-flow rapid test we detected 12 positive cases out of the 55 (21.8%) COVID-19 patients versus zero positive cases in a control group of 70 healthy individuals (P = 2.5E-5). This includes 9 cases of positive immunoglobulin M (IgM), 2 cases of positive immunoglobulin G (IgG), and 1 case of positive IgM as well as IgG antibodies. ELISA testing for dengue was positive in 2 additional subjects using envelope protein directed antibodies. Out of 95 samples obtained from patients diagnosed with dengue before September 2019, SARS-CoV-2 serology targeting the S protein was positive/equivocal in 21 (22%) (16 IgA, 5 IgG) versus 4 positives/equivocal in 102 controls (4%) (P = 1.6E-4). Subsequent in-silico analysis revealed possible similarities between SARS-CoV-2 epitopes in the HR2 domain of the spike protein and the dengue envelope protein. CONCLUSIONS: Our findings support possible cross-reactivity between dengue virus and SARS-CoV-2, which can lead to false-positive dengue serology among COVID-19 patients and vice versa. This can have serious consequences for both patient care and public health.

A comparative genomics methodology reveals a widespread family of membrane-disrupting T6SS effectors.

Gram-negative bacteria deliver effectors via the type VI secretion system (T6SS) to outcompete their rivals. Each bacterial strain carries a different arsenal of effectors; the identities of many remain unknown. Here, we present an approach to identify T6SS effectors encoded in bacterial genomes of interest, without prior knowledge of the effectors' domain content or genetic neighborhood. Our pipeline comprises a comparative genomics analysis followed by screening using a surrogate T6SS+ strain. Using this approach, we identify an antibacterial effector belonging to the T6SS1 of Vibrio parahaemolyticus, representing a widespread family of T6SS effectors sharing a C-terminal domain that we name Tme (Type VI membrane-disrupting effector). Tme effectors function in the periplasm where they intoxicate bacteria by disrupting membrane integrity. We believe our approach can be scaled up to identify additional T6SS effectors in various bacterial genera.

Vibrio pore-forming leukocidin activates pyroptotic cell death via the NLRP3 inflammasome.

Cell death mechanisms are central to combat infections and to drive inflammation. The inflammasome controls infection through activation of caspase-1 leading to either IL-1ß dependent inflammation, or pyroptotic cell death in infected cells. Hemolysins, which are pore-forming toxins (PFTs), alter the permeability of the host target membrane, often leading to cell death. We previously discovered a leukocidin domain-containing PFT produced by the Gram-negative bacterium Vibrio proteolyticus, named VPRH. VPRH constitutes a distinct, understudied class within the leukocidin superfamily, which is distributed among several photogenic Vibrios. Since PFTs of other pathogens were shown to activate the inflammasome pathway, we hypothesized that VPRH-induced cell death is mediated by direct activation of the inflammasome in mammalian immune host cells. Indeed, we found that VPRH induced a two-step cell death in macrophages. The first, a rapid step, was mediated by activating the NLRP3 inflammasome, leading to caspase-1 activation that resulted in IL-1ß secretion and pyroptosis. The second step was independent of the inflammasome; however, its mechanism remains unknown. This study sets the foundation for better understanding the immunological consequences of inflammasome activation by a new leukocidin class of toxins, which may be shared between marine bacteria and give rise to new pathogenic isolates.

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.

Flipping the dogma - phosphatidylserine in non-apoptotic cell death.

The exposure of phosphatidylserine (PS) on the outer plasma membrane has long been considered a unique feature of apoptotic cells. Together with other "eat me" signals, it enables the recognition and phagocytosis of dying cells (efferocytosis), helping to explain the immunologically-silent nature of apoptosis. Recently, however, PS exposure has also been reported in non-apoptotic forms of regulated inflammatory cell death, such as necroptosis, challenging previous dogma. In this review, we outline the evidence for PS exposure in non-apoptotic cells and extracellular vesicles (EVs), and discuss possible mechanisms based on our knowledge of apoptotic-PS exposure. In addition, we examine the outcomes of non-apoptotic PS exposure, including the reversibility of cell death, efferocytosis, and consequent inflammation. By examining PS biology, we challenge the established approach of distinguishing apoptosis from other cell death pathways by AnnexinV staining of PS externalization. Finally, we re-evaluate how PS exposure is thought to define apoptosis as an immunologically silent process distinct from other non-apoptotic and inflammatory cell death pathways. Ultimately, we suggest that a complete understanding of how regulated cell death processes affect the immune system is far from being fully elucidated.

NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis.

Cancer-Associated Fibroblasts (CAFs) were shown to orchestrate tumour-promoting inflammation in multiple malignancies, including breast cancer. However, the molecular pathways that govern the inflammatory role of CAFs are poorly characterised. In this study we found that fibroblasts sense damage-associated molecular patterns (DAMPs), and in response activate the NLRP3 inflammasome pathway, resulting in instigation of pro-inflammatory signalling and secretion of IL-1ß. This upregulation was evident in CAFs in mouse and in human breast carcinomas. Moreover, CAF-derived inflammasome signalling facilitated tumour growth and metastasis, which was attenuated when NLRP3 or IL-1ß were specifically ablated. Functionally, CAF-derived inflammasome promoted tumour progression and metastasis by modulating the tumour microenvironment towards an immune suppressive milieu and by upregulating the expression of adhesion molecules on endothelial cells. Our findings elucidate a mechanism by which CAFs promote breast cancer progression and metastasis, by linking the physiological tissue damage response of fibroblasts with tumour-promoting inflammation.