Mechanistic basis for potassium efflux-driven activation of the human NLRP1 inflammasome.

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple nonhematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAKα, p38, and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAKα knockout, or pharmacologic inhibitors of ZAKα and p38 kinase activities. By surveying a panel of ionophores, we show that electroneutrality of ion movement is essential to activate ZAKα-driven RSR and a greater extent of K+ depletion is necessary to activate ZAKα-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux, and innate immunity.

Regulated cell death in neutrophils: From apoptosis to NETosis and pyroptosis.

Neutrophils are among the most abundant immune cells, representing about 50%- 70% of all circulating leukocytes in humans. Neutrophils rapidly infiltrate inflamed tissues and play an essential role in host defense against infections. They exert microbicidal activity through a variety of specialized effector mechanisms, including phagocytosis, production of reactive oxygen species, degranulation and release of secretory vesicles containing broad-spectrum antimicrobial factors. In addition to their homeostatic turnover by apoptosis, recent studies have revealed the mechanisms by which neutrophils undergo various forms of regulated cell death. In this review, we will discuss the different modes of regulated cell death that have been described in neutrophils, with a particular emphasis on the current understanding of neutrophil pyroptosis and its role in infections and autoinflammation.

Druggable redox pathways against Mycobacterium abscessus in cystic fibrosis patient-derived airway organoids.

Mycobacterium abscessus (Mabs) drives life-shortening mortality in cystic fibrosis (CF) patients, primarily because of its resistance to chemotherapeutic agents. To date, our knowledge on the host and bacterial determinants driving Mabs pathology in CF patient lung remains rudimentary. Here, we used human airway organoids (AOs) microinjected with smooth (S) or rough (R-)Mabs to evaluate bacteria fitness, host responses to infection, and new treatment efficacy. We show that S Mabs formed biofilm, and R Mabs formed cord serpentines and displayed a higher virulence. While Mabs infection triggers enhanced oxidative stress, pharmacological activation of antioxidant pathways resulted in better control of Mabs growth and reduced virulence. Genetic and pharmacological inhibition of the CFTR is associated with better growth and higher virulence of S and R Mabs. Finally, pharmacological activation of antioxidant pathways inhibited Mabs growth, at least in part through the quinone oxidoreductase NQO1, and improved efficacy in combination with cefoxitin, a first line antibiotic. In conclusion, we have established AOs as a suitable human system to decipher mechanisms of CF-driven respiratory infection by Mabs and propose boosting of the NRF2-NQO1 axis as a potential host-directed strategy to improve Mabs infection control.

EEF2-inactivating toxins engage the NLRP1 inflammasome and promote epithelial barrier disruption.

Human airway and corneal epithelial cells, which are critically altered during chronic infections mediated by Pseudomonas aeruginosa, specifically express the inflammasome sensor NLRP1. Here, together with a companion study, we report that the NLRP1 inflammasome detects exotoxin A (EXOA), a ribotoxin released by P. aeruginosa type 2 secretion system (T2SS), during chronic infection. Mechanistically, EXOA-driven eukaryotic elongation factor 2 (EEF2) ribosylation and covalent inactivation promote ribotoxic stress and subsequent NLRP1 inflammasome activation, a process shared with other EEF2-inactivating toxins, diphtheria toxin and cholix toxin. Biochemically, irreversible EEF2 inactivation triggers ribosome stress-associated kinases ZAKα- and P38-dependent NLRP1 phosphorylation and subsequent proteasome-driven functional degradation. Finally, cystic fibrosis cells from patients exhibit exacerbated P38 activity and hypersensitivity to EXOA-induced ribotoxic stress-dependent NLRP1 inflammasome activation, a process inhibited by the use of ZAKα inhibitors. Altogether, our results show the importance of P. aeruginosa virulence factor EXOA at promoting NLRP1-dependent epithelial damage and identify ZAKα as a critical sensor of virulence-inactivated EEF2.

A druggable copper-signalling pathway that drives inflammation.

Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2-4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.

Establishing 20S Proteasome Genetic, Translational and Post-Translational Status from Precious Biological and Patient Samples with Top-Down MS.

The mammalian 20S catalytic core of the proteasome is made of 14 different subunits (α1-7 and ß1-7) but exists as different subtypes depending on the cell type. In immune cells, for instance, constitutive catalytic proteasome subunits can be replaced by the so-called immuno-catalytic subunits, giving rise to the immunoproteasome. Proteasome activity is also altered by post-translational modifications (PTMs) and by genetic variants. Immunochemical methods are commonly used to investigate these PTMs whereby protein-tagging is necessary to monitor their effect on 20S assembly. Here, we present a new miniaturized workflow combining top-down and bottom-up mass spectrometry of immunopurified 20S proteasomes that analyze the proteasome assembly status as well as the full proteoform footprint, revealing PTMs, mutations, single nucleotide polymorphisms (SNPs) and induction of immune-subunits in different biological samples, including organoids, biopsies and B-lymphoblastoid cell lines derived from patients with proteasome-associated autoinflammatory syndromes (PRAAS). We emphasize the benefits of using top-down mass spectrometry in preserving the endogenous conformation of protein modifications, while enabling a rapid turnaround (1 h run) and ensuring high sensitivity (1-2 pmol) and demonstrate its capacity to semi-quantify constitutive and immune proteasome subunits.

Collective Sex and Committed Relationships: A Qualitative Exploration of Relationship Desires Among Gay Men Who Attend Sex Clubs.

Studies have documented gay men's engagement in collective sex (e.g., group sex in public or commercial environments) but little attention has been paid to these men's relationship desires or agreements. We report on qualitative interviews with 20 gay men who attended private sex clubs in New York City, asking how participants navigated sometimes conflicting desires for collective sex and committed relationships. Participants felt that collective sex was either a) incompatible with relationships, which should be monogamous; b) complementary to a primary non-monogamous relationship; or c) at least as important as relationships. Gay men attending sex clubs attribute different value to collective sex and to committed relationships, and experience different challenges in satisfying their desires for intimacy. Resources could help gay men navigating these apparently conflicting desires and making suitable choices regarding their relationships and/or sexual agreements.

Attitudes Toward HIV-Positive Status Disclosure Among U=U-Aware Sexual and Gender Minority Individuals in the USA: a Consensual Qualitative Research Approach.

Introduction: The U=U (i.e., undetectable equals untransmittable) campaign is founded upon biomedical advancements that have positioned HIV as a manageable condition with effectively zero risk of transmission. In spite of these developments, attitudes of sexual and gender minority populations regarding the necessity of seropositive status disclosure remain unexamined. Methods: The current study analyzed qualitative data regarding the necessity of seropositive status disclosure from 62 sexual minority men as well as transgender and gender non-conforming individuals who have sex with men from 2020 to 2021. Results: The majority of participants believed disclosure to be necessary and invoked several social and structural factors that informed their attitudes. Participants cited HIV criminalization laws, the ethics of non-disclosure, and disclosure as a means of educating sex partners when appraising the necessity of disclosure. Participants also presented concerns regarding U=U efficacy and HIV stigma. Conclusions: Findings indicate that the disclosure of seropositive status to sex partners is still important to U=U-aware sexual and gender minority individuals. The majority of the study sample, irrespective of HIV status, believed seropositive status disclosure was necessary in advance of sex. Policy Implications: Findings suggest opportunities for public health messaging to remediate concerns about U=U efficacy, combat misinformation, and clarify out-of-date information on HIV criminalization.

EM-Driven Unsupervised Learning for Efficient Motion Segmentation.

In this paper, we present a CNN-based fully unsupervised method for motion segmentation from optical flow. We assume that the input optical flow can be represented as a piecewise set of parametric motion models, typically, affine or quadratic motion models. The core idea of our work is to leverage the Expectation-Maximization (EM) framework in order to design in a well-founded manner a loss function and a training procedure of our motion segmentation neural network that does not require either ground-truth or manual annotation. However, in contrast to the classical iterative EM, once the network is trained, we can provide a segmentation for any unseen optical flow field in a single inference step and without estimating any motion models. We investigate different loss functions including robust ones and propose a novel efficient data augmentation technique on the optical flow field, applicable to any network taking optical flow as input. In addition, our method is able by design to segment multiple motions. Our motion segmentation network was tested on four benchmarks, DAVIS2016, SegTrackV2, FBMS59, and MoCA, and performed very well, while being fast at test time.

Sexual and Gender Minority Individuals' Interest in Sexual Health Services at Collective Sex Venues in New York City.

Sexual and gender minority individuals who attend collective sex venues (CSVs; establishments where people can have sex in groups or the presence of others) are at elevated risk for HIV and STIs. On-site sexual health interventions have been attempted at CSVs, but attendees' interest in receiving such services is under-investigated. This paper presents results from a 2020 online cross-sectional survey completed by 342 sexual and gender minority individuals who attended CSVs in New York City. Interest in services such as on-site testing for STIs, testing vans near CSVs, and informational referrals was overall high, particularly among younger participants. Among participants who reported being HIV negative, those of younger age and those who were not using PrEP reported being more likely to take an HIV test if it would be offered at CSVs. In open-text survey responses, participants expressed interest in CSVs providing free prevention services such as HIV/STI testing, PEP, PrEP, and STI medications or vaccination, as well as in ways to improve norms surrounding condom use and consent at these venues. Some participants expressed barriers to on-site services such as privacy concerns, preexisting access to health services, an emphasis on personal responsibility, and negative reactions to the presence of service providers. However, some participants also felt that these services could be delivered in a positive, acceptable, and non-judgmental way, especially by involving CSV organizers and attendees in their implementation. Findings from this study can inform future initiatives to develop sexual health interventions at CSVs.

Interest in Sex Work-Related Resources Among Gay, Bisexual, and Other Men Who Have Sex with Men Engaged in Sex Work.

Introduction: Practicing sex work can present considerable risks to an individual's sexual health and overall well-being. Though resources tailored for sex workers can help mitigate such risks, little is known about male sex workers' interest in them. Methods: From 2018 to 2020, we conducted in-depth telephone interviews with 180 U.S. men who engaged in sex work with clients met online. We inquired about what sex work-related resources they were aware of or had used, the specific content of resources they would want, and whether they would use such resources or not. Results: Few participants had used sex work-related resources, but many had found material related to sex work through organizations and online media. While some participants expressed interest in resources to help make their engagement in sex work safer and more profitable, others were interested in resources that would help them address the conditions that had led them to sex work in the first place. Participants also discussed some barriers to resource utilization such as low perceived need, privacy concerns, and low credibility of the material. Conclusions & Policy Implications: Results show that there is substantial interest in sex work-related resources among men engaged in the practice. However, programs should carefully consider potential barriers to utilization when developing these resources.

Antiviral and Anti-Inflammatory Activities of Fluoxetine in a SARS-CoV-2 Infection Mouse Model.

The coronavirus disease 2019 (COVID-19) pandemic continues to cause significant morbidity and mortality worldwide. Since a large portion of the world's population is currently unvaccinated or incompletely vaccinated and has limited access to approved treatments against COVID-19, there is an urgent need to continue research on treatment options, especially those at low cost and which are immediately available to patients, particularly in low- and middle-income countries. Prior in vitro and observational studies have shown that fluoxetine, possibly through its inhibitory effect on the acid sphingomyelinase/ceramide system, could be a promising antiviral and anti-inflammatory treatment against COVID-19. In this report, we evaluated the potential antiviral and anti-inflammatory activities of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and against variants of concern in vitro, i.e., SARS-CoV-2 ancestral strain, Alpha B.1.1.7, Gamma P1, Delta B1.617 and Omicron BA.5. Fluoxetine, administrated after SARS-CoV-2 infection, significantly reduced lung tissue viral titres and expression of several inflammatory markers (i.e., IL-6, TNFα, CCL2 and CXCL10). It also inhibited the replication of all variants of concern in vitro. A modulation of the ceramide system in the lung tissues, as reflected by the increase in the ratio HexCer 16:0/Cer 16:0 in fluoxetine-treated mice, may contribute to explain these effects. Our findings demonstrate the antiviral and anti-inflammatory properties of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and its in vitro antiviral activity against variants of concern, establishing fluoxetine as a very promising candidate for the prevention and treatment of SARS-CoV-2 infection and disease pathogenesis.

GSDMD drives canonical inflammasome-induced neutrophil pyroptosis and is dispensable for NETosis.

Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1ß secretion and neutrophil cell death remain unclear. Here, we show that neutrophil-targeted expression of the disease-associated gain-of-function Nlrp3A350V mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1ß in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1ß are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91Phox -deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively.

Topical Aspirin Administration Improves Cutaneous Wound Healing in Diabetic Mice Through a Phenotypic Switch of Wound Macrophages Toward an Anti-inflammatory and Proresolutive Profile Characterized by LXA4 Release.

Patients with diabetes present a persistent inflammatory process, leading to impaired wound healing. Since nonhealing diabetic wound management shows limited results, the introduction of advanced therapies targeting and correcting the inflammatory status of macrophages in chronic wounds could be an effective therapeutic strategy to stop the sustained inflammation and to return to a healing state. In an excisional skin injury in a diet-induced diabetic murine model, we demonstrate that topical administration of low-dose aspirin (36 µg/wound/day) improves cutaneous wound healing by increasing wound closure through the promotion of the inflammation resolution program of macrophages. This treatment increased efferocytosis of wound macrophages from aspirin-treated diabetic mice compared with untreated diabetic mice. We also show that aspirin treatment of high-fat-fed mice oriented the phenotype of wound macrophages toward an anti-inflammatory and proresolutive profile characterized by a decrease of LTB4 production. The use of diabetic mice deficient for 5-LOX or 12/15-LOX demonstrated that these two enzymes of acid arachidonic metabolism are essential for the beneficial effect of aspirin on wound healing. Thus, aspirin treatment modified the balance between pro- and anti-inflammatory eicosanoids by promoting the synthesis of proresolving LXA4 through 5-LOX, LTA4, 12/15-LOX signaling. In conclusion, the restoration of an anti-inflammatory and proresolutive phenotype of wound macrophages by the topical administration of low-dose aspirin represents a promising therapeutic approach in chronic wounds.

Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa.

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1ß secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1ß production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.

Perceived Stigma and Stigma Management Strategies Among Online Male Sex Workers.

Technological advances like the Internet and Internet-enabled devices, such as smartphones, and the dating and hookup websites and apps available to the users of them, have transformed the nature, organization, and practice of sex work in fundamental ways. Some scholars have argued that these changes have contributed to a normalization of male exchange sex (i.e., providing sex in exchange for money, drugs, shelter, or goods), and in so doing, have diminished the stigma historically associated with it. However, little empirical research has focused on how male sex workers (MSWs), including those engaged in what might be called informal or incidental or casual sex work and primarily use dating/hookup websites and apps not designed for commercial to meet clients experience and manage stigma. To help fill this gap, we analyzed interview data from 180 MSWs who engaged in exchange sex and met their client on dating/hookup websites and apps. Most participants felt that sex work was still highly stigmatized in society at large, but many also felt it was generally accepted-if not completely normalized-within the gay community. Nevertheless, many struggled with the emotional impact of engaging in a stigmatized practice and most employed one or more of the following stigma management strategies: information management, distancing, discrediting the discreditors, asserting no other option existed, and challenging or reframing stereotypes and narratives. These findings indicate that MSWs, even those engaged in informal or incidental sex work, who meet clients on dating/hookup websites and apps are still strongly affected by sex work-related stigma and seek to manage it in various ways. Future research should investigate the sources of internalized stigma among this under-studied population of sex workers.

Analysis of Bacteria-Triggered Inflammasome: Activation in Neutrophils by Immunoblot.

Detection of microbes relies on the expression of germline-encoded pattern recognition receptors (PRRs). While PRRs can directly sense conserved pattern expressed by various microbes, they can also induce effector-triggered immunity (ETI) by sensing pathogenic alterations of cellular homeostasis. One consequence of ETI is the death of the infected cell through the induction of inflammasome-dependent cell death, namely, pyroptosis. Such process can be easily studied in macrophages and epithelial cells, yet neutrophils encode an arsenal of proteolytic enzymes that imped easy and reliable study of ETI-triggered inflammasome response. Here, we describe an immunoblotting methodology to study both ETI- and PRR-driven inflammasome responses in neutrophils upon bacterial infections. This method is also transposable to other microbial pathogen- and toxin-induced inflammasome response in neutrophils.

Attitudes Toward Peer-Delivered Sexual-Health Services Among New York City Sexual and Gender Minority Individuals Who Have Sex with Men and Attend Collective Sex Venues.

Collective sex venues such as sex clubs are strategic sites to promote sexual health among sexual and gender minority individuals. We present qualitative findings from a multiple-method study on the acceptability of sexual-health services at collective sex venues in New York City (NYC) among attendees who identified as men, transgender, or gender non-conforming. In a survey used for sample selection (n = 342), most respondents (82.7%) agreed that "having outreach workers at sex venues is a good thing." Interviewees (n = 30) appreciated how on-site services could promote sexual health in their community. They felt peer workers should be familiar with collective sex venues and share demographic characteristics with attendees. Some participants felt workers should keep some boundaries from attendees, while others felt they could be fully integrated in the environment, suggesting that either peer outreach or popular-opinion leader types of interventions could be feasible.

Intersectional Experienced Stigma and Psychosocial Syndemic Conditions in a Sample of Black Men Who Have Sex with Men Engaged in Sex Work (BMSM-SW) from Six US Cities.

Black men who have sex with men (BMSM) in the United States experience a disproportionate burden of violence, substance use, physical and mental health conditions relative to other racial groups. BMSM who engage in sex work (BMSM-SW) experience a high burden of psychosocial conditions, sexually transmitted infections, including HIV, and intersectional stigma. This analysis characterizes remuneration and client typologies for BMSM-SW, documents intersectional stigma experienced by BMSM-SW relative to other BMSM, and explores the impact of experienced intersectional stigma on the relationship between sex work engagement and psychosocial syndemic conditions (violence, polydrug use, and depression symptoms). Results show that a majority of BMSM-SW in the sample had female clients and that sex workers were more likely than other BMSM to hire another sex worker. BMSM-SW were more likely than other BMSM to report stigma attributed to race; sexuality; HIV status; socioeconomic status; and "other" attributes, and were more likely to report experiencing stigma across all settings assessed (schools; healthcare; employment; housing; police/courts; and in public/community). Intersectional stigma mediated the relationship between sex work engagement and psychosocial syndemic conditions, accounting for 49% (95% CI: 47.6-50.0%) of the relationship. Interventions for BMSM-SW should include resilience-building components to counteract the effects of intersectional stigma.

Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells.

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.