BackgroundCFTR modulator therapies have positive clinical outcomes, yet chronic inflammation and bacterial infections persist in people with CF (pwCF). How elexacaftor–tezacaftor–ivacaftor (ETI) fails to improve innate immune signaling responsible for bacterial clearance and inflammation resolution remains unknown.MethodsWe used an unbiased proteomics approach to measure the effect of ETI on inflammatory proteins. Plasma from 20 pediatric pwCF and 20 non-CF (NCF) was collected during routine examination and 3 months after ETI initiation. Protein screening was performed with an inflammation panel (Target 96, Olink®). Bioinformatics analysis was used to determine changes in protein expression.ResultsThere were significantly fewer pulmonary exacerbations after ETI initiation, along with sustained improvement in lung function and reduced bacterial colonization. Unpaired analysis of CF pre-ETI and NCF resulted in 34 significantly different proteins. Of these, CCL20, MMP-10, EN-RAGE, and AXIN1 had a log2 fold change of 1.2 or more. There was a modest shift in overall CF protein profiles post-ETI toward the NCF cluster. Unpaired analysis of protein differential expression between NCF and CF post-ETI identified a total of 24 proteins significantly impacted by ETI therapy (p-value ≤ 0.05), with only CCL20 having a log2 fold change higher than 1.2. Paired analysis (CF pre- and CF post-ETI) of differential protein expression demonstrated significant expression changes of MMP-10, EN-RAGE, and IL-17A. Pathway analysis identified significantly impacted pathways such as the NF-κB pathway.ConclusionThis study showed that ETI in a pediatric cohort had a modest effect on several inflammatory proteins with potential as biomarkers. Pathways significantly impacted by ETI can be further studied for future therapies to combat persistent inflammation and dysregulated immunity.

Interferon‐induced transmembrane protein 3 (IFITM3) is an antiviral protein that alters cell membranes to block fusion of viruses. Conflicting reports identified opposing effects of IFITM3 on SARS‐CoV‐2 infection of cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with SARS‐CoV‐2 experience extreme weight loss and lethality compared to mild infection in wild‐type (WT) mice. KO mice have higher lung viral titers and increases in inflammatory cytokine levels, immune cell infiltration, and histopathology. Mechanistically, we observe disseminated viral antigen staining throughout the lung and pulmonary vasculature in KO mice, as well as increased heart infection, indicating that IFITM3 constrains dissemination of SARS‐CoV‐2. Global transcriptomic analysis of infected lungs shows upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS‐CoV‐2 infection and overall demonstrate that IFITM3 is protective in SARS‐CoV‐2 infections in vivo .

The Many Hosts of Mycobacteria (MHM) meeting series brings together basic scientists, clinicians and veterinarians to promote robust discussion and dissemination of recent advances in our knowledge of numerous mycobacterial diseases, including human and bovine tuberculosis (TB), nontuberculous mycobacteria (NTM) infection, Hansen's disease (leprosy), Buruli ulcer and Johne's disease. The 9th MHM conference (MHM9) was held in July 2022 at The Ohio State University (OSU) and centered around the theme of "Confounders of Mycobacterial Disease." Confounders can and often do drive the transmission of mycobacterial diseases, as well as impact surveillance and treatment outcomes. Various confounders were presented and discussed at MHM9 including those that originate from the host (comorbidities and coinfections) as well as those arising from the environment (e.g., zoonotic exposures), economic inequality (e.g. healthcare disparities), stigma (a confounder of leprosy and TB for millennia), and historical neglect (a confounder in Native American Nations). This conference report summarizes select talks given at MHM9 highlighting recent research advances, as well as talks regarding the historic and ongoing impact of TB and other infectious diseases on Native American Nations, including those in Southwestern Alaska where the regional TB incidence rate is among the highest in the Western hemisphere.

Background Abnormal macrophage function caused by dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) is a critical contributor to chronic airway infections and inflammation in people with cystic fibrosis (PWCF). Elexacaftor/tezacaftor/ivacaftor (ETI) is a new CFTR modulator therapy for PWCF. Host-pathogen and clinical responses to CFTR modulators are poorly described. We sought to determine how ETI impacts macrophage CFTR function, resulting effector functions and relationships to clinical outcome changes.Methods Clinical information and/or biospecimens were obtained at ETI initiation and 3, 6, 9 and 12 months post-ETI in 56 PWCF and compared with non-CF controls. Peripheral blood monocyte-derived macrophages (MDMs) were isolated and functional assays performed.Results ETI treatment was associated with increased CF MDM CFTR expression, function and localisation to the plasma membrane. CF MDM phagocytosis, intracellular killing of CF pathogens and efferocytosis of apoptotic neutrophils were partially restored by ETI, but inflammatory cytokine production remained unchanged. Clinical outcomes including increased forced expiratory volume in 1 s (+10%) and body mass index (+1.0 kg center dot m-2) showed fluctuations over time and were highly individualised. Significant correlations between post-ETI MDM CFTR function and sweat chloride levels were observed. However, MDM CFTR function correlated with clinical outcomes better than sweat chloride.Conclusion ETI is associated with unique changes in innate immune function and clinical outcomes.

Objectives: Structural or mucus hypersecretory pulmonary diseases such as cystic fibrosis (CF), wherein viscous mucus accumulates and clearance functions are impaired, predispose people to lung infection by inhaled bacteria that form biofilm aggregates. Nontuberculous mycobacteria (NTM), primarily Mycobacterium abscessus and Mycobacterium avium, are the growing cause of these lung infections and are extremely challenging to treat due to antibiotic recalcitrance. Better therapeutic approaches are urgently needed. We developed a humanized monoclonal antibody (HuTipMab) directed against a biofilm structural linchpin, the bacterial DNABII proteins, that rapidly disrupts biofilms and generates highly vulnerable newly released bacteria (NRel). Methods: HuTipMab's ability to recognize HupB, NTM's DNABII homologue was determined by ELISA. Relative ability of HuTipMab to disrupt biofilms formed by lab-passaged and clinical isolates of NTM was assessed by CLSM. Relative sensitivity of NTM NRel to antibiotic killing compared to when grown planktonically was evaluated by plate count. Results: HuTipMab recognized HupB and significantly disrupted NTM biofilms in a time- and dose-dependent manner. Importantly, NTM NRel of lab-passaged and clinical isolates were now highly sensitive to killing by amikacin and azithromycin. Conclusions: If successful, this combinatorial treatment strategy would empower existing antibiotics to more effectively kill NTM newly released from a biofilm by HuTipMab and thereby both improve clinical outcomes and perhaps decrease length of antibiotic treatment for people that are NTM culture-positive.

neutrophils change their fate and function in PwCF will help in designing strategies to clear recurrent infections in PwCF.

Significance We report the discovery of fundamental roles for the noncanonical inflammasome molecule Caspase-4/11 in promoting pathological inflammatory and prothrombotic pathways in severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2) infections. Our work demonstrates that Caspase-11 has a broader role in immune responses beyond its previously appreciated effects in bacterial infections. Further, we show that Caspase-11–deficient mice infected with SARS–CoV-2 fare significantly better in terms of overall illness, lung inflammation, and thrombosis than wild-type (WT) mice, thus implicating Caspase-11 as a new therapeutic target for preventing or treating COVID-19.

Bacterial biofilms are often defined as communities of surface-attached bacteria and are typically depicted with a classic mushroom-shaped structure characteristic of Pseudomonas aeruginosa. However, it has become evident that this is not how all biofilms develop, especially in vivo, in clinical and industrial settings, and in the environment, where biofilms often are observed as non-surface-attached aggregates. In this Review, we describe the origin of the current five-step biofilm development model and why it fails to capture many aspects of bacterial biofilm physiology. We aim to present a simplistic developmental model for biofilm formation that is flexible enough to include all the diverse scenarios and microenvironments where biofilms are formed. With this new expanded, inclusive model, we hereby introduce a common platform for developing an understanding of biofilms and anti-biofilm strategies that can be tailored to the microenvironment under investigation. In this Review, Bjarnsholt and colleagues propose a revised conceptual model of the biofilm life cycle that encompasses the three major steps of biofilm formation — aggregation, growth and disaggregation — independently of surfaces, and initiation from single-cell planktonic bacteria, and thus represents a broader range of biofilm systems.

Nontuberculous mycobacteria (NTM) are an increasingly common cause of respiratory infection in people with cystic fibrosis (PwCF). Relative to those with no history of NTM infection (CF-NTMNEG), PwCF and a history of NTM infection (CF-NTMPOS) are more likely to develop severe lung disease and experience complications over the course of treatment. In other mycobacterial infections (e.g., tuberculosis), an overexuberant immune response causes pathology and compromises organ function; however, since the immune profiles of CF-NTMPOS and CF-NTMNEG airways are largely unexplored, it is unknown which, if any, immune responses distinguish these cohorts or concentrate in damaged tissues. Here, we evaluated lung lobe-specific immune profiles of 3 cohorts (CF-NTMPOS, CF-NTMNEG, and non-CF adults) and found that CF-NTMPOS airways are distinguished by a hyperinflammatory cytokine profile. Importantly, the CF-NTMPOS airway immune profile was dominated by B cells, classical macrophages, and the cytokines that support their accumulation. These and other immunological differences between cohorts, including the near absence of NK cells and complement pathway members, were enriched in the most damaged lung lobes. The implications of these findings for our understanding of lung disease in PwCF are discussed, as are how they may inform the development of host-directed therapies to improve NTM disease treatment.

Biofilms are multicellular microbial aggregates that can be associated with host mucosal epithelia in the airway, gut, and genitourinary tract. The host environment plays a critical role in the establishment of these microbial communities in both health and disease. These host mucosal microenvironments however are distinct histologically, functionally, and regarding nutrient availability. This review discusses the specific mucosal epithelial microenvironments lining the airway, focusing on: i) biofilms in the human respiratory tract and the unique airway microenvironments that make it exquisitely suited to defend against infection, and ii) how airway pathophysiology and dysfunctional barrier/clearance mechanisms due to genetic mutations, damage, and inflammation contribute to biofilm infections. The host cellular responses to infection that contribute to resolution or exacerbation, and insights about evaluating and therapeutically targeting airway-associated biofilm infections are briefly discussed. Since so many studies have focused on Pseudomonas aeruginosa in the context of cystic fibrosis (CF) or on Haemophilus influenzae in the context of upper and lower respiratory diseases, these bacteria are used as examples. However, there are notable differences in diseased airway microenvironments and the unique pathophysiology specific to the bacterial pathogens themselves.

Both Pseudomonas aeruginosa and Mycobacterium abscessus are bacteria that cause pulmonary infection in people with inflammatory lung disease, including individuals with cystic fibrosis (CF). These bacterial species inhabit the same environmental reservoirs (soil and water) and can be coisolated in the lungs of people with CF. We investigated the interaction of these bacteria and found an antagonistic interaction favoring P. aeruginosa that was observed in biofilms but not in planktonic cultures. This antagonism extended to multiple P. aeruginosa strains and against Mycobacterium smegmatis. We tested known P. aeruginosa mutants for genes that can play roles in interbacterial contact-dependent (type III and type VI secretion systems) and contact-independent (quorum sensing, type II secretion) antagonism pathways to interrogate the mechanism of action. Our results indicate that well-known mechanisms of interbacterial competition are not responsible for the antagonism of P. aeruginosa toward M. abscessus, suggesting a novel antibacterial strategy. IMPORTANCE The biofilm lifestyle is favored by many organisms, and understanding interbacterial interactions that occur between coisolated bacterial species can provide new information regarding bacterial defense mechanisms and antibacterial targets. This may also provide insights into possible interbacterial interactions impacting host immunity during coinfection. Here, we investigate an antagonistic interaction favoring P. aeruginosa over M. abscessus exclusively in dual-species biofilms and not in liquid coculture.

SARS-CoV-2 is a worldwide health concern, and new treatment strategies are needed 1. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4), and its mouse homologue, caspase-11 (CASP11), are upregulated in SARS-CoV-2 infections, and that CASP4 expression correlates with severity of SARS-CoV-2 infection in humans. SARS-CoV-2-infected Casp11-/- mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) and gasdermin-D knock out (Gsdmd-/-) mice. GSDMD is a downstream effector of CASP11 and CASP1. Notably, viral titers were similar in the three genotypes. Global transcriptomics of SARS-CoV-2-infected WT, Casp11-/- and Gsdmd-/- lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11-/- mice. We confirmed that protein levels of inflammatory mediators IL-1β, IL6, and CXCL1, and neutrophil functions, were reduced in Casp11-/- lungs. Additionally, Casp11-/- lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11, promotes detrimental SARS-CoV-2-associated inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

Influenza virus infections increase susceptibility to secondary bacterial infections, such as pneumococcal pneumonia, resulting in increased morbidity and mortality. Influenza-induced tissue damage is hypothesized to increase susceptibility to Streptococcus pneumoniae infection by increasing adherence to the respiratory epithelium. Using a mouse model of influenza infection followed by S. pneumoniae infection, we found that an influenza infection does not increase the number of pneumococci initially present within the trachea, but does inhibit pneumococcal clearance by 2 hours after infection. To determine whether influenza damage increases pneumococcal adherence, we developed a novel murine tracheal explant system to determine influenza-induced tissue damage and subsequent pneumococcal adherence. Murine tracheas were kept viable ex vivo as shown by microscopic examination of ciliary beating and cellular morphology using continuous media flow for up to 8 days. Tracheas were infected with influenza virus for 0.5-5 days ex vivo, and influenza-induced tissue damage and the early stages of repair to the epithelium were assessed histologically. A prior influenza infection did not increase pneumococcal adherence, even when the basement membrane was maximally denuded or during the repopulation of the basement membrane with undifferentiated epithelial cells. We measured mucociliary clearance in vivo and found it was decreased in influenza-infected mice. Together, our results indicate that exposure of the tracheal basement membrane contributes minimally to pneumococcal adherence. Instead, an influenza infection results in decreased tracheal mucociliary velocity and initial clearance of pneumococci, leading to an increased pneumococcal burden as early as 2 hours after pneumococcal infection.

Mycobacterium abscessus is emerging as a cause of recalcitrant chronic pulmonary infections, particularly in people with cystic fibrosis (CF). Biofilm formation has been implicated in the pathology of this organism, however the role of biofilm formation in infection is unclear. Two colony-variants of M. abscessus are routinely isolated from CF samples, smooth ( Ma Sm ) and rough ( Ma Rg ). These two variants display distinct colony morphologies due to the presence ( Ma Sm ) or absence ( Ma Rg ) of cell wall glycopeptidolipids (GPLs). We hypothesized that Ma Sm and Ma Rg variant biofilms might have different mechanical properties. To test this hypothesis, we performed uniaxial mechanical indentation, and shear rheometry on Ma Sm and Ma Rg colony-biofilms. We identified that Ma Rg biofilms were significantly stiffer than Ma Sm under a normal force, while Ma Sm biofilms were more pliant compared to Ma Rg , under both normal and shear forces. Furthermore, using theoretical indices of mucociliary and cough clearance, we identified that M. abscessus biofilms may be more resistant to mechanical forms of clearance from the lung, compared to another common pulmonary pathogen, Pseudomonas aeruginosa. Thus, the mechanical properties of M. abscessus biofilms may contribute to the persistent nature of pulmonary infections caused by this organism.

Rapid and specific antibody testing is crucial for improved understanding, control, and treatment of COVID-19 pathogenesis. Herein, we describe and apply a rapid, sensitive, and accurate virus neutralization assay for SARS-CoV-2 antibodies. The assay is based on an HIV-1 lentiviral vector that contains a secreted intron Gaussia luciferase (Gluc) or secreted nano-luciferase reporter cassette, pseudotyped with the SARS-CoV-2 spike (S) glycoprotein, and is validated with a plaque-reduction assay using an authentic, infectious SARS-CoV-2 strain. The assay was used to evaluate SARS-CoV-2 antibodies in serum from individuals with a broad range of COVID-19 symptoms; patients included those in the intensive care unit (ICU), health care workers (HCWs), and convalescent plasma donors. The highest neutralizing antibody titers were observed among ICU patients, followed by general hospitalized patients, HCWs, and convalescent plasma donors. Our study highlights a wide phenotypic variation in human antibody responses against SARS-CoV-2 and demonstrates the efficacy of a potentially novel lentivirus pseudotype assay for high-throughput serological surveys of neutralizing antibody titers in large cohorts.

Interferon-induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. Here we show that SARS-CoV-2 Spike-pseudotyped virus and genuine SARS-CoV-2 infections are generally restricted by expression of human IFITM1, IFITM2, and IFITM3, using both gain- and loss-of-function approaches. Mechanistically, restriction of SARS-CoV-2 occurred independently of IFITM3 S-palmitoylation sites, indicating a restrictive capacity that is distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the human IFITM3 endocytosis-promoting YxxΦ motif converted human IFITM3 into an enhancer of SARS-CoV-2 infection, and cell-to-cell fusion assays confirmed the ability of endocytic mutants to enhance Spike-mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which reportedly increases plasma membrane fusion versus endosome fusion of SARS-CoV-2, attenuated IFITM3 restriction and converted amphipathic helix mutants into strong enhancers of infection. In sum, these data uncover new pro- and anti-viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity-based mechanisms used for endosomal virus restriction. Indeed, the net effect of IFITM3 on SARS-CoV-2 infections may be a result of these opposing activities, suggesting that shifts in the balance of these activities could be coopted by viruses to escape this important first line innate defense mechanism.

Mycobacteria are important causes of disease in human and animal hosts. Diseases caused by mycobacteria include leprosy, tuberculosis (TB), nontuberculous mycobacteria (NTM) infections and Buruli Ulcer. To better understand and treat mycobacterial disease, clinicians, veterinarians and scientists use a range of discipline-specific approaches to conduct basic and applied research, including conducting epidemiological surveys, patient studies, wildlife sampling, animal models, genetic studies and computational simulations. To foster the exchange of knowledge and collaboration across disciplines, the Many Hosts of Mycobacteria (MHM) conference series brings together clinical, veterinary and basic scientists who are dedicated to advancing mycobacterial disease research. Started in 2007, the MHM series recently held its 8th conference at the Albert Einstein College of Medicine (Bronx, NY). Here, we review the diseases discussed at MHM8 and summarize the presentations on research advances in leprosy, NTM and Buruli Ulcer, human and animal TB, mycobacterial disease comorbidities, mycobacterial genetics and 'omics, and animal models. A mouse models workshop, which was held immediately after MHM8, is also summarized. In addition to being a resource for those who were unable to attend MHM8, we anticipate this review will provide a benchmark to gauge the progress of future research concerning mycobacteria and their many hosts.

ABSTRACT Mycobacterium abscessus has emerged as an important pathogen in people with chronic inflammatory lung diseases such as cystic fibrosis, and recent reports suggest that it may be transmissible by fomites. M. abscessus exhibits two major colony morphology variants: a smooth morphotype (MaSm) and a rough morphotype (MaRg). Biofilm formation, prolonged intracellular survival, and colony variant diversity can each contribute to the persistence of M. abscessus and other bacterial pathogens in chronic pulmonary diseases. A prevailing paradigm of chronic M. abscessus infection is that MaSm is a noninvasive, biofilm-forming, persistent phenotype and MaRg an invasive phenotype that is unable to form biofilms. We show that MaRg is hyperaggregative and forms biofilm-like aggregates, which, like MaSm biofilm aggregates, are significantly more tolerant than planktonic variants to acidic pHs, hydrogen peroxide (H2O2), and treatment with amikacin or azithromycin. We further show that both variants are recalcitrant to antibiotic treatment inside human macrophage-like cells and that MaRg is more refractory than MaSm to azithromycin. Our results indicate that biofilm-like aggregation and protracted intracellular survival may each contribute to the persistence of this problematic pathogen in the face of antimicrobial agents regardless of morphotype. Biofilms of each M. abscessus variant are rapidly killed, however, by acetic acid, which may help to prevent local fomite transmission.

Autophagy and inflammasome complex assembly are physiological processes that control homeostasis, inflammation, and immunity. Autophagy is a ubiquitous pathway that degrades cytosolic macromolecules or organelles, as well as intracellular pathogens. Inflammasomes are multi-protein complexes that assemble in the cytosol of cells upon detection of pathogen- or danger-associated molecular patterns. A critical outcome of inflammasome assembly is the activation of the cysteine protease caspase-1, which activates the pro-inflammatory cytokine precursors pro-IL-1β and pro-IL-18. Studies on chronic inflammatory diseases, heart diseases, Alzheimer's disease, and multiple sclerosis revealed that autophagy and inflammasomes intersect and regulate each other. In the context of infectious diseases, however, less is known about the interplay between autophagy and inflammasome assembly, although it is becoming evident that pathogens have evolved multiple strategies to inhibit and/or subvert these pathways and to take advantage of their intricate crosstalk. An improved appreciation of these pathways and their subversion by diverse pathogens is expected to help in the design of anti-infective therapeutic interventions.