The FAM13A Long Isoform Regulates Cilia Movement and Coordination in Airway Mucociliary Transport.

Single nucelotide polymorphisms (SNPs) at the FAM13A locus are among the most commonly reported risk alleles associated with chronic obstructive pulmonary disease (COPD) and other respiratory diseases; however, the physiological role of FAM13A is unclear. In humans, two major protein isoforms are expressed at the FAM13A locus: "long" and "short," but their functions remain unknown, partly because of a lack of isoform conservation in mice. We performed in-depth characterization of organotypic primary human airway epithelial cell subsets and show that multiciliated cells predominantly express the FAM13A long isoform containing a putative N-terminal Rho GTPase-activating protein (RhoGAP) domain. Using purified proteins, we directly demonstrate the RhoGAP activity of this domain. In Xenopus laevis, which conserve the long-isoform, Fam13a deficiency impaired cilia-dependent embryo motility. In human primary epithelial cells, long-isoform deficiency did not affect multiciliogenesis but reduced cilia coordination in mucociliary transport assays. This is the first demonstration that FAM13A isoforms are differentially expressed within the airway epithelium, with implications for the assessment and interpretation of SNP effects on FAM13A expression levels. We also show that the long FAM13A isoform coordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance.

A common polymorphism in the Intelectin-1 gene influences mucus plugging in severe asthma.

By incompletely understood mechanisms, type 2 (T2) inflammation present in the airways of severe asthmatics drives the formation of pathologic mucus which leads to airway mucus plugging. Here we investigate the molecular role and clinical significance of intelectin-1 (ITLN-1) in the development of pathologic airway mucus in asthma. Through analyses of human airway epithelial cells we find that ITLN1 gene expression is highly induced by interleukin-13 (IL-13) in a subset of metaplastic MUC5AC+ mucus secretory cells, and that ITLN-1 protein is a secreted component of IL-13-induced mucus. Additionally, we find ITLN-1 protein binds the C-terminus of the MUC5AC mucin and that its deletion in airway epithelial cells partially reverses IL-13-induced mucostasis. Through analysis of nasal airway epithelial brushings, we find that ITLN1 is highly expressed in T2-high asthmatics, when compared to T2-low children. Furthermore, we demonstrate that both ITLN-1 gene expression and protein levels are significantly reduced by a common genetic variant that is associated with protection from the formation of mucus plugs in T2-high asthma. This work identifies an important biomarker and targetable pathways for the treatment of mucus obstruction in asthma.

In vitro platform to model the function of ionocytes in the human airway epithelium.

BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.

Effects of the G-quadruplex-binding drugs quarfloxin and CX-5461 on the malaria parasite Plasmodium falciparum.

Plasmodium falciparum is the deadliest causative agent of human malaria. This parasite has historically developed resistance to most drugs, including the current frontline treatments, so new therapeutic targets are needed. Our previous work on guanine quadruplexes (G4s) in the parasite's DNA and RNA has highlighted their influence on parasite biology, and revealed G4 stabilising compounds as promising candidates for repositioning. In particular, quarfloxin, a former anticancer agent, kills blood-stage parasites at all developmental stages, with fast rates of kill and nanomolar potency. Here we explored the molecular mechanism of quarfloxin and its related derivative CX-5461. In vitro, both compounds bound to P. falciparum-encoded G4 sequences. In cellulo, quarfloxin was more potent than CX-5461, and could prevent establishment of blood-stage malaria in vivo in a murine model. CX-5461 showed clear DNA damaging activity, as reported in human cells, while quarfloxin caused weaker signatures of DNA damage. Both compounds caused transcriptional dysregulation in the parasite, but the affected genes were largely different, again suggesting different modes of action. Therefore, CX-5461 may act primarily as a DNA damaging agent in both Plasmodium parasites and mammalian cells, whereas the complete antimalarial mode of action of quarfloxin may be parasite-specific and remains somewhat elusive.

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.

Single-Cell and Population Transcriptomics Reveal Pan-epithelial Remodeling in Type 2-High Asthma.

The type 2 cytokine-high asthma endotype (T2H) is characterized by IL-13-driven mucus obstruction of the airways. To further investigate this incompletely understood pathobiology, we characterize IL-13 effects on human airway epithelial cell cultures using single-cell RNA sequencing, finding that IL-13 generates a distinctive transcriptional state for each cell type. Specifically, we discover a mucus secretory program induced by IL-13 in all cell types which converts both mucus and defense secretory cells into a metaplastic state with emergent mucin production and secretion, while leading to ER stress and cell death in ciliated cells. The IL-13-remodeled epithelium secretes a pathologic, mucin-imbalanced, and innate immunity-depleted proteome that arrests mucociliary motion. Signatures of IL-13-induced cellular remodeling are mirrored by transcriptional signatures characteristic of the nasal airway epithelium within T2H versus T2-low asthmatic children. Our results reveal the epithelium-wide scope of T2H asthma and present candidate therapeutic targets for restoring normal epithelial function.

Criticality of plasma membrane lipids reflects activation state of macrophage cells.

Signalling is of particular importance in immune cells, and upstream in the signalling pathway many membrane receptors are functional only as complexes, co-locating with particular lipid species. Work over the last 15 years has shown that plasma membrane lipid composition is close to a critical point of phase separation, with evidence that cells adapt their composition in ways that alter the proximity to this thermodynamic point. Macrophage cells are a key component of the innate immune system, are responsive to infections and regulate the local state of inflammation. We investigate changes in the plasma membrane's proximity to the critical point as a response to stimulation by various pro- and anti-inflammatory agents. Pro-inflammatory (interferon γ, Kdo 2-Lipid A, lipopolysaccharide) perturbations induce an increase in the transition temperature of giant plasma membrane vesicles; anti-inflammatory interleukin 4 has the opposite effect. These changes recapitulate complex plasma membrane composition changes, and are consistent with lipid criticality playing a master regulatory role: being closer to critical conditions increases membrane protein activity.

Motile cilia hydrodynamics: entrainment versus synchronization when coupling through flow.

Coordinated motion of cilia is a fascinating and vital aspect of very diverse forms of eukaryotic life, enabling swimming and propulsion of fluid across cellular epithelia. There are many questions still unresolved, and broadly they fall into two classes. (i) The mechanism of how cilia physically transmit forces onto each other. It is not known for many systems if the forces are mainly of hydrodynamical origin, or if elastic forces within the cytoskeleton are important. (ii) In those systems where we know that forces are purely hydrodynamical, we do not have a framework for linking our understanding of how each cilium behaves in isolation to the collective properties of two or more cilia. In this work, we take biological data of cilia dynamics from a variety of organisms as an input for an analytical and numerical study. We calculate the relative importance of external flows versus internal cilia flows on cilia coupling. This study contributes to both the open questions outlined above: firstly, we show that it is, in general, incorrect to infer cilium-cilium coupling strength on the basis of experiments with external flows, and secondly, we show a framework to recapitulate the dynamics of single cilia (the waveform) showing classes that correspond to biological systems with the same physiological activity (swimming by propulsion, versus forming collective waves). This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Activation of Toll-like receptors nucleates assembly of the MyDDosome signaling hub.

Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling.

Assessing the Collective Dynamics of Motile Cilia in Cultures of Human Airway Cells by Multiscale DDM.

The technique of differential dynamic microscopy is extended here, showing that it can provide a powerful and objective method of video analysis for optical microscopy videos of in vitro samples of live human bronchial epithelial ciliated cells. These cells are multiciliated, with motile cilia that play key physiological roles. It is shown that the ciliary beat frequency can be recovered to match conventional analysis, but in a fully automated fashion. Furthermore, it is shown that the properties of spatial and temporal coherence of cilia beat can be recovered and distinguished, and that if a collective traveling wave (the metachronal wave) is present, this has a distinct signature and its wavelength and direction can be measured.

Lipopolysaccharide-induced NF-κB nuclear translocation is primarily dependent on MyD88, but TNFα expression requires TRIF and MyD88.

TLR4 signalling through the MyD88 and TRIF-dependent pathways initiates translocation of the transcription factor NF-κB into the nucleus. In cell population studies using mathematical modeling and functional analyses, Cheng et al. suggested that LPS-driven activation of MyD88, in the absence of TRIF, impairs NF-κB translocation. We tested the model proposed by Cheng et al. using real-time single cell analysis in macrophages expressing EGFP-tagged p65 and a TNFα promoter-driven mCherry. Following LPS stimulation, cells lacking TRIF show a pattern of NF-κB dynamics that is unaltered from wild-type cells, but activation of the TNFα promoter is impaired. In macrophages lacking MyD88, there is minimal NF-κB translocation to the nucleus in response to LPS stimulation, and there is no activation of the TNFα promoter. These findings confirm that signalling through MyD88 is the primary driver for LPS-dependent NF-κB translocation to the nucleus. The pattern of NF-κB dynamics in TRIF-deficient cells does not, however, directly reflect the kinetics of TNFα promoter activation, supporting the concept that TRIF-dependent signalling plays an important role in the transcription of this cytokine.

Interaction between colloidal particles on an oil-water interface in dilute and dense phases.

The interaction between micron-sized charged colloidal particles at polar/non-polar liquid interfaces remains surprisingly poorly understood for a relatively simple physical chemistry system. By measuring the pair correlation function g(r) for different densities of polystyrene particles at the decane-water interface, and using a powerful predictor-corrector inversion scheme, effective pair-interaction potentials can be obtained up to fairly high densities, and these reproduce the experimental g(r) in forward simulations, so are self consistent. While at low densities these potentials agree with published dipole-dipole repulsion, measured by various methods, an apparent density dependence and long range attraction are obtained when the density is higher. This condition is thus explored in an alternative fashion, measuring the local mobility of colloids when confined by their neighbors. This method of extracting interaction potentials gives results that are consistent with dipolar repulsion throughout the concentration range, with the same magnitude as in the dilute limit. We are unable to rule out the density dependence based on the experimental accuracy of our data, but we show that incomplete equilibration of the experimental system, which would be possible despite long waiting times due to the very strong repulsions, is a possible cause of artefacts in the inverted potentials. We conclude that to within the precision of these measurements, the dilute pair potential remains valid at high density in this system.

The frequency and duration of Salmonella-macrophage adhesion events determines infection efficiency.

Salmonella enterica causes a range of important diseases in humans and a in a variety of animal species. The ability of bacteria to adhere to, invade and survive within host cells plays an important role in the pathogenesis of Salmonella infections. In systemic salmonellosis, macrophages constitute a niche for the proliferation of bacteria within the host organism. Salmonella enterica serovar Typhimurium is flagellated and the frequency with which this bacterium collides with a cell is important for infection efficiency. We investigated how bacterial motility affects infection efficiency, using a combination of population-level macrophage infection experiments and direct imaging of single-cell infection events, comparing wild-type and motility mutants. Non-motile and aflagellate bacterial strains, in contrast to wild-type bacteria, collide less frequently with macrophages, are in contact with the cell for less time and infect less frequently. Run-biased Salmonella also collide less frequently with macrophages but maintain contact with macrophages for a longer period of time than wild-type strains and infect the cells more readily. Our results suggest that uptake of S. Typhimurium by macrophages is dependent upon the duration of contact time of the bacterium with the cell, in addition to the frequency with which the bacteria collide with the cell.

Actin polymerization as a key innate immune effector mechanism to control Salmonella infection.

Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1ß recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1ß production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.

Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex.

Pathogen recognition by nucleotide-binding oligomerization domain-like receptor (NLR) results in the formation of a macromolecular protein complex (inflammasome) that drives protective inflammatory responses in the host. It is thought that the number of inflammasome complexes forming in a cell is determined by the number of NLRs being activated, with each NLR initiating its own inflammasome assembly independent of one another; however, we show here that the important foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) simultaneously activates at least two NLRs, whereas only a single inflammasome complex is formed in a macrophage. Both nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 are simultaneously present in the same inflammasome, where both NLRs are required to drive IL-1ß processing within the Salmonella-infected cell and to regulate the bacterial burden in mice. Superresolution imaging of Salmonella-infected macrophages revealed a macromolecular complex with an outer ring of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain and an inner ring of NLRs, with active caspase effectors containing the pro-IL-1ß substrate localized internal to the ring structure. Our data reveal the spatial localization of different components of the inflammasome and how different members of the NLR family cooperate to drive robust IL-1ß processing during Salmonella infection.

Phagocytosis dynamics depends on target shape.

A complete understanding of phagocytosis requires insight into both its biochemical and physical aspects. One of the ways to explore the physical mechanism of phagocytosis is to probe whether and how the target properties (e.g., size, shape, surface states, stiffness, etc.) affect their uptake. Here we report an imaging-based method to explore phagocytosis kinetics, which is compatible with real-time imaging and can be used to validate existing reports using fixed and stained cells. We measure single-event engulfment time from a large number of phagocytosis events to compare how size and shape of targets determine their engulfment. The data shows an increase in the average engulfment time for increased target size, for spherical particles. The uptake time data on nonspherical particles confirms that target shape plays a more dominant role than target size for phagocytosis: Ellipsoids with an eccentricity of 0.954 and much smaller surface areas than spheres were taken up five times more slowly than spherical targets.

Dynamics of Salmonella infection of macrophages at the single cell level.

Salmonella enterica causes a range of diseases. Salmonellae are intracellular parasites of macrophages, and the control of bacteria within these cells is critical to surviving an infection. The dynamics of the bacteria invading, surviving, proliferating in and killing macrophages are central to disease pathogenesis. Fundamentally important parameters, however, such as the cellular infection rate, have not previously been calculated. We used two independent approaches to calculate the macrophage infection rate: mathematical modelling of Salmonella infection experiments, and analysis of real-time video microscopy of infection events. Cells repeatedly encounter salmonellae, with the bacteria often remain associated with the macrophage for more than ten seconds. Once Salmonella encounters a macrophage, the probability of that bacterium infecting the cell is remarkably low: less than 5%. The macrophage population is heterogeneous in terms of its susceptibility to the first infection event. Once infected, a macrophage can undergo further infection events, but these reinfection events occur at a lower rate than that of the primary infection.

The nonlinear mechanical response of the red blood cell.

We measure the dynamical mechanical properties of human red blood cells. A single cell response is measured with optical tweezers. We investigate both the stress relaxation following a fast deformation and the effect of varying the strain rate. We find a power-law decay of the stress as a function of time, down to a plateau stress, and a power-law increase of the cell's elasticity as a function of the strain rate. Interestingly, the exponents of these quantities violate the linear superposition principle, indicating a nonlinear response. We propose that this is due to the breaking of a fraction of the crosslinks during the deformation process. The soft glassy rheology model accounts for the relation between the exponents we observe experimentally. This picture is consistent with recent models of bond remodeling in the red blood cell's molecular structure. Our results imply that the blood cell's mechanical behavior depends critically on the deformation process.

Critical fluctuations in plasma membrane vesicles.

We demonstrate critical behavior in giant plasma membrane vesicles (GPMVs) that are isolated directly from living cells. GPMVs contain two liquid phases at low temperatures and one liquid phase at high temperatures and exhibit transition temperatures in the range of 15 to 25 degrees C. In the two-phase region, line tensions linearly approach zero as temperature is increased to the transition. In the one-phase region, micrometer-scale composition fluctuations occur and become increasingly large and long-lived as temperature is decreased to the transition. These results indicate proximity to a critical point and are quantitatively consistent with established theory. Our observations of robust critical fluctuations suggest that the compositions of mammalian plasma membranes are tuned to reside near a miscibility critical point and that heterogeneity corresponding to < 50 nm-sized compositional fluctuations are present in GPMV membranes at physiological temperatures. Our results provide new insights for plasma membrane heterogeneity that may be related to functional lipid raft domains in live cells.