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Neutrophils rapidly infiltrate sites of infection and possess several microbicidal strategies, such as neutrophil extracellular traps release and phagocytosis. Enhanced neutrophil infiltration is associated with higher susceptibility to Leishmania infection, but neutrophil effector response contribution to this phenotype is uncertain. Here, we show that neutrophils from susceptible BALB/c mice (B/c) produce more NETs in response to Leishmania major than those from resistant C57BL/6 mice (B6), which are more phagocytic. The absence of neutrophil elastase contributes to phagocytosis regulation. Microarray analysis shows enrichment of genes involved in NET formation (mpo, pi3kcg, il1b) in B/c, while B6 shows upregulation of genes involved in phagocytosis and cell death (Arhgap12, casp9, mlkl, FasL). scRNA-seq in L. major-infected B6 showed heterogeneity in the pool of intralesional neutrophils, and we identified the N1 subset as the putative subpopulation involved with phagocytosis. In vivo, imaging validates NET formation in infected B/c ears where NETing neutrophils were mainly uninfected cells. NET digestion in vivo augmented parasite lymphatic drainage. Hence, a balance between NET formation and phagocytosis in neutrophils may contribute to the divergent phenotype observed in these mice., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Recent work by our laboratory and others indicates that co-display of multiple antigens on protein-based nanoparticles may be key to induce cross-reactive antibodies that provide broad protection against disease. To reach the ultimate goal of a universal vaccine for seasonal influenza, a mosaic influenza nanoparticle vaccine (FluMos-v1) was developed for clinical trial (NCT04896086). FluMos-v1 is unique in that it is designed to co-display four recently circulating haemagglutinin (HA) strains; however, current vaccine analysis techniques are limited to nanoparticle population analysis, thus, are unable to determine the valency of an individual nanoparticle. For the first time, we demonstrate by total internal reflection fluorescence microscopy and supportive physical-chemical methods that the co-display of four antigens is indeed achieved in single nanoparticles. Additionally, we have determined percentages of multivalent (mosaic) nanoparticles with four, three, or two HA proteins. The integrated imaging and physicochemical methods we have developed for single nanoparticle multivalency will serve to further understand immunogenicity data from our current FluMos-v1 clinical trial., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The demand for a vaccine for coronavirus disease 2019 (COVID-19) highlighted gaps in our understanding of the requirements for B cell responses to antigens, particularly to membrane-presented antigens, as occurs in vivo. We found that human B cell responses to membrane-presented antigens required the function of Piezo1, a plasma membrane mechanosensitive cation channel. Simply making contact with a glass probe induced calcium (Ca 2+ ) fluxes in B cells that were blocked by the Piezo1 inhibitor GsMTx4. When placed on glass surfaces, the plasma membrane tension of B cells increased, which stimulated Ca 2+ influx and spreading of B cells over the glass surface, which was blocked by the Piezo1 inhibitor OB-1. B cell responses to membrane-presented antigens but not to soluble antigens were inhibited both by Piezo1 inhibitors and by siRNA-mediated knockdown of Piezo1. Thus, the activation of Piezo1 defines an essential event in B cell activation to membrane-presented antigens that may be exploited to improve the efficacy of vaccines.

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder caused by biallelic mutations in the lysosomal trafficking regulator (LYST) gene. Even though enlarged lysosomes and/or lysosome-related organelles (LROs) are the typical cellular hallmarks of CHS, they have not been investigated in human neuronal models. Moreover, how and why the loss of LYST function causes a lysosome phenotype in cells has not been elucidated. We report that the LYST-deficient human neuronal model exhibits lysosome depletion accompanied by hyperelongated tubules extruding from enlarged autolysosomes. These results have also been recapitulated in neurons differentiated from CHS patients' induced pluripotent stem cells (iPSCs), validating our model system. We propose that LYST ensures the correct fission/scission of the autolysosome tubules during autophagic lysosome reformation (ALR), a crucial process to restore the number of free lysosomes after autophagy. We further demonstrate that LYST is recruited to the lysosome membrane, likely to facilitate the fission of autolysosome tubules. Together, our results highlight the key role of LYST in maintaining lysosomal homeostasis following autophagy and suggest that ALR dysregulation is likely associated with the neurodegenerative CHS phenotype., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Intracellular malaria parasites export many proteins into their host cell, inserting several into the erythrocyte plasma membrane to enable interactions with their external environment. While static techniques have identified some surface-exposed proteins, other candidates have eluded definitive localization and membrane topology determination. Moreover, both export kinetics and the mechanisms of membrane insertion remain largely unexplored. We introduce Reporter of Insertion and Surface Exposure (RISE), a method for continuous nondestructive tracking of antigen exposure on infected cells. RISE utilizes a small 11-amino acid (aa) HiBit fragment of NanoLuc inserted into a target protein and detects surface exposure through high-affinity complementation to produce luminescence. We tracked the export and surface exposure of CLAG3, a parasite protein linked to nutrient uptake, throughout the Plasmodium falciparum cycle in human erythrocytes. Our approach revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved the luminescence signal, but other changes abolished the surface signal, revealing that both size and charge of the extracellular epitope influence membrane insertion. Marked cell-to-cell variation with larger inserts containing multiple HiBit epitopes suggests complex regulation of CLAG3 insertion at the host membrane. Quantitative, continuous tracking of CLAG3 surface exposure thus reveals multiple factors that determine this protein's trafficking and insertion at the host erythrocyte membrane. The RISE assay will enable study of surface antigens from divergent intracellular pathogens. IMPORTANCE Malaria parasites invade and replicate within red blood cells of their human or animal hosts to avoid immune detection. At the same time, these parasites insert their own proteins into the host membrane to scavenge plasma nutrients, facilitate immune evasion, and perform other essential activities. As there is broad interest in developing vaccines and antimalarial therapies against these surface-exposed antigens, robust methods are needed to examine how and when parasite proteins insert at the host membrane. We therefore developed and used Reporter of Insertion and Surface Exposure (RISE) to track parasite antigen exposure. Using RISE, we followed the time course of membrane insertion for CLAG3, a conserved protein linked to a nutrient uptake channel on infected erythrocytes. We found that CLAG3 insertion occurs at specific parasite stages and that this insertion is required for the formation of the nutrient uptake channel. We also varied the size and charge of the extracellular domain to define constraints on protein insertion at the host membrane. Single-cell imaging revealed that some cells continued to export CLAG3 even with large extracellular loops, suggesting sophisticated strategies used by malaria parasites to control their interactions with host plasma.

During lymphocyte maturation and differentiation, cells undergo a series of proliferative stages interrupted with stages of low activity. The rapid proliferation stages are marked by changes in metabolic outputs-adapting to energy demands by either hindering or utilizing metabolic pathways. As such, it is necessary to view these changes in real time; however, current strategies for metabolomics are time consuming and very rarely provide a holistic profile of the cellular metabolism while also characterizing mitochondrial metabolism. Here, we devised a fluorescence lifetime imaging microscopy (FLIM) strategy to image mitochondrial metabolic profiles in lymphocytes as they go through changes in metabolic activity. Our method provides not only a comprehensive view of cellular metabolism but also narrow in mitochondrial contributions while also efficiently excluding non-viable cells with and without the use of a viability dye. Our novel imaging strategy offers a reliable tool to study changes in mitochondrial metabolism., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Artemisinin and its semisynthetic derivatives (ART) are fast acting, potent antimalarials; however, their use in malaria treatment is frequently confounded by recrudescences from bloodstream Plasmodium parasites that enter into and later reactivate from a dormant persister state. Here, we provide evidence that the mitochondria of dihydroartemisinin (DHA)-exposed persisters are dramatically altered and enlarged relative to the mitochondria of young, actively replicating ring forms. Restructured mitochondrial-nuclear associations and an altered metabolic state are consistent with stress from reactive oxygen species. New contacts between the mitochondria and nuclei may support communication pathways of mitochondrial retrograde signaling, resulting in transcriptional changes in the nucleus as a survival response. Further characterization of the organelle communication and metabolic dependencies of persisters may suggest strategies to combat recrudescences of malaria after treatment. IMPORTANCE The major first-line treatment for malaria, especially the deadliest form caused by Plasmodium falciparum, is combination therapy with an artemisinin-based drug (ART) plus a partner drug to assure complete cure. Without an effective partner drug, ART administration alone can fail because of the ability of small populations of blood-stage malaria parasites to enter into a dormant state and survive repeated treatments for a week or more. Understanding the nature of parasites in dormancy (persisters) and their ability to wake and reestablish actively propagating parasitemias (recrudesce) after ART exposure may suggest strategies to improve treatment outcomes and counter the threats posed by parasites that develop resistance to partner drugs. Here, we show that persisters have dramatically altered mitochondria and mitochondrial-nuclear interactions associated with features of metabolic quiescence. Restructured associations between the mitochondria and nuclei may support signaling pathways that enable the ART survival responses of dormancy.

Fluorescence lifetime imaging (FLIM) measures the characteristic time that a molecule remains in an excited state prior to emitting a photon and returning to the ground state. It is a state-of-the-art and noninvasive technique that has the potential to obtain signature physiological information during malaria blood-stage infection. The use of autofluorescence signals from intrinsic fluorophores obviates the need to tag the cells with synthetic molecules or to modify their gene expression. Furthermore, it permits time-lapse interrogation of the changes that occur from invasion to the point when the parasite takes over the host for its own survival mechanisms, as well as changes in the health of the parasite due to extrinsically applied metabolic disruptors. In this chapter, we present a protocol to investigate the autofluorescence lifetime signals of both normal red blood cells (RBC) and P. falciparum-infected RBCs. The data shared with this protocol reveals that there is a significant overall increase in autofluorescence lifetime in infected erythrocytes compared to the healthy uninfected ones. We include a metabolic experiment that confirms that the signals obtained from this imaging technique are key metabolites in energetics of the parasites. Furthermore, facilitating these protocols makes it possible to identify infected RBC based on FLIM signals alone, which presents a huge potential for the study of energetic effects of antimalarials and fast, noninvasive diagnosing.

Cytoskeletal integrity is essential for neuronal complexity and functionality. Certain inherited neurological diseases are associated with mutated genes that directly or indirectly compromise cytoskeletal stability. While the large size and complexity of the neurons grown in culture poses certain challenges for imaging, live-cell imaging is an excellent approach to determine the morphological consequences of such mutants. This protocol details the use of spinning disk confocal microscopy and image analysis tools to evaluate branching and neurite length of healthy iPSC-derived glutamatergic neurons that express specific fluorescent proteins. The protocols can be adapted to neuronal cell lines of choice by the investigator.

Malaria parasites increase their host erythrocyte's permeability to various nutrients, fueling intracellular pathogen development and replication. The plasmodial surface anion channel (PSAC) mediates this uptake and is linked to the parasite-encoded RhopH complex, consisting of CLAG3, RhopH2, and RhopH3. While interactions between these subunits are well established, it is not clear whether they remain associated from their synthesis in developing merozoites through erythrocyte invasion and trafficking to the host membrane. Here, we explored protein-protein interactions between RhopH subunits using live-cell imaging and Förster resonance energy transfer (FRET) experiments. Using the green fluorescent protein (GFP) derivatives mCerulean and mVenus, we generated single- and double-tagged parasite lines for fluorescence measurements. While CLAG3-mCerulean served as an efficient FRET donor for RhopH2-mVenus within rhoptry organelles, mCerulean targeted to this organelle via a short signal sequence produced negligible FRET. Upon merozoite egress and reinvasion, these tagged RhopH subunits were deposited into the new host cell's parasitophorous vacuole; these proteins were then exported and trafficked to the erythrocyte membrane, where CLAG3 and RhopH2 remained fully associated. Fluorescence intensity measurements identified stoichiometric increases in exported RhopH protein when erythrocytes are infected with two parasites; whole-cell patch-clamp revealed a concomitant increase in PSAC functional copy number and a dose effect for RhopH contribution to ion and nutrient permeability. These studies establish live-cell FRET imaging in human malaria parasites, reveal that RhopH subunits traffic to their host membrane destination without dissociation, and suggest quantitative contribution to PSAC formation. IMPORTANCE Malaria parasites grow within circulating red blood cells and uptake nutrients through a pore on their host membrane. Here, we used gene editing to tag CLAG3 and RhopH2, two proteins linked to the nutrient pore, with fluorescent markers and tracked these proteins in living infected cells. After their synthesis in mature parasites, imaging showed that both proteins are packaged into membrane-bound rhoptries. When parasites ruptured their host cells and invaded new red blood cells, these proteins were detected within a vacuole around the parasite before they migrated and inserted in the surface membrane of the host cell. Using simultaneous labeling of CLAG3 and RhopH2, we determined that these proteins interact tightly during migration and after surface membrane insertion. Red blood cells infected with two parasites had twice the protein at their surface and a parallel increase in the number of nutrient pores. Our work suggests that these proteins directly facilitate parasite nutrient uptake from human plasma.

Chronic human infectious diseases, including malaria, are associated with a large expansion of a phenotypically and transcriptionally distinct subpopulation of B cells distinguished by their high expression of a variety of inhibitory receptors including FcγRIIB. Because these B cells, termed atypical memory B cells (MBCs), are unable to respond to soluble antigens, it was suggested that they contributed to the poor acquisition of immunity in chronic infections. Here, we show that the high expression of FcγRIIB restricts atypical MBC responses to membrane-associated antigens that function to actively exclude FcγRIIB from the B cell immune synapse and include the co-receptor CD19, allowing B cell antigen receptor signaling and differentiation toward plasma cells. Thus, chronic infectious diseases result in the expansion of B cells that robustly respond to antigens that associate with cell surfaces, such as antigens in immune complexes, but are unable to respond to fully soluble antigens, such as self-antigens., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

During pregnancy, Plasmodium falciparum -infected erythrocytes (IE) accumulate in the intervillous spaces of the placenta by binding to chondroitin sulfate A (CSA) and elicit inflammatory responses that are associated with poor pregnancy outcomes. Primigravidae lack immunity to IE that sequester in the placenta and thus are susceptible to placental malaria (PM). Women become resistant to PM over successive pregnancies as antibodies to placental IE are acquired. Here, we assayed plasma collected at delivery from Malian and Tanzanian women of different parities for total antibody levels against recombinant VAR2CSA antigens (FCR3 allele), and for surface reactivity and binding inhibition and opsonizing functional activities against IE using two CSA-binding laboratory isolates (FCR3 and NF54). Overall, antibody reactivity to VAR2CSA recombinant proteins and to CSA-binding IE was higher in multigravidae than in primigravidae. However, plasma from Malian gravid women reacted more strongly with FCR3 whereas Tanzanian plasma preferentially reacted with NF54. Further, acquisition of functional antibodies was variant dependent: binding inhibition of P. falciparum strain NF54 ( P  < 0.001) but not of the strain FCR3 increased significantly with parity, while only opsonizing activity against FCR3 ( P  < 0.001) increased significantly with parity. In addition, opsonizing and binding inhibition activities of plasma of multigravidae were significantly correlated in assays of FCR3 ( r  = 0.4, P  = 0.01) but not of NF54 isolates; functional activities did not correlate in plasma from primigravidae. These data suggest that IE surface-expressed epitopes involved in each functional activity differ among P. falciparum strains. Consequently, geographic bias in circulating strains may impact antibody functions. Our study has implications for the development of PM vaccines aiming to achieve broad protection against various parasite strains., (Copyright © 2019 American Society for Microbiology.)

Protective antibody responses to vaccination or infection depend on affinity maturation, a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind, gather, and present antigen to T follicular helper (T fh ) cells. Here, we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with naïve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen affinity- and T fh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic, actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens, whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of naïve B cells. Thus, intrinsic properties of human GC B cells set thresholds for affinity selection., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

This updated unit compares three methods to acquire Förster Resonance Energy Transfer (FRET) data in living cells using a confocal microscope: Acceptor photobleaching, Acceptor-sensitized emission FRET, and Donor fluorescence lifetime imaging. Detailed protocols for live cell husbandry, image acquisition, and data analysis are provided. In addition to providing instructions for manufacturer's analysis tool sets, we provide an easy-to-use, MATLAB-based code to calculate FRET efficiency from data obtained using the Acceptor photobleaching or Acceptor-sensitized emission method, which can be freely downloaded. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite-host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action., Competing Interests: GA, GH, JM, JD, DN, JB, SR, OD, BT, LM, SP, PD, PC, SD, EL No competing interests declared, LT L.M. Thomas is an employee of Pfizer Inc, with ownership of stocks in Pfizer Inc.

The development of vaccines for infectious diseases for which we currently have none, including HIV, will likely require the use of adjuvants that strongly promote germinal center responses and somatic hypermutation to produce broadly neutralizing antibodies. Here we compared the outcome of immunization with the T-cell dependent antigen, NP-conjugated to chicken gamma globulin (NP-CGG) adjuvanted with the toll-like receptor 9 (TLR9) ligands, CpG-A or CpG-B, alone or conjugated with the cationic lipid carrier, DOTAP. We provide evidence that only NP-CGG adjuvanted with DOTAP-CpG-B was an effective vaccine in mice resulting in robust germinal center responses, isotype switching and high affinity NP-specific antibodies. The effectiveness of DOTAP-CpG-B as an adjuvant was dependent on the expression of the TLR9 signaling adaptor MyD88 in immunized mice. These results indicate DOTAP-CpG-B but not DOTAP-CpG-A is an effective adjuvant for T cell-dependent protein antigen-based vaccines., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)