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In vitro studies are crucial for our understanding of the human macrophage immune functions. However, traditional in vitro culture media poorly reflect the metabolic composition of blood, potentially affecting the outcomes of these studies. Here, we analyzed the impact of a physiological medium on human induced pluripotent stem cell (iPSC)-derived macrophages (iPSDM) function. Macrophages cultured in a human plasma-like medium (HPLM) were more permissive to Mycobacterium tuberculosis (Mtb) replication and showed decreased lipid metabolism with increased metabolic polarization. Functionally, we discovered that HPLM-differentiated macrophages showed different metabolic organelle content and activity. Specifically, HPLM-differentiated macrophages displayed reduced lipid droplet and peroxisome content, increased lysosomal proteolytic activity, and increased mitochondrial activity and dynamics. Inhibiting or inducing lipid droplet formation revealed that lipid droplet content is a key factor influencing macrophage permissiveness to Mtb. These findings underscore the importance of using physiologically relevant media in vitro for accurately studying human macrophage function., Importance: This work compellingly demonstrates that the choice of culture medium significantly influences M. tuberculosis replication outcomes, thus emphasizing the importance of employing physiologically relevant media for accurate in vitro host-pathogen interaction studies. We anticipate that our work will set a precedent for future research with clinical relevance, particularly in evaluating antibiotic efficacy and resistance in cellulo ., Competing Interests: The authors declare no conflict of interest.

Mitochondrial membranes define distinct structural and functional compartments. Cristae of the inner mitochondrial membrane (IMM) function as independent bioenergetic units that undergo rapid and transient remodelling, but the significance of this compartmentalized organization is unknown 1 . Using super-resolution microscopy, here we show that cytosolic IMM vesicles, devoid of outer mitochondrial membrane or mitochondrial matrix, are formed during resting state. These vesicles derived from the IMM (VDIMs) are formed by IMM herniation through pores formed by voltage-dependent anion channel 1 in the outer mitochondrial membrane. Live-cell imaging showed that lysosomes in proximity to mitochondria engulfed the herniating IMM and, aided by the endosomal sorting complex required for transport machinery, led to the formation of VDIMs in a microautophagy-like process, sparing the remainder of the organelle. VDIM formation was enhanced in mitochondria undergoing oxidative stress, suggesting their potential role in maintenance of mitochondrial function. Furthermore, the formation of VDIMs required calcium release by the reactive oxygen species-activated, lysosomal calcium channel, transient receptor potential mucolipin 1, showing an interorganelle communication pathway for maintenance of mitochondrial homeostasis. Thus, IMM compartmentalization could allow for the selective removal of damaged IMM sections via VDIMs, which should protect mitochondria from localized injury. Our findings show a new pathway of intramitochondrial quality control., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The management of end-stage kidney disease (ESKD) has been constantly evolving over the last decade with the development of targeted approaches. In this field, telemedicine and remote monitoring are based on the availability of new cyclers that allow for bidirectional communication (between patient and physician) and for the application of the Sharesource cloud-based platform. These technologies allow patients with ESKD to undergo automated peritoneal dialysis (APD) at home. However, these approaches are not well standardized and largely applied yet. Therefore, this study aimed to elaborate a protocol for the utilization of the Sharesource platform to facilitate the practical management of patients treated with APD. A series of expert meetings were held between September 2022 and January 2023 in Italy. The participants (ten nephrologists and five nurses) from nine Italian public dialysis centers shared their opinions, examined the current scientific literature in the field, and reviewed the key characteristics of the Sharesource system to achieve a common position on this topic. A detailed and practical document containing experts' opinions and suggestions on the use of the Sharesource platform for the management of patients treated with APD was produced. This expert opinion might represent a new useful instrument in clinical practice for managing patients undergoing home-based peritoneal dialysis (PD) through the Sharesource platform, which is valid not only for Italy. These recommendations pave the way to novel patient-centered and personalized therapeutic approaches for ESKD and highlight the advantages of telemedicine and remote monitoring in the management of patients with ESKD undergoing PD and its positive impact on their quality of life.

Single-organelle resolution approaches have the potential to advance our knowledge of the heterogeneity of lysosome function. Challenging population-based models, we propose a "lysosome states" concept that links single lysosomes to function., Competing Interests: MGG is an Academic Editor at PLOS Biology., (Copyright: © 2024 Bussi, Gutierrez. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21). DS is a gene dosage disorder that results in multiple phenotypes including congenital heart defects. This clinically important cardiac pathology is the result of a third copy of one or more of the approximately 230 genes on Hsa21, but the identity of the causative dosage-sensitive genes and hence mechanisms underlying this cardiac pathology remain unclear. Here, we show that hearts from human fetuses with DS and embryonic hearts from the Dp1Tyb mouse model of DS show reduced expression of mitochondrial respiration genes and cell proliferation genes. Using systematic genetic mapping, we determined that three copies of the dual-specificity tyrosine phosphorylation-regulated kinase 1A ( Dyrk1a ) gene, encoding a serine/threonine protein kinase, are associated with congenital heart disease pathology. In embryos from Dp1Tyb mice, reducing Dyrk1a gene copy number from three to two reversed defects in cellular proliferation and mitochondrial respiration in cardiomyocytes and rescued heart septation defects. Increased dosage of DYRK1A protein resulted in impairment of mitochondrial function and congenital heart disease pathology in mice with DS, suggesting that DYRK1A may be a useful therapeutic target for treating this common human condition.

Peroxisomes are organelles involved in many metabolic processes including lipid metabolism, reactive oxygen species (ROS) turnover, and antimicrobial immune responses. However, the cellular mechanisms by which peroxisomes contribute to bacterial elimination in macrophages remain elusive. Here, we investigated peroxisome function in iPSC-derived human macrophages (iPSDM) during infection with Mycobacterium tuberculosis (Mtb). We discovered that Mtb-triggered peroxisome biogenesis requires the ESX-1 type 7 secretion system, critical for cytosolic access. iPSDM lacking peroxisomes were permissive to Mtb wild-type (WT) replication but were able to restrict an Mtb mutant missing functional ESX-1, suggesting a role for peroxisomes in the control of cytosolic but not phagosomal Mtb. Using genetically encoded localization-dependent ROS probes, we found peroxisomes increased ROS levels during Mtb WT infection. Thus, human macrophages respond to the infection by increasing peroxisomes that generate ROS primarily to restrict cytosolic Mtb. Our data uncover a peroxisome-controlled, ROS-mediated mechanism that contributes to the restriction of cytosolic bacteria., (© 2023 Pellegrino et al.)

Endomembrane damage represents a form of stress that is detrimental for eukaryotic cells 1,2 . To cope with this threat, cells possess mechanisms that repair the damage and restore cellular homeostasis 3-7 . Endomembrane damage also results in organelle instability and the mechanisms by which cells stabilize damaged endomembranes to enable membrane repair remains unknown. Here, by combining in vitro and in cellulo studies with computational modelling we uncover a biological function for stress granules whereby these biomolecular condensates form rapidly at endomembrane damage sites and act as a plug that stabilizes the ruptured membrane. Functionally, we demonstrate that stress granule formation and membrane stabilization enable efficient repair of damaged endolysosomes, through both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms. We also show that blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, a human pathogen that exploits endomembrane damage to survive within the host., (© 2023. The Author(s).)

Autophagy is a cellular innate-immune defence mechanism against intracellular microorganisms, including Mycobacterium tuberculosis (Mtb). How canonical and non-canonical autophagy function to control Mtb infection in phagosomes and the cytosol remains unresolved. Macrophages are the main host cell in humans for Mtb. Here we studied the contributions of canonical and non-canonical autophagy in the genetically tractable human induced pluripotent stem cell-derived macrophages (iPSDM), using a set of Mtb mutants generated in the same genetic background of the common lab strain H37Rv. We monitored replication of Mtb mutants that are either unable to trigger canonical autophagy (Mtb ΔesxBA) or reportedly unable to block non-canonical autophagy (Mtb ΔcpsA) in iPSDM lacking either ATG7 or ATG14 using single-cell high-content imaging. We report that deletion of ATG7 by CRISPR-Cas9 in iPSDM resulted in increased replication of wild-type Mtb but not of Mtb ΔesxBA or Mtb ΔcpsA. We show that deletion of ATG14 resulted in increased replication of both Mtb wild type and the mutant Mtb ΔesxBA. Using Mtb reporters and quantitative imaging, we identified a role for ATG14 in regulating fusion of phagosomes containing Mtb with lysosomes, thereby enabling intracellular bacteria restriction. We conclude that ATG7 and ATG14 are both required for restricting Mtb replication in human macrophages., (© 2023. The Author(s).)

Transient lysosomal damage after infection with cytosolic pathogens or silica crystals uptake results in protease leakage. Whether limited leakage of lysosomal contents into the cytosol affects the function of cytoplasmic organelles is unknown. Here, we show that sterile and non-sterile lysosomal damage triggers a cell death independent proteolytic remodelling of the mitochondrial proteome in macrophages. Mitochondrial metabolic reprogramming required leakage of lysosomal cathepsins and was independent of mitophagy, mitoproteases and proteasome degradation. In an in vivo mouse model of endomembrane damage, live lung macrophages that internalised crystals displayed impaired mitochondrial function. Single-cell RNA-sequencing revealed that lysosomal damage skewed metabolic and immune responses in alveolar macrophages subsets with increased lysosomal content. Functionally, drug modulation of macrophage metabolism impacted host responses to Mycobacterium tuberculosis infection in an endomembrane damage dependent way. This work uncovers an inter-organelle communication pathway, providing a general mechanism by which macrophages undergo mitochondrial metabolic reprograming after endomembrane damage., (© 2022. The Author(s).)

Mycobacterium tuberculosis segregates within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a live dual-imaging approach to monitor host subcellular acidification and M. tuberculosis intrabacterial pH. By combining this approach with pharmacological and genetic perturbations, we show that M. tuberculosis can maintain its intracellular pH independently of the surrounding pH in human macrophages. Importantly, unlike bedaquiline (BDQ), isoniazid (INH), or rifampicin (RIF), the drug pyrazinamide (PZA) displays antibacterial efficacy by disrupting M. tuberculosis intrabacterial pH homeostasis in cellulo . By using M. tuberculosis mutants, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo . We anticipate this imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. IMPORTANCE We still do not completely understand why tuberculosis (TB) treatment requires the combination of several antibiotics for up to 6 months. M. tuberculosis is a facultative intracellular pathogen, and it is still unknown whether heterogenous and dynamic intracellular populations of bacteria in different cellular environments affect antibiotic efficacy. By developing a dual live imaging approach to monitor mycobacterial pH homeostasis, host cell environment, and antibiotic action, we show here that intracellular localization of M. tuberculosis affects the efficacy of one first-line anti-TB drug. Our observations can be applicable to the treatment of other intracellular pathogens and help to inform the development of more effective combined therapies for tuberculosis that target heterogenous bacterial populations within the host.

The development of neuroinflammation, as well as the progression of several neurodegenerative diseases, has been associated with the activation and mobilization of the peripheral immune system due to systemic inflammation. However, the mechanism by which this occurs remains unclear. Here, we addressed the effect of systemic sterile induced-co-expression of IL-12 and IL-18, in the establishment of a novel cytokine-mediated model of neuroinflammation. Following peripheral hydrodynamic shear of IL-12 plus IL-18 cDNAs in C57BL/6 mice, we induced systemic and persistent level of IL-12, which in turn promoted the elevation of circulating pro-inflammatory cytokines TNF-α and IFN-γ, accompanied with splenomegaly. Moreover, even though we identified an increased gene expression of both TNF-α and IFN-γ in the brain, we observed that only IFN-γ, but not TNF-α signaling through its type I receptor, was required to induce both the trafficking of leukocytes from the periphery toward the brain and upregulate MHC-II in microglia and inflammatory monocytes. Therefore, only TNF-α was shown to be dispensable, revealing an IFN-γ-dependent activation of microglia and recruitment of leukocytes, particularly of highly activated inflammatory monocytes. Taken together, our results argue for a systemic cytokine-mediated establishment and development of neuroinflammation, having identified IFN-γ as a potential target for immunomodulation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Xenophagy is an important cellular defence mechanism against cytosol-invading pathogens, such as Mycobacterium tuberculosis (Mtb). Activation of xenophagy in macrophages targets Mtb to autophagosomes; however, how Mtb is targeted to autophagosomes in human macrophages at a high spatial and temporal resolution is unknown. Here, we use human induced pluripotent stem cell-derived macrophages (iPSDMs) to study the human macrophage response to Mtb infection and the role of the ESX-1 type VII secretion system. Using RNA-seq, we identify ESX-1-dependent transcriptional responses in iPSDMs after infection with Mtb. This analysis revealed differential inflammatory responses and dysregulated pathways such as eukaryotic initiation factor 2 (eIF2) signalling and protein ubiquitylation. Moreover, live-cell imaging revealed that Mtb infection in human macrophages induces dynamic ESX-1-dependent, LC3B-positive tubulovesicular autophagosomes (LC3-TVS). Through a correlative live-cell and focused ion beam scanning electron microscopy (FIB SEM) approach, we show that upon phagosomal rupture, Mtb induces the formation of LC3-TVS, from which the bacterium is able to escape to reside in the cytosol. Thus, iPSDMs represent a valuable model for studying spatiotemporal dynamics of human macrophage-Mtb interactions, and Mtb is able to evade capture by autophagic compartments., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The application of nanoparticles for drug or gene delivery promises benefits in the form of single-cell-specific therapeutic and diagnostic capabilities. Many methods of cell transfection rely on unspecific means to increase the transport of genetic material into cells. Targeted transport is in principle possible with magnetically propelled micromotors, which allow responsive nanoscale actuation and delivery. However, many commonly used magnetic materials (e.g., Ni and Co) are not biocompatible, possess weak magnetic remanence (Fe 3 O 4 ), or cannot be implemented in nanofabrication schemes (NdFeB). Here, it is demonstrated that co-depositing iron (Fe) and platinum (Pt) followed by one single annealing step, without the need for solution processing, yields ferromagnetic FePt nanomotors that are noncytotoxic, biocompatible, and possess a remanence and magnetization that rival those of permanent NdFeB micromagnets. Active cell targeting and magnetic transfection of lung carcinoma cells are demonstrated using gradient-free rotating millitesla fields to drive the FePt nanopropellers. The carcinoma cells express enhanced green fluorescent protein after internalization and cell viability is unaffected by the presence of the FePt nanopropellers. The results establish FePt, prepared in the L1 0 phase, as a promising magnetic material for biomedical applications with superior magnetic performance, especially for micro- and nanodevices., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

T helper 17 (Th17) cells are pathogenic in many inflammatory diseases, but also support the integrity of the intestinal barrier in a non-inflammatory manner. It is unclear what distinguishes inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals. Here, we compared the characteristics of Th17 cells differentiating in response to commensal bacteria (SFB) to those differentiating in response to a pathogen (Citrobacter rodentium). Homeostatic Th17 cells exhibited little plasticity towards expression of inflammatory cytokines, were characterized by a metabolism typical of quiescent or memory T cells, and did not participate in inflammatory processes. In contrast, infection-induced Th17 cells showed extensive plasticity towards pro-inflammatory cytokines, disseminated widely into the periphery, and engaged aerobic glycolysis in addition to oxidative phosphorylation typical for inflammatory effector cells. These findings will help ensure that future therapies directed against inflammatory Th17 cells do not inadvertently damage the resident gut population., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tuberculosis (TB) caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb) remains one of the deadliest infectious diseases with over a billion deaths in the past 200 years (Paulson 2013). TB causes more deaths worldwide than any other single infectious agent, with 10.4 million new cases and close to 1.7 million deaths in 2017. The obstacles that make TB hard to treat and eradicate are intrinsically linked to the intracellular lifestyle of Mtb. Mtb needs to replicate within human cells to disseminate to other individuals and cause disease. However, we still do not completely understand how Mtb manages to survive within eukaryotic cells and why some cells are able to eradicate this lethal pathogen. Here, we summarise the current knowledge of the complex host cell-pathogen interactions in TB and review the cellular mechanisms operating at the interface between Mtb and the human host cell, highlighting the technical and methodological challenges to investigating the cell biology of human host cell-Mtb interactions., (© FEMS 2019.)

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Acute brain injury leads to the recruitment and activation of immune cells including resident microglia and infiltrating peripheral myeloid cells (MC), which contribute to the inflammatory response involved in neuronal damage. We previously reported that TLR2 stimulation by peptidoglycan (PGN) from Staphylococcus aureus, in vitro and in vivo , induced microglial cell activation followed by autophagy induction. In this report, we evaluated if phosphatidyl-inositol-3 kinase (PI3K) pharmacological inhibitors LY294200 and 3-methyladenine (3-MA) can modulate the innate immune response to PGN in the central nervous system. We found that injection of PGN into the mouse brain parenchyma (caudate putamen) triggered an inflammatory reaction, which involved activation of microglial cells, recruitment of infiltrating MC to injection site, production of pro-inflammatory mediators, and neuronal injury. In addition, we observed the accumulation of LC3B + CD45 + cells and colocalization of LC3B and lysosomal-associated membrane protein 1 in brain cells. Besides, we found that pharmacological inhibitors of PI3K, including the classical autophagy inhibitor 3-MA, reduced the recruitment of MC, microglial cell activation, and neurotoxicity induced by brain PGN injection. Collectively, our results suggest that PI3K pathways and autophagic response may participate in the PGN-induced microglial activation and MC recruitment to the brain. Thus, inhibition of these pathways could be therapeutically targeted to control acute brain inflammatory conditions.

Brain-resident microglia and peripheral migratory leukocytes play essential roles in shaping the immune response in the central nervous system. These cells activate and migrate in response to chemokines produced during active immune responses and may contribute to the progression of neuroinflammation. Herein, we addressed the participation of type I-II interferons in the response displayed by microglia and inflammatory monocytes to comprehend the contribution of these cytokines in the establishment and development of a neuroinflammatory process. Following systemic lipopolysaccharide (LPS) challenge, we found glial reactivity and an active recruitment of CD45 hi leukocytes close to CD31 + vascular endothelial cells in circumventricular organs. Isolated CD11b + CD45 hi Ly6C hi Ly6G - -primed inflammatory monocytes were able to induce T cell proliferation, unlike CD11b + CD45 lo microglia. Moreover, ex vivo re-stimulation with LPS exhibited an enhancement of T cell proliferative response promoted by inflammatory monocytes. These myeloid cells also proved to be recruited in a type I interferon-dependent fashion as opposed to neutrophils, unveiling a role of these cytokines in their trafficking. Together, our results compares the phenotypic and functional features between tissue-resident vs peripheral recruited cells in an inflamed microenvironment, identifying inflammatory monocytes as key sentinels in a LPS-induced murine model of neuroinflammation.

Autophagy is a fundamental cellular homeostatic mechanism, whereby cells autodigest parts of their cytoplasm for removal or turnover. Neurodegenerative disorders are associated with autophagy dysregulation, and drugs modulating autophagy have been successful in several animal models. Microglial cells are phagocytes in the central nervous system (CNS) that become activated in pathological conditions and determine the fate of other neural cells. Here, we studied the effects of autophagy on the production of pro-inflammatory molecules in microglial cells and their effects on neuronal cells. We observed that both trehalose and rapamycin activate autophagy in BV2 microglial cells and down-regulate the production of pro-inflammatory cytokines and nitric oxide (NO), in response to LPS and alpha-synuclein. Autophagy also modulated the phosphorylation of p38 and ERK1/2 MAPKs in BV2 cells, which was required for NO production. These actions of autophagy modified the impact of microglial activation on neuronal cells, leading to suppression of neurotoxicity. Our results demonstrate a novel role for autophagy in the regulation of microglial cell activation and pro-inflammatory molecule secretion, which may be important for the control of inflammatory responses in the CNS and neurotoxicity.