Randall L. Lindquist

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b+ cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an “oxidative stress memory” both in the periphery and CNS compartments, in chronic neuroinflammation.

Ongoing Oxidative Stress Causes Subclinical Neuronal Dysfunction in the Recovery Phase of EAE

Most multiple sclerosis (MS) patients develop over time a secondary progressive disease course, characterized histologically by axonal loss and atrophy. In early phases of the disease, focal inflammatory demyelination leads to functional impairment, but the mechanism of chronic progression in MS is still under debate. Reactive oxygen species generated by invading and resident central nervous system (CNS) macrophages have been implicated in mediating demyelination and axonal damage, but demyelination and neurodegeneration proceed even in the absence of obvious immune cell infiltration, during clinical recovery in chronic MS. Here, we employ intravital NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX1–4, DUOX1, 2) and, thus, to identify the cellular source of oxidative stress in the CNS of mice affected by experimental autoimmune encephalomyelitis (EAE) in the remission phase of the disease. This directly affects neuronal function in vivo, as monitored by cellular calcium levels using intravital FRET–FLIM, providing a possible mechanism of disease progression in MS.

Certain types of iron oxide nanoparticles are not suited to passively target inflammatory cells that infiltrate the brain in response to stroke

Intravenous administration of iron oxide nanoparticles during the acute stage of experimental stroke can produce signal intensity changes in the ischemic region. This has been attributed, albeit controversially, to the infiltration of iron-laden blood-borne macrophages. The properties of nanoparticles that render them most suitable for phagocytosis is a matter of debate, as is the most relevant timepoint for administration. Both of these questions are examined in the present study. Imaging experiments were performed in mice with 30 minutes of middle cerebral artery occlusion (MCAO). Iron oxide nanoparticles with different charges and sizes were used, and mice received 300 μmol Fe/kg intravenously: either superparamagnetic iron oxide nanoparticles (SPIOs), ultrasmall SPIOs, or very small SPIOs. The particles were administered 7 days before MCAO, at the time of reperfusion, or 72 hours after MCAO. Interestingly, there was no observable signal change in the ischemic brains that could be attributed to iron. Furthermore, no Prussian blue-positive cells were found in the brains or blood leukocytes, despite intense staining in the livers and spleens. This implies that the nanoparticles selected for this study are not phagocytosed by blood-borne leukocytes and do not enter the ischemic mouse brain.

Synergistic Strategy for Multicolor Two-photon Microscopy: Application to the Analysis of Germinal Center Reactions In Vivo

Simultaneous detection of multiple cellular and molecular players in their native environment, one of the keys to a full understanding of immune processes, remains challenging for in vivo microscopy. Here, we present a synergistic strategy for spectrally multiplexed in vivo imaging composed of (i) triple two-photon excitation using spatiotemporal synchronization of two femtosecond lasers, (ii) a broad set of fluorophores with emission ranging from blue to near infrared, (iii) an effective spectral unmixing algorithm. Using our approach, we simultaneously excite and detect seven fluorophores expressed in distinct cellular and tissue compartments, plus second harmonics generation from collagen fibers in lymph nodes. This enables us to visualize the dynamic interplay of all the central cellular players during germinal center reactions. While current in vivo imaging typically enables recording the dynamics of 4 tissue components at a time, our strategy allows a more comprehensive analysis of cellular dynamics involving 8 single-labeled compartments. It enables to investigate the orchestration of multiple cellular subsets determining tissue function, thus, opening the way for a mechanistic understanding of complex pathophysiologic processes in vivo. In the future, the design of transgenic mice combining a larger spectrum of fluorescent proteins will reveal the full potential of our method.

Unbiased classification of mosquito blood cells by single-cell genomics and high-content imaging

Significance Mosquito blood cells are central players of immunity against the vector-borne pathogens that devastate the lives of millions of people worldwide. However, their molecular identity and classification remain controversial. By applying single-cell RNA sequencing and high-content imaging flow cytometry, we defined the molecular fingerprint of a subset of mosquito blood cells and characterized two transcriptionally distinct blood cell populations that resemble previously described cell types. Surprisingly, cell population analyses at a single-cell level uncovered an active molecular transfer between the two cell types that may contribute to cellular diversity and plasticity seen across biological systems. Mosquito blood cells are immune cells that help control infection by vector-borne pathogens. Despite their importance, little is known about mosquito blood cell biology beyond morphological and functional criteria used for their classification. Here, we combined the power of single-cell RNA sequencing, high-content imaging flow cytometry, and single-molecule RNA hybridization to analyze a subset of blood cells of the malaria mosquito Anopheles gambiae. By demonstrating that blood cells express nearly half of the mosquito transcriptome, our dataset represents an unprecedented view into their transcriptional program. Analyses of differentially expressed genes identified transcriptional signatures of two cell types and provide insights into the current classification of these cells. We further demonstrate the active transfer of a cellular marker between blood cells that may confound their identification. We propose that cell-to-cell exchange may contribute to cellular diversity and functional plasticity seen across biological systems.

NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes

The balance between various cellular subsets of the innate and adaptive immune system and microbiota in the gastrointestinal tract is carefully regulated to maintain tolerance to the normal flora and dietary antigens, while protecting against pathogens. The intestinal epithelial cells and the network of dendritic cells and macrophages in the lamina propria are crucial lines of defense that regulate this balance. The complex relationship between the myeloid compartment (dendritic cells and macrophages) and lymphocyte compartment (T cells and innate lymphoid cells), as well as the impact of the epithelial cell layer have been studied in depth in recent years, revealing that the regulatory and effector functions of both innate and adaptive immune compartments exhibit more plasticity than had been previously appreciated. However, little is known about the metabolic activity of these cellular compartments, which is the basic function underlying all other additional tasks the cells perform. Here we perform intravital NAD(P)H fluorescence lifetime imaging in the small intestine of fluorescent reporter mice to monitor the NAD(P)H-dependent metabolism of epithelial and myeloid cells. The majority of myeloid cells which comprise the surveilling network in the lamina propria have a low metabolic activity and remain resting even upon stimulation. Only a few myeloid cells, typically localized at the tip of the villi, are metabolically active and are able to activate NADPH oxidases upon stimulation, leading to an oxidative burst. In contrast, the epithelial cells are metabolically highly active and, although not considered professional phagocytes, are also able to activate NADPH oxidases, leading to massive production of reactive oxygen species. Whereas the oxidative burst in myeloid cells is mainly catalyzed by the NOX2 isotype, in epithelial cells other isotypes of the NADPH oxidases family are involved, especially NOX4. They are constitutively expressed by the epithelial cells, but activated only on demand to ensure rapid defense against pathogens. This minimizes the potential for inadvertent damage from resting NOX activation, while maintaining the capacity to respond quickly if needed.

Single-cell RNA-sequencing redefines blood cell type classification in mosquitoes

Mosquito blood cells are ancestral immune cells that help control infection by vector-borne pathogens. Despite their importance, little is known about mosquito blood cell biology beyond the ambiguous morphological and functional criteria used for their classification. Here we combined the power of single-cell RNA-sequencing, imaging flow cytometry and single-molecule RNA hybridization to analyze blood cells of the malaria mosquito Anopheles gambiae. By demonstrating that blood cells express nearly half of the mosquito transcriptome, our dataset represents an unprecedented view into their transcriptional machinery. Analyses of differentially expressed genes identified transcriptional signatures of two distinct cell types that challenge the current morphology-based classification of these cells. We further demonstrated an active transfer of a cellular marker between blood cells that confounds their identity. We propose that cell-to-cell exchange is broadly relevant for cell type classification and may account for the remarkable cellular diversity observed in nature.

Mosquito lipids regulate Plasmodium sporogony and infectivity to the mammalian host

Malaria is a human parasitic disease that is transmitted by a mosquito vector. Plasmodium parasites, the causative agents, differ in their infectivity and virulence to the mammalian host, but the mechanistic underpinnings of this variation remain unknown. As mosquitoes provide a nutrient-rich niche for development of transmissible stages, we examined the role of lipids in parasite development and infectivity by disrupting lipid trafficking in mosquito adults. We show that depleting the major mosquito lipoprotein lipophorin deprives parasites of neutral lipids, arrests oocysts growth and impairs sporozoite formation. Importantly, lipid deficiency decreases parasite mitochondrial membrane potential and severely compromises sporozoite infectivity and virulence in the mammalian host. Our findings demonstrate the requirement of mosquito lipids for Plasmodium metabolism, and uncover the mitochondrial contribution to parasite infectivity and virulence. By drawing a connection between vector nutrition and malaria virulence, our results redefine the paradigm of vector-host-pathogen interactions.Malaria is a fatal human parasitic disease transmitted by a mosquito vector. The evolution of within-host malaria virulence has been the focus of many empirical and theoretical studies. However, the vector’s contribution to virulence evolution is not well understood. Here we explored how within-vector resource exploitation impacts evolutionary trajectories of within-host Plasmodium virulence. We developed a nested model of within-vector dynamics and malaria epidemiology, which predicted that non-competitive resource exploitation within-vector restricts within-host parasite virulence. To validate our model, we experimentally manipulated mosquito lipid trafficking and gauged within-vector parasite development, within-host infectivity and virulence. We found that mosquito-derived lipids determine within-host parasite virulence by shaping development and metabolic activity of transmissible sporozoites. Our findings uncover the role of within-vector environment in regulating within-host Plasmodium virulence and identify Plasmodium metabolic traits that may contribute to the evolution of malaria virulence.

Tracking Plasma Cell Differentiation in Living Mice with Two-Photon Microscopy

Due to the multitude of cell types involved in the differentiation of plasma cells during the germinal center reaction, and due to a lack of in vitro systems, which recapitulate germinal centers, the most suitable way to study plasma cell generation in germinal centers is in vivo. In this chapter we describe how to induce humoral immune responses to defined model antigens and how to visualize and track plasma cells and their interactions with other cells in the lymph nodes of living mice.

Imaging of magnetic microfield distortions allows sensitive single-cell detection

Cell tracking with magnetic resonance imaging (MRI) is mostly performed using superparamagnetic iron oxide (SPIO) nanoparticle-labeled cells. However, negative contrast in T2*-weighted imaging is inherently problematic as a homogeneous background signal is required to visualize the negative signal. In a magnetic field, SPIO-labeled cells develop their own magnetization, distorting the main field. We show here a method to visualize these distortions and use them to identify single cells with increased sensitivity and certainty compared to T2* images. We labeled HeLa cells with SPIOs, suspended labeled cells in agarose to make phantoms, and performed high-resolution gradient-echo MRI. Phase images were processed to enhance the visibility of single cells. To quantify SPIO content, we generated a map of frequency differences. MRI of cell phantoms showed that single cells could be detected at concentrations ranging from 200 to 10,000 cells mL(-1). Postprocessing of the magnetic resonance phase images reveals characteristic microfield distortions, increasing dramatically the sensitivity of cell recognition, compared to unprocessed T2* images. Calculating frequency shifts and comparing microfield distortions to simulations permit estimation of the nanoparticle load of single cells. We expect the ability to detect and quantify the iron load of single cells to prove useful in studies of cell trafficking, especially in rare cell populations.