Origin of monocytes and macrophages in a committed progenitor.

Monocytes, macrophages and dendritic cells (DCs) are developmentally related regulators of the immune system that share the monocyte-macrophage DC progenitor (MDP) as a common precursor. Unlike differentiation into DCs, the distal pathways for differentiation into monocytes and monocyte-derived macrophages are not fully elucidated. We have now demonstrated the existence of a clonogenic, monocyte- and macrophage-restricted progenitor cell derived from the MDP. This progenitor was a Ly6C(+) proliferating cell present in the bone marrow and spleen that generated the major monocyte subsets and macrophages, but not DCs or neutrophils. By in-depth quantitative proteomics, we characterized changes in the proteome during monocyte differentiation, which provided insight into the molecular principles of developing monocytes, such as their functional maturation. Thus, we found that monocytes and macrophages were renewed independently of DCs from a committed progenitor.

Automatic extraction of actin networks in plants.

The actin cytoskeleton is essential in eukaryotes, not least in the plant kingdom where it plays key roles in cell expansion, cell division, environmental responses and pathogen defence. Yet, the precise structure-function relationships of properties of the actin network in plants are still to be unravelled, including details of how the network configuration depends upon cell type, tissue type and developmental stage. Part of the problem lies in the difficulty of extracting high-quality, quantitative measures of actin network features from microscopy data. To address this problem, we have developed DRAGoN, a novel image analysis algorithm that can automatically extract the actin network across a range of cell types, providing seventeen different quantitative measures that describe the network at a local level. Using this algorithm, we then studied a number of cases in Arabidopsis thaliana, including several different tissues, a variety of actin-affected mutants, and cells responding to powdery mildew. In many cases we found statistically-significant differences in actin network properties. In addition to these results, our algorithm is designed to be easily adaptable to other tissues, mutants and plants, and so will be a valuable asset for the study and future biological engineering of the actin cytoskeleton in globally-important crops.

DNA Methylation of α-Synuclein Intron 1 Is Significantly Decreased in the Frontal Cortex of Parkinson's Individuals with GBA1 Mutations.

Parkinson's disease (PD) is a common movement disorder, estimated to affect 4% of individuals by the age of 80. Mutations in the glucocerebrosidase 1 (GBA1) gene represent the most common genetic risk factor for PD, with at least 7-10% of non-Ashkenazi PD individuals carrying a GBA1 mutation (PD-GBA1). Although similar to idiopathic PD, the clinical presentation of PD-GBA1 includes a slightly younger age of onset, a higher incidence of neuropsychiatric symptoms, and a tendency to earlier, more prevalent and more significant cognitive impairment. The pathophysiological mechanisms underlying PD-GBA1 are incompletely understood, but, as in idiopathic PD, α-synuclein accumulation is thought to play a key role. It has been hypothesized that this overexpression of α-synuclein is caused by epigenetic modifications. In this paper, we analyze DNA methylation levels at 17 CpG sites located within intron 1 and the promoter of the α-synuclein (SNCA) gene in three different brain regions (frontal cortex, putamen and substantia nigra) in idiopathic PD, PD-GBA1 and elderly non-PD controls. In all three brain regions we find a tendency towards a decrease in DNA methylation within an eight CpG region of intron 1 in both idiopathic PD and PD-GBA1. The trend towards a reduction in DNA methylation was more pronounced in PD-GBA1, with a significant decrease in the frontal cortex. This suggests that PD-GBA1 and idiopathic PD have distinct epigenetic profiles, and highlights the importance of separating idiopathic PD and PD-GBA1 cases. This work also provides initial evidence that different genetic subtypes might exist within PD, each characterized by its own pathological mechanism. This may have important implications for how PD is diagnosed and treated.

Clinicopathologic Features, Treatment Response, and Outcomes of Immune Checkpoint Inhibitor-Related Esophagitis.

BACKGROUND: Although immune checkpoint inhibitors (ICIs) have provided practice-changing outcomes in treating many cancers, ICI-related gastrointestinal toxicity can limit their use. Upper gastrointestinal toxicity is not common nor as well described as lower gastrointestinal toxicity. We aimed to characterize the clinical presentation, endoscopic and histologic features, treatment response, and outcomes of ICI-related esophagitis. METHODS: We retrospectively studied patients at The University of Texas MD Anderson Cancer Center in whom esophagitis developed after receiving ICIs from June 2011 through January 2020. We included patients with endoscopic evidence of esophagitis and excluded those with other obvious causes of esophagitis. A chi-square test was used to assess associations between categorical variables. The Mann-Whitney U test was used to compare differences between continuous variables. RESULTS: Of 657 consecutive patients who underwent esophagogastroduodenoscopy (EGD) during or within 6 months of completing ICI-based therapy, 21 (3%) had esophagitis deemed to be from ICIs. Of these patients, 1 (5%) received an inhibitor of CTLA-4 alone, 15 (71%) received anti-PD-1 or PD-L1 monotherapy, and 5 (24%) received a combination of these. Median time from ICI initiation to onset of esophagitis was 4 months. Upon evaluation with EGD, only 3 patients (14%) had isolated esophageal involvement; 18 (86%) had concurrent involvement of the stomach, duodenum, or both. Most patients (67%) were treated with proton pump inhibitors, and 4 (19%) received steroids (prednisone or budesonide). The mortality rate was 38% (median follow-up, 15 months). CONCLUSIONS: Esophagitis associated with ICI use is rare. The diagnosis is one of exclusion because its clinical presentation appears similar to that of inflammation resulting from other causes. It often occurs in conjunction with other upper gastrointestinal toxicity. Symptoms are mild and respond well to nonimmunosuppressive treatment, with few severe complications.

Ion channel noise shapes the electrical activity of endocrine cells.

Endocrine cells in the pituitary gland typically display either spiking or bursting electrical activity, which is related to the level of hormone secretion. Recent work, which combines mathematical modelling with dynamic clamp experiments, suggests the difference is due to the presence or absence of a few large-conductance potassium channels. Since endocrine cells only contain a handful of these channels, it is likely that stochastic effects play an important role in the pattern of electrical activity. Here, for the first time, we explicitly determine the effect of such noise by studying a mathematical model that includes the realistic noisy opening and closing of ion channels. This allows us to investigate how noise affects the electrical activity, examine the origin of spiking and bursting, and determine which channel types are responsible for the greatest noise. Further, for the first time, we address the role of cell size in endocrine cell electrical activity, finding that larger cells typically display more bursting, while the smallest cells almost always only exhibit spiking behaviour.

A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes.

Peroxisomes are dynamic organelles which fulfil essential roles in lipid and ROS metabolism. Peroxisome movement and positioning allows interaction with other organelles and is crucial for their cellular function. In mammalian cells, such movement is microtubule-dependent and mediated by kinesin and dynein motors. The mechanisms of motor recruitment to peroxisomes are largely unknown, as well as the role this plays in peroxisome membrane dynamics and proliferation. Here, using a combination of microscopy, live-cell imaging analysis and mathematical modelling, we identify a role for Mitochondrial Rho GTPase 1 (MIRO1) as an adaptor for microtubule-dependent peroxisome motility in mammalian cells. We show that MIRO1 is targeted to peroxisomes and alters their distribution and motility. Using a peroxisome-targeted MIRO1 fusion protein, we demonstrate that MIRO1-mediated pulling forces contribute to peroxisome membrane elongation and proliferation in cellular models of peroxisome disease. Our findings reveal a molecular mechanism for establishing peroxisome-motor protein associations in mammalian cells and provide new insights into peroxisome membrane dynamics in health and disease.

Receptor Models of Phagocytosis: The Effect of Target Shape.

Phagocytosis is a remarkably complex process, requiring simultaneous organisation of the cell membrane, the cytoskeleton, receptors and various signalling molecules. As can often be the case, mathematical modelling is able to penetrate some of this complexity, identifying the key biophysical components and generating understanding that would take far longer with a purely experimental approach. This chapter will review a particularly important class of phagocytosis model, championed in recent years, that primarily focuses on the role of receptors during the engulfment process. These models are pertinent to a host of unsolved questions in the subject, including the rate of cup growth during uptake, the role of both intra- and extracellular noise, and the precise differences between phagocytosis and other forms of endocytosis. In particular, this chapter will focus on the effect of target shape and orientation, including how these influence the rate and final outcome of phagocytic engulfment.

Re-examining the Nature and Function of Self-Reactive T cells.

Recent studies have leveraged MHC tetramer and TCR sequencing approaches towards a more precise characterization of the peripheral T cell repertoire, providing important insight into both the contribution of self-reactive T cells to the overall repertoire and their function. The peripheral T cell repertoire of healthy individuals contains a high frequency of diverse, self-reactive T cells. Furthermore, self-reactive T cells can perform essential beneficial physiological functions. We review these recent findings here, and discuss their implications to the current understanding of peripheral tolerance and the role of self-reactive T cells in autoimmune disease. We outline gaps in understanding, and argue that an important step forward is to revise the definition of self-reactive T cells to incorporate new concepts regarding the nature and physiological functions of different populations of T cells capable of recognizing self-antigens.

Upper gastrointestinal symptoms and associated endoscopic and histological features in patients receiving immune checkpoint inhibitors.

Background: Immune checkpoint inhibitors (ICIs) have demonstrated effectiveness in treating many malignancies. Gastrointestinal (GI) adverse events are commonly reported; however, few reports describe upper GI tract toxic effects. We aimed to describe clinical features of upper GI injury related to ICI. Methods: We studied consecutive patients who received ICIs between April 2011 and March 2018 and developed upper GI symptoms requiring esophagogastroduodenoscopy (EGD). Results: Sixty patients developed upper GI symptoms between ICI initiation and 6 months after the last infusion. Among patients who had both EGD and colonoscopy (n = 38), 21 had endoscopic evidence of inflammation involving both the upper and lower GI tract. Overall, histological signs of inflammation of the stomach were evident in 83% of patients, but inflammation of the duodenum in 38%. Total of 42 patients had other risk factors of gastritis, i.e., chemotherapy, radiotherapy, and non-steroidal anti-inflammatory drugs. Only isolated gastric inflammation was seen on endoscopy in patients without these risk factors. The rates of ulceration were similar in the cohorts with and without other risk factors for gastritis. Isolated upper GI inflammation was related to anti-PD-1/L1 in 47% of patients. Immunosuppressive therapy in our cohort with upper GI toxicity consisted of steroids (42%) and infliximab or vedolizumab (23%). Most isolated upper GI symptoms were treated with proton pump inhibitors (65%) or H2 blockers (35%). Conclusion: We observed a correlation between ICI use and onset of upper GI inflammation even when other risk factors were excluded. Gastric involvement was evident more often than duodenal involvement on endoscopic and histological level.

Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis.

Phagocytosis and receptor-mediated endocytosis are vitally important particle uptake mechanisms in many cell types, ranging from single-cell organisms to immune cells. In both processes, engulfment by the cell depends critically on both particle shape and orientation. However, most previous theoretical work has focused only on spherical particles and hence disregards the wide-ranging particle shapes occurring in nature, such as those of bacteria. Here, by implementing a simple model in one and two dimensions, we compare and contrast receptor-mediated endocytosis and phagocytosis for a range of biologically relevant shapes, including spheres, ellipsoids, capped cylinders, and hourglasses. We find a whole range of different engulfment behaviors with some ellipsoids engulfing faster than spheres, and that phagocytosis is able to engulf a greater range of target shapes than other types of endocytosis. Further, the 2D model can explain why some nonspherical particles engulf fastest (not at all) when presented to the membrane tip-first (lying flat). Our work reveals how some bacteria may avoid being internalized simply because of their shape, and suggests shapes for optimal drug delivery.

How cells engulf: a review of theoretical approaches to phagocytosis.

Phagocytosis is a fascinating process whereby a cell surrounds and engulfs particles such as bacteria and dead cells. This is crucial both for single-cell organisms (as a way of acquiring nutrients) and as part of the immune system (to destroy foreign invaders). This whole process is hugely complex and involves multiple coordinated events such as membrane remodelling, receptor motion, cytoskeleton reorganisation and intracellular signalling. Because of this, phagocytosis is an excellent system for theoretical study, benefiting from biophysical approaches combined with mathematical modelling. Here, we review these theoretical approaches and discuss the recent mathematical and computational models, including models based on receptors, models focusing on the forces involved, and models employing energetic considerations. Along the way, we highlight a beautiful connection to the physics of phase transitions, consider the role of stochasticity, and examine links between phagocytosis and other types of endocytosis. We cover the recently discovered multistage nature of phagocytosis, showing that the size of the phagocytic cup grows in distinct stages, with an initial slow stage followed by a much quicker second stage starting around half engulfment. We also address the issue of target shape dependence, which is relevant to both pathogen infection and drug delivery, covering both one-dimensional and two-dimensional results. Throughout, we pay particular attention to recent experimental techniques that continue to inform the theoretical studies and provide a means to test model predictions. Finally, we discuss population models, connections to other biological processes, and how physics and modelling will continue to play a key role in future work in this area.

The Contained Self-Reactive Peripheral T Cell Repertoire: Size, Diversity, and Cellular Composition.

Individual self-reactive T cells have been discovered in both humans and mice. It is difficult to assess the entire contained self-reactive peripheral T cell repertoire in healthy individuals because regulatory T cells (Tregs) can render these cells anergic and, therefore, functionally indistinguishable. We addressed this issue by removing regulatory T cells, thereby allowing us to characterize the exposed self-reactive T cells. This resulted in activation of approximately 4% of both CD4(+) and CD8(+) T cells. Activation and division of these cells was not a bystander product of Ag-independent signals but required TCR stimulation. Analysis of TCR sequences showed that these responding cells were polyclonal and encompassed a broad range of structural TCR diversity. Adoptive transfer of naive and effector/memory T cell populations showed that even the naive T cell pool contained self-reactive T cell precursors. In addition, transfer of mature thymocytes showed that this response was an intrinsic T cell property rather than a peripheral adaptation. Finally, we found that the unexpectedly strong contribution of the naive CD5(low) T cell pool showed that the overall self-reactive response has not only a diverse polyclonal TCR repertoire, but also comprises a broad range of affinities for self.

Transcription Factor 7 Limits Regulatory T Cell Generation in the Thymus.

Regulatory T cells (Tregs) differentiate in the thymus, but the mechanisms that control this process are not fully understood. We generated a comprehensive quantitative and differential proteome of murine Tregs and conventional T cells. We identified 5225 proteins, 164 of which were differentially expressed in Tregs. Together with the comparative analysis of proteome and gene expression data, we identified TCF7 as a promising candidate. Genetic elimination of transcription factor 7 (TCF7) led to increased fractions of Tregs in the thymus. Reduced levels of TCF7, found in the heterozygote, resulted in a greater potential for Treg precursors to differentiate into the Treg lineage. In contrast, activation of TCF7 through ß-catenin had the opposite effect. TCF7 levels influenced the required TCR signaling strength of Treg precursors, and TCF7 deficiency broadened the repertoire and allowed lower TCR affinities to be recruited into the Treg lineage. FOXP3 was able to repress TCF7 protein expression. In summary, we propose a regulatory role for TCF7 in limiting access to the Treg lineage.

Accurate encoding and decoding by single cells: amplitude versus frequency modulation.

Cells sense external concentrations and, via biochemical signaling, respond by regulating the expression of target proteins. Both in signaling networks and gene regulation there are two main mechanisms by which the concentration can be encoded internally: amplitude modulation (AM), where the absolute concentration of an internal signaling molecule encodes the stimulus, and frequency modulation (FM), where the period between successive bursts represents the stimulus. Although both mechanisms have been observed in biological systems, the question of when it is beneficial for cells to use either AM or FM is largely unanswered. Here, we first consider a simple model for a single receptor (or ion channel), which can either signal continuously whenever a ligand is bound, or produce a burst in signaling molecule upon receptor binding. We find that bursty signaling is more accurate than continuous signaling only for sufficiently fast dynamics. This suggests that modulation based on bursts may be more common in signaling networks than in gene regulation. We then extend our model to multiple receptors, where continuous and bursty signaling are equivalent to AM and FM respectively, finding that AM is always more accurate. This implies that the reason some cells use FM is related to factors other than accuracy, such as the ability to coordinate expression of multiple genes or to implement threshold crossing mechanisms.

Spatiotemporal analysis of different mechanisms for interpreting morphogen gradients.

During development, multicellular organisms must accurately control both temporal and spatial aspects of tissue patterning. This is often achieved using morphogens, signaling molecules that form spatially varying concentrations and so encode positional information. Typical analysis of morphogens assumes that spatial information is decoded in steady state by measuring the value of the morphogen concentration. However, recent experimental work suggests that both pre-steady-state readout and measurement of spatial and temporal derivatives of the morphogen concentration can play important roles in defining boundaries. Here, we undertake a detailed theoretical and numerical study of the accuracy of patterning-both in space and time-in models where readout is provided not by the morphogen concentration but by its spatial and temporal derivatives. In both cases we find that accurate patterning can be achieved, with sometimes even smaller errors than directly reading the morphogen concentration. We further demonstrate that such models provide other potential benefits to the system, such as the ability to switch on and off gene response with a high degree of spatiotemporal accuracy. Finally, we discuss how such derivatives might be calculated biologically and examine these models in relation to Sonic Hedgehog signaling in the vertebrate central nervous system. We show that, when coupled to a downstream transcriptional network, pre-steady-state measurement of the temporal change in the Shh morphogen is a plausible mechanism for determining precise gene boundaries in both space and time.

Transcriptional control of regulatory T cells.

Regulatory T cells (Tregs) constitute unique T cell lineage that plays a key role for immunological tolerance. Tregs are characterized by the expression of the forkhead box transcription factor Foxp3, which acts as a lineage-specifying factor by determining the unique suppression profile of these immune cells. Here, we summarize the recent progress in understanding how Foxp3 expression itself is epigenetically and transcriptionally controlled, how the Treg-specific signature is achieved and how unique properties of Treg subsets are defined by other transcription factors. Finally, we will discuss recent studies focusing on the molecular targeting of Tregs to utilize the specific properties of this unique cell type in therapeutic settings.

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces.

In cells that exhibit apical growth, mechanisms that regulate cell polarity are crucial for determination of cellular shape and for the adaptation of growth to intrinsic and extrinsic cues. Broadly conserved pathways control cell polarity in eukaryotes, but less is known about polarly growing prokaryotes. An evolutionarily ancient form of apical growth is found in the filamentous bacteria Streptomyces, and is directed by a polarisome-like complex involving the essential protein DivIVA. We report here that this bacterial polarization machinery is regulated by a eukaryotic-type Ser/Thr protein kinase, AfsK, which localizes to hyphal tips and phosphorylates DivIVA. During normal growth, AfsK regulates hyphal branching by modulating branch-site selection and some aspect of the underlying polarisome-splitting mechanism that controls branching of Streptomyces hyphae. Further, AfsK is activated by signals generated by the arrest of cell wall synthesis and directly communicates this to the polarisome by hyperphosphorylating DivIVA. Induction of high levels of DivIVA phosphorylation by using a constitutively active mutant AfsK causes disassembly of apical polarisomes, followed by establishment of multiple hyphal branches elsewhere in the cell, revealing a profound impact of this kinase on growth polarity. The function of AfsK is reminiscent of the phoshorylation of polarity proteins and polarisome components by Ser/Thr protein kinases in eukaryotes.

The mechanism of phagocytosis: two stages of engulfment.

Despite being of vital importance to the immune system, the mechanism by which cells engulf relatively large solid particles during phagocytosis is still poorly understood. From movies of neutrophil phagocytosis of polystyrene beads, we measure the fractional engulfment as a function of time and demonstrate that phagocytosis occurs in two distinct stages. During the first stage, engulfment is relatively slow and progressively slows down as phagocytosis proceeds. However, at approximately half-engulfment, the rate of engulfment increases dramatically, with complete engulfment attained soon afterwards. By studying simple mathematical models of phagocytosis, we suggest that the first stage is due to a passive mechanism, determined by receptor diffusion and capture, whereas the second stage is more actively controlled, perhaps with receptors being driven toward the site of engulfment. We then consider a more advanced model that includes signaling and captures both stages of engulfment. This model predicts that there is an optimum ligand density for quick engulfment. Further, we show how this model explains why nonspherical particles engulf quickest when presented tip-first. Our findings suggest that active regulation may be a later evolutionary innovation, allowing fast and robust engulfment even for large particles.

Quantitative dynamics of telomere bouquet formation.

The mechanism by which homologous chromosomes pair during meiosis, as a prelude to recombination, has long been mysterious. At meiosis, the telomeres in many organisms attach to the nuclear envelope and move together to form the telomere bouquet, perhaps to facilitate the homologous search. It is believed that diffusion alone is not sufficient to account for the formation of the bouquet, and that some directed movement is also required. Here we consider the formation of the telomere bouquet in a wheat-rye hybrid both experimentally and using mathematical modelling. The large size of the wheat nucleus and wheat's commercial importance make chromosomal pairing in wheat a particularly interesting and important process, which may well shed light on pairing in other organisms. We show that, prior to bouquet formation, sister chromatid telomeres are always attached to a hemisphere of the nuclear membrane and tend to associate in pairs. We study a mutant lacking the Ph1 locus, a locus ensuring correct homologous chromosome pairing, and discover that bouquet formation is delayed in the wild type compared to the mutant. Further, we develop a mathematical model of bouquet formation involving diffusion and directed movement, where we show that directed movement alone is sufficient to explain bouquet formation dynamics.

Mechanistic basis of branch-site selection in filamentous bacteria.

Many filamentous organisms, such as fungi, grow by tip-extension and by forming new branches behind the tips. A similar growth mode occurs in filamentous bacteria, including the genus Streptomyces, although here our mechanistic understanding has been very limited. The Streptomyces protein DivIVA is a critical determinant of hyphal growth and localizes in foci at hyphal tips and sites of future branch development. However, how such foci form was previously unknown. Here, we show experimentally that DivIVA focus-formation involves a novel mechanism in which new DivIVA foci break off from existing tip-foci, bypassing the need for initial nucleation or de novo branch-site selection. We develop a mathematical model for DivIVA-dependent growth and branching, involving DivIVA focus-formation by tip-focus splitting, focus growth, and the initiation of new branches at a critical focus size. We quantitatively fit our model to the experimentally-measured tip-to-branch and branch-to-branch length distributions. The model predicts a particular bimodal tip-to-branch distribution results from tip-focus splitting, a prediction we confirm experimentally. Our work provides mechanistic understanding of a novel mode of hyphal growth regulation that may be widely employed.