Biology subjects

Patel, S.

Publications and source records attributed to Patel, S..

At least 217 records · Page 12Linked to original sources

bioRxiv2019

An engineered CRISPR/Cas9 mouse line for simultaneous readout of lineage histories and gene expression profiles in single cells

Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Particularly in the context of stem cell biology, analysis of single cell lineages in their native state has elucidated novel fates and highlighted heterogeneity of function. Coupling of such ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CARLIN (for CRISPR Array Repair LINeage tracing) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures based on in vivo stem cell function without a need for markers or cell sorting.

developmental biology↗

bioRxiv2019

Automated Lifespan Determination Across Caenorhabditis Strains and Species Reveals Assay-Specific Effects of Chemical Interventions

The goal of the Caenorhabditis Intervention Testing Program is to identify robust and reproducible pro-longevity interventions that are efficacious across genetically diverse cohorts in the Caenorhabditis genus. The project design features multiple experimental replicates collected by three different laboratories. Our initial effort employed fully manual survival assays. With an interest in increasing throughput, we explored automation with flatbed scanner-based Automated Lifespan Machines (ALMs). We used ALMs to measure survivorship of 22 Caenorhabditis strains spanning three species. Additionally, we tested five chemicals that we previously found extended lifespan in manual assays. Overall, we found similar sources of variation among trials for the ALM and our previous manual assays, verifying reproducibility of outcome. Survival assessment was generally consistent between the manual and the ALM assays, although we did observe radically contrasting results for certain compound interventions. We found that particular lifespan outcome differences could be attributed to protocol elements such as enhanced light exposure of specific compounds in the ALM, underscoring that differences in technical details can influence outcomes and therefore interpretation. Overall, we demonstrate that the ALMs effectively reproduce a large, conventionally scored dataset from a diverse test set, independently validating ALMs as a robust and reproducible approach towards aging-intervention screening.

physiology↗

bioRxiv2019

Regulation of thymocyte β-selection, development and positive selection by glycogen synthase kinase-3

Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine kinase, that exists as two isoforms in mammals, GSK-3 and GSK-3{beta}, that are key downstream mediators of the phosphatidylinositol 3 kinase, Wnt, Notch and other pathways. Here, we report that simultaneous inactivation of both GSK-3 and GSK-3{beta} during early thymocyte ontogeny has profound effects on both {beta}-selection and positive selection, key checkpoints essential to producing functionally mature {beta} T cells. Conditional GSK-3/{beta} knockout animals (LckCre+ GSK-3{beta}fl/fl) possessed pre-double positive (pre-DP) thymocytes (CD4-CD8-CD117-CD25-) with compromised TCR{beta} chain expression along with elevated levels of {beta}-catenin and reduced Notch activity. {beta}-selection was impaired allowing pre-DP thymocytes to differentiate to DP thymocytes (CD4+CD8+) while bypassing strict requirements for productive TCR{beta} chain rearrangements and functional expression. Also impaired was the requisite pre-TCR and Notch-mediated expansion that normally precedes differentiation to the DP stage. Consequently, LckCre+ GSK-3{beta}fl/fl mice initially generated fewer DP thymocytes that expressed significantly reduced levels of mature TCR. The aberrant DP thymocytes expressed high levels of the pro-survival Bcl-2 family member Mcl-1, failed positive selection and accumulated as CD4hiCD8lo positive selection intermediates resulting in loss of both mature CD4 and CD8 lineages. LckCre+ GSK-3{beta}fl/fl mice succumbed to oligoclonal peripheral lymphomas with high penetrance. These data reveal essential roles for GSK-3 in several checkpoints of early T cell development.

immunology↗

bioRxiv2019

GDF15 and the beneficial actions of metformin in pre-diabetes

Metformin, the worlds most prescribed anti-diabetic drug, is also effective in preventing Type 2 diabetes in people at high risk, by lowering body weight, fat mass and circulating insulin levels through mechanisms that are incompletely understood. Recent observational studies reporting the association of metformin use and circulating levels of GDF15 led us to hypothesize that GDF15, which signals through a specific receptor complex in the hindbrain to reduce body weight, might mediate these effects. We measured GDF15 in people without diabetes from a randomized placebo-controlled trial of metformin. Over 18 months, participants allocated metformin lost significant weight and levels of GDF15 were persistently elevated compared to placebo. The change in plasma GDF15 in this study correlated positively with weight loss. In wild-type mice, oral metformin increased circulating GDF15 with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to high fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GFRAL. In obese, high fat-fed mice, the effects of metformin to reduce body weight were reversed by a GFRAL antagonist antibody. Metformin had effects on both energy intake and energy expenditure that required GDF15. The insulin sensitising effects of metformin determined by insulin tolerance were abolished in mice lacking GDF15. Metformin significantly reduced fasting glucose and insulin levels in wild type but not in mice lacking GDF15. In summary, metformin increases the circulating levels of GDF15, which appears to be necessary for many of its actions as a metabolic chemopreventive agent.

physiology↗

bioRxiv2019

Human Umbilical Cord blood monocytes, but not adult blood monocytes, rescue brain cells from hypoxic-ischemic injury: Mechanistic and therapeutic implications

Cord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC.

neuroscience↗

bioRxiv2019

Toxoplasma gondii infection drives conversion of NK Cells into ILC1s

Innate lymphoid cells (ILCs) were originally classified based on their cytokine profiles, placing natural killer (NK) cells and ILC1s together, but recent studies support their separation into different lineages at steady-state. However, tumors may induce NK cell conversion into ILC1-like cells that are limited to the tumor microenvironment and whether this conversion occurs beyond this environment remains unknown. Here we describe Toxoplasma gondii infection converts NK cells into cells resembling steady-state ILC1s that are heterogeneous and distinct from both steady-state NK cells and ILC1s in uninfected mice. Most toxoplasma-induced ILC1s were Eomes-dependent, indicating that NK cells can give rise to Eomes- Tbet-dependent ILC1-like cells that circulate widely and persist independent of ongoing infection. Moreover, these changes appear permanent, as supported by epigenetic analyses. Thus, these studies markedly expand current concepts of NK cells, ILCs, and their potential conversion.

immunology↗

bioRxiv2019

Leveraging non-human primate multisensory neurons and circuits in assessing consciousness theory

Both the Global Neuronal Workspace (GNW) and Integrated Information Theory (IIT) posit that highly complex and interconnected networks engender perceptual awareness. GNW specifies that activity recruiting fronto-parietal networks will elicit a subjective experience, while IIT is more concerned with the functional architecture of networks than with activity within it. Here, we argue that according to IIT mathematics, circuits converging on integrative vs. convergent yet non-integrative neurons should support a greater degree of consciousness. We test this hypothesis by analyzing a dataset of neuronal responses collected simultaneously from primary somatosensory cortex (S1) and ventral premotor cortex (vPM) in non-human primates presented with auditory, tactile, and audio-tactile stimuli as they are progressively anesthetized with Propofol. We first describe the multisensory (audio-tactile) characteristics of S1 and vPM neurons (mean and dispersion tendencies, as well as noise-correlations), and functionally label these neurons as convergent or integrative according to their spiking responses. Then, we characterize how these different pools of neurons behave as a function of consciousness. At odds with the IIT mathematics, results suggest that convergent neurons more readily exhibit properties of consciousness (neural complexity and noise correlation) and are more impacted during the loss of consciousness than integrative neurons. Lastly, we provide support for the GNW by showing that neural ignition (i.e., same trial co-activation of S1 and vPM) was more frequent in conscious than unconscious states. Overall, we contrast GNW and IIT within the same single-unit activity dataset, and support the GNW.

neuroscience↗

bioRxiv2019

Eco-Evolutionary Feedbacks between Predator’s Linkage Disequilibrium and Prey Densities Maintain Diversity

Diversity occurs at multiple scales. Within a single population, there is diversity in genotypes and phenotypes. At a larger scale, within ecological communities, there is diversity in species. There have been a number of studies to investigate how diversity at these two scales influence each other through what has been termed eco-evolutionary feedbacks. Here we study a three-species ecological module called apparent competition, in which the predator is evolving in a trait that determines its interaction with two prey species. Unlike previous studies on apparent competition, which employed evolutionary frameworks with greatly simplified genetics, we study a multilocus eco-evolutionary model in which the predators evolving trait is determined by two diallelic loci. We ask how eco-evolutionary feedbacks with these two loci affect the coexistence of the prey species and the maintenance of polymorphisms within the predator species. In doing so, we uncover a novel eco-evolutionary feedback between the prey species and the linkage disequilibrium of the two loci of the predator. Through a stability analysis, we demonstrate how these feedbacks affect polymorphisms at both loci and, among others, may generate stable cycling.

evolutionary biology↗

bioRxiv2019

Antagonistic coevolution between multiple quantitative traits: matching interactions can be subsets of difference interactions

AO_SCPLOWBSTRACTC_SCPLOWCoevolution is one of the major drivers of complex dynamics in population ecology. Historically, antagonistic coevolution in victim-exploiter systems has been a topic of special interest, and involves traits with various genetic architectures (e.g., the number of genes involved) and effects on interactions. For example, exploiters may need to have traits that \"match\" those of victims for successful exploitation (i.e., a matching interaction), or traits that exceed those of victims (i.e., a difference interaction). Different models exist which are appropriate for different types of traits, including Mendelian (discrete) and quantitative (continuous) traits. For models with multiple Mendelian traits, recent studies have shown that antagonistic coevolutionary patterns that appear as matching interactions can arise due to multiple difference interactions with costs of having large trait values. Here we generalize their findings to quantitative traits and show, analogously, that the multidimensional difference interactions with costs sometimes behave qualitatively the same as matching interactions. While previous studies in quantitative genetics have used the dichotomy between matching and difference frameworks to explore coevolutionary dynamics, we suggest that exploring multidimensional trait space is important to examine the generality of results obtained from one-dimensional traits.

evolutionary biology↗

bioRxiv2018

Destabilizing evolutionary and eco-evolutionary feedbacks drive eco-evo cycles in empirical systems

We develop a method to identify how ecological, evolutionary, and eco-evolutionary feedbacks influence system stability. We apply our method to nine empirically-parameterized eco-evolutionary models of exploiter-victim systems from the literature and identify which particular feedbacks cause some systems to converge to a steady state or to exhibit sustained oscillations. We find that ecological feedbacks involving the interactions between all species and evolutionary and eco-evolutionary feedbacks involving only the interactions between exploiter species (predators or pathogens) are typically stabilizing. In contrast, evolutionary and eco-evolutionary feedbacks involving the interactions between victim species (prey or hosts) are destabilizing more often than not. We also find that while eco-evolutionary feedbacks rarely altered system stability from what would be predicted from just ecological and evolutionary feedbacks, eco-evolutionary feedbacks have the potential to alter system stability at faster or slower speeds of evolution. As the number of empirical studies demonstrating eco-evolutionary feedbacks increases, we can continue to apply these methods to determine whether the patterns we observe are common in other empirical communities.

ecology↗

bioRxiv2018

Mechanistic insights from replica exchange molecular dynamics simulations into mutation induced disordered-to-ordered transition in Hahellin, a βγ-crystallin

Intrinsically disordered proteins (IDPs) form a special category because they lack a unique well-folded 3D structure under physiological conditions. They play crucial role in cell signaling, regulatory functions and responsible for several diseases. Although, they are abundant in nature, only a small fraction of it has been characterized till date. Such proteins adopt a range of conformations and can undergo transformation from disordered-to-ordered state or vice-versa upon binding to ligand. Insights of such conformational transition is perplexing in several cases. In the present study, we characterized disordered as well as ordered states and the factors contributing the transitions through a mutational study by employing replica exchange molecular dynamics simulation on a {beta}{gamma}-crystallin. Most of the proteins within this superfamily are inherently ordered. However, Hahellin, although a member of {beta}{gamma}-crystallin, it is intrinsically disordered in its apo-form which takes a well-ordered {beta}{gamma}-crystallin fold upon binding to Ca2+. It is intriguing that the mutation at the 5th position of the canonical motif to Arg increases the domain stability in several ordered microbial {beta}{gamma}-crystallins with concomitant loss in Ca2+ binding affinity. We carried out similar Ser to Arg mutations at 5th position of the canonical motif for the first time in an intrinsically disordered protein to understand the mechanistic insights of conformational transition. Our study revealed that newly formed ionic and hydrogen bonding interactions at the canonical Ca2+ binding sites play crucial role in transforming the disordered conformation into ordered {beta}{gamma}-crystallin.\n\nAuthor summaryIntrinsically disordered proteins lack a unique ordered 3D structure under physiological condition. Although, they are abundant in nature, only a small fraction of these proteins has been characterized till date due to adaptation of multiple conformations and methodological limitation. {beta}{gamma}-crystallins are inherently ordered, however recently a small number of proteins within this superfamily have been identified as intrinsically disordered protein. Hahellin is one such protein which is intrinsically disordered in its apo-form but takes a well-ordered {beta}{gamma}-crystallin fold upon binding to Ca2+. In the present study, we decipher the underlying mechanism of disordered-to-ordered transition in Hahellin by mutations, employing replica exchange molecular dynamics simulations. Earlier experimental studies reported an increase in stabilization of the ordered {beta}{gamma}-crystallion upon mutation to Arg at 5th position of the canonical Ca2+ binding motifs, N/D-N/D-X1-X2-S/T-S. We performed similar Ser to Arg mutation in an intrinsically disordered Hahellin to get the mechanistic insights of the conformational transition in the absence of Ca2+. Our study revealed that several newly formed ionic and hydrogen bonding interactions contributed by the mutant residues are responsible for both intra- and inter-motif rigidification, resulting in overall stability of {beta}{gamma}-crystallin domain.

biophysics↗

bioRxiv2018

Genome-wide scan and fine-mapping of rare nonsynonymous associations implicates intracellular lipolysis genes in fat distribution and cardio-metabolic risk

Difficulties in identifying causal variants and genes underlying genetic associations have limited the translational potential of genetic studies of body fat distribution, an important, partly-heritable risk factor for cardio-metabolic disease. Rare variant associations facilitate fine-mapping of causal alleles, but their contribution to fat distribution is understudied. We performed a genome-wide scan of rare nonsynonymous variants for body mass index-adjusted waist-to-hip-ratio (BMI-adjusted WHR; a widely-used measure of fat distribution) in 450,562 European ancestry individuals, followed by systematic Bayesian fine-mapping at six genome-wide (p<5x10-08; main-analysis) and two subthreshold signals (significant at a Bonferroni-corrected p<1.3x10-06). We found strong statistical evidence of causal association for nonsynonymous alleles in CALCRL (p.L87P, pconditional=5.9x10-12; posterior-probability of association [PPA]=52%), PLIN1 (p.L90P, pconditional=5.5x10-13; PPA>99%), PDE3B (p.R783X, pconditional=6.2x10-15; PPA>99%), ACVR1C (p.I195T; pconditional=5.4x10-12; PPA>99%), and FGF1 (p.G21E, pconditional=1.6x10-07; PPA=98%). Alleles at the four likely-causal main-analysis genes affected fat distribution primarily via larger hip-rather than smaller waist-circumference and six of nine conditionally-independent WHR-lowering index-variants were associated with protection from cardiovascular or metabolic disease. All four genes are expressed in adipose tissue and have been linked with the regulation of intracellular lipolysis, which controls fat retention in mature cells. Targeted follow-up analyses of key intracellular-lipolysis genes revealed associations for a variant in the initiator of intracellular lipolysis PNPLA2 (p.N252K) with higher BMI-adjusted-WHR and higher cardio-metabolic risk. This study provides human genetic evidence of a link between intracellular lipolysis, fat-distribution and its cardio-metabolic complications in the general population.

genetics↗

bioRxiv2018

Gfi1-expressing Paneth cells revert to stem cells following intestinal injury

BackgroundAimChemotherapy drugs harm rapidly dividing normal healthy cells such as those lining the gastrointestinal tract, causing morbidity and mortality that complicates medical treatment modalities. Growth Factor-Independent 1 (GFI1) is a zinc finger transcriptional repressor implicated in the differentiation of secretory precursors into Paneth and goblet cells in the intestinal epithelium. We hypothesize that stimulating the reversion of Gfi1+ secretory cells into stem cells will improve intestinal epithelial regeneration and mitigate injury.\n\nMethodsGfi1 reporter mice (Gfi1cre/+; ROSA26 LSL-YFP) were treated with Doxorubicin, radiation, anti-CD3 antibody, and rotavirus to induce intestinal injury. Mice and intestinal organoids (enteroids) were used to investigate cellular repair mechanisms following injury.\n\nResultsUnder homeostatic conditions, Gfi1-lineage cells are Paneth and goblet cells, which were non-proliferative and not part of the stem cell pool. After injury, Gfi1+ secretory cells can re-enter the cell cycle and give rise to all cell lineages of the intestinal epithelium including stem cells. Reversion of Gfi1-lineage cells was observed in other injury model systems, including irradiation and anti-CD3 treatment, but not in ISC-sparing rotavirus infection. Our results also demonstrated that PI3kinase/AKT activation improved cell survival, and elevated WNT signaling increased the efficiency of Gfi1+ cell reversion upon injury.\n\nConclusionsThese findings indicate that Gfi1+ secretory cells display plasticity and reacquire stemness following severe damage. Moreover, PI3kinase/AKT and WNT are key regulators involved in injury-induced regeneration. Our studies identified potential therapeutic intervention strategies to mitigate the adverse effects of chemotherapy-induced damage to normal tissues and improve the overall effectiveness of cancer chemotherapy.

cell biology↗

bioRxiv2018

Growth Factor Independent 1 is a tumor suppressor gene in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer death in the United States, causing about 50,000 deaths each year. Growth Factor-Independent 1 (GFI1) is a critical zinc finger transcriptional repressor responsible for controlling secretory cell differentiation in the small intestine and colon. GFI1 plays a significant role in the development of human malignancies, including leukemia, lung cancer and prostate cancer. However, the role of GFI1 in CRC progression is largely unknown. Our results demonstrate that RNA and protein expression of GFI1 are reduced in advanced stages of non-mucinous CRC. Subcutaneous tumor models demonstrated that the re-expression of GFI1 in 4 different human CRC cell lines inhibits tumor growth by 25-60%. To further investigate the role of Gfi1 in de novo colorectal tumorigenesis, we developed transgenic mice harboring a deletion of Gfi1 in the distal intestine driven by the CDX2cre (Gfi1F/F; CDX2cre/+) and crossed them with ApcMin/+ mice (ApcMin/+; Gfi1F/F; CDX2cre/+). Loss of Gfi1 significantly increased the total number of colorectal adenomas compared to littermate controls with an APC mutation alone. Furthermore, we found that compound (ApcMin/+; Gfi1F/F; CDX2cre/+) mice develop both adenomas as well as carcinoid-like tumors expressing the neuroendocrine marker chromogranin A, a feature that has not been previously described in APC-mutant tumors in mice. Collectively, these results demonstrate that Gfi1 deficiency promotes colorectal tumorigenesis, and suggest that loss of Gfi1 may promote formation of carcinoid cancers of the large intestines.\n\nSignificanceThese findings reveal that GFI1 functions as a tumor suppressor gene in colorectal tumorigenesis.

cancer biology↗

bioRxiv2018

A non-canonical lysosome biogenesis pathway generates Golgi-associated lysosomes during epidermal differentiation

Keratinocytes maintain epidermis integrity and function including physical and antimicrobial barrier through cellular differentiation. This process is predicted to be controlled by calcium ion gradient and nutritional stress. Keratinocytes undergo proteome changes during differentiation, which enhances the intracellular organelle digestion to sustain the stress conditions. However, the molecular mechanism between epidermal differentiation and organelle homeostasis is poorly understood. Here, we used primary neonatal human epidermal keratinocytes to study the link between cellular differentiation, signaling pathways and organelle turnover. Upon addition of calcium chloride (2 mM) to the culture medium, keratinocytes increased their cell size and the expression of differentiation markers. Moreover, differentiated keratinocytes showed enhanced lysosome biogenesis that was dependent on ATF6-arm of UPR signaling but independent of mTOR-MiT/TFE transcription factors. Furthermore, chemical inhibition of mTOR has increased keratinocyte differentiation and relocalized the MiT/TFE TFs to the lysosome membranes, indicating that autophagy activation promotes the epidermal differentiation. Interestingly, differentiation of keratinocytes resulted in dispersal of fragmented Golgi and lysosomes, and the later organelles showed colocalization with Golgi-tethering proteins, suggesting that these lysosomes possibly originated from Golgi, hence named as Golgi-associated lysosomes (GALs). Consistent to this prediction, inhibition of Golgi function using brefeldin A completely abolished the formation of GALs and the keratinocyte differentiation. Thus, ER stress regulates the biogenesis of GALs, which maintains keratinocyte differentiation and epidermal homeostasis.

cell biology↗

bioRxiv2018

Helicase Promotes Replication Re-initiation from an RNA Transcript

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind the DNA, resulting in a gap in the newly replicated DNA. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances the fork, the DNAP also moves forward processively, via its association with the helicase. The presence of the DNAP, in turn, increases both helicases processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicases novel role in replication re-initiation, independent of replication restart proteins or primase.

biophysics↗

bioRxiv2018

TRPML1-mediated Modulation of Dense-core Granules Tunes Functional Potential in NK Cells

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. We found that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show accumulation of granzyme B, localized in dense-core secretory lysosomes, converged close to the centrosome. This discrete morphological phenotype persists in self-KIR+ NK cells independently of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. The granzymeB dense, large secretory lysosomes in self-KIR+ NK cells were efficiently released upon target cell recognition, contributing to their enhanced cytotoxic capacity. Secretory lysosomes are part of the acidic lysosomal compartment, which has been shown to channel calcium and mediate intracellular signalling in several cell types. Interference of signaling from acidic Ca2+ stores in primary NK cells reduced both target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1 led to increased levels of granzyme B and enhanced functional potential. These results indicate an intrinsic role for lysosomal homeostasis in NK cell education.

immunology↗

bioRxiv2018

Microbiome inhibition of IRAK-4 by trimethylamine mediates metabolic and immune benefits in high-fat-diet-induced insulin resistance

The global type 2 diabetes epidemic is a major health crisis and there is a critical need for innovative strategies to fight it. Although the microbiome plays important roles in the onset of insulin resistance (IR) and low-grade inflammation, the microbial compounds regulating these phenomena remain to be discovered. Here, we reveal that the microbiome inhibits a central kinase, eliciting immune and metabolic benefits. Through a series of in vivo experiments based on choline supplementation, blocking trimethylamine (TMA) production then administering TMA, we demonstrate that TMA decouples inflammation and IR from obesity in the context of high-fat diet (HFD) feeding. Through in vitro kinome screens, we reveal TMA specifically inhibits Interleukin-1 Receptor-associated Kinase 4 (IRAK4), a central kinase integrating signals from various toll-like receptors and cytokine receptors. TMA blunts TLR4 signalling in primary human hepatocytes and peripheral blood monocytic cells, and improves mouse survival after a lipopolysaccharide-induced septic shock. Consistent with this, genetic deletion and chemical inhibition of IRAK4 result in similar metabolic and immune improvements in HFD. In summary, TMA appears to be a key microbial compound inhibiting IRAK4 and mediating metabolic and immune effects with benefits upon HFD. Thereby we highlight the critical contribution of the microbial signalling metabolome in homeostatic regulation of host disease and the emerging role of the kinome in microbial-mammalian chemical crosstalk.

systems biology↗