Insular cortex neurons encode and retrieve specific immune responses.

Increasing evidence indicates that the brain regulates peripheral immunity, yet whether and how the brain represents the state of the immune system remains unclear. Here, we show that the brain's insular cortex (InsCtx) stores immune-related information. Using activity-dependent cell labeling in mice (FosTRAP), we captured neuronal ensembles in the InsCtx that were active under two different inflammatory conditions (dextran sulfate sodium [DSS]-induced colitis and zymosan-induced peritonitis). Chemogenetic reactivation of these neuronal ensembles was sufficient to broadly retrieve the inflammatory state under which these neurons were captured. Thus, we show that the brain can store and retrieve specific immune responses, extending the classical concept of immunological memory to neuronal representations of inflammatory information.

Optogenetic activation of local colonic sympathetic innervations attenuates colitis by limiting immune cell extravasation.

The sympathetic nervous system is composed of an endocrine arm, regulating blood adrenaline and noradrenaline, and a local arm, a network of fibers innervating immune organs. Here, we investigated the impact of the local arm of the SNS in an inflammatory response in the colon. Intra-rectal insertion of an optogenetic probe in mice engineered to express channelrhodopsin-2 in tyrosine hydroxylase cells activated colonic sympathetic fibers. In contrast to systemic application of noradrenaline, local activation of sympathetic fibers attenuated experimental colitis and reduced immune cell abundance. Gene expression profiling showed decreased endothelial expression of the adhesion molecule MAdCAM-1 upon optogenetic stimulation; this decrease was sensitive to adrenergic blockers and 6-hydroxydopamine. Antibody blockade of MAdCAM-1 abrogated the optogenetic effect on immune cell extravasation into the colon and the pathology. Thus, sympathetic fibers control colonic inflammation by regulating immune cell extravasation from circulation, a mechanism likely relevant in multiple organs.

Early Cardiac Remodeling Promotes Tumor Growth and Metastasis.

BACKGROUND: Recent evidence suggests that cancer and cardiovascular diseases are associated. Chemotherapy drugs are known to result in cardiotoxicity, and studies have shown that heart failure and stress correlate with poor cancer prognosis. However, whether cardiac remodeling in the absence of heart failure is sufficient to promote cancer is unknown. METHODS: To investigate the effect of early cardiac remodeling on tumor growth and metastasis colonization, we used transverse aortic constriction (TAC), a model for pressure overload-induced cardiac hypertrophy, and followed it by cancer cell implantation. RESULTS: TAC-operated mice developed larger primary tumors with a higher proliferation rate and displayed more metastatic lesions compared with controls. Serum derived from TAC-operated mice potentiated cancer cell proliferation in vitro, suggesting the existence of secreted tumor-promoting factors. Using RNA-sequencing data, we identified elevated mRNA levels of periostin in the hearts of TAC-operated mice. Periostin levels were also found to be high in the serum after TAC. Depletion of periostin from the serum abrogated the proliferation of cancer cells; conversely, the addition of periostin enhanced cancer cell proliferation in vitro. This is the first study to show that early cardiac remodeling nurtures tumor growth and metastasis and therefore promotes cancer progression. CONCLUSIONS: Our study highlights the importance of early diagnosis and treatment of cardiac remodeling because it may attenuate cancer progression and improve cancer outcome.

Adaptation of Salmonella enterica Serovar Senftenberg to Linalool and Its Association with Antibiotic Resistance and Environmental Persistence.

A clinical isolate of Salmonella enterica serovar Senftenberg, isolated from an outbreak linked to the herb Ocimum basilicum L. (basil), has been shown to be resistant to basil oil and to the terpene alcohol linalool. To better understand how human pathogens might develop resistance to linalool and to investigate the association of this resistance with resistance to different antimicrobial agents, selective pressure was applied to the wild-type strain by sequential exposure to increasing concentrations of linalool. The results demonstrated that S Senftenberg adapted to linalool with a MIC increment of at least 8-fold, which also resulted in better resistance to basil oil and better survival on harvested basil leaves. Adaptation to linalool was shown to confer cross protection against the antibiotics trimethoprim, sulfamethoxazole, piperacillin, chloramphenicol, and tetracycline, increasing their MICs by 2- to 32-fold. The improved resistance was shown to correlate with multiple phenotypes that included changes in membrane fatty acid composition, induced efflux, reduced influx, controlled motility, and the ability to form larger aggregates in the presence of linalool. The adaptation to linalool obtained in vitro did not affect survival on the basil phyllosphere in planta and even diminished survival in soil, suggesting that development of extreme resistance to linalool may be accompanied by a loss of fitness. Altogether, this report notes the concern regarding the ability of human pathogens to develop resistance to commercial essential oils, a resistance that is also associated with cross-resistance to antibiotics and may endanger public health.IMPORTANCE Greater consumer awareness and concern regarding synthetic chemical additives have led producers to control microbial spoilage and hazards by the use of natural preservatives, such as plant essential oils with antimicrobial activity. This report establishes, however, that these compounds may provoke the emergence of resistant human pathogens. Herein, we demonstrate the acquisition of resistance to basil oil by Salmonella Senftenberg. Exposure to linalool, a component of basil oil, resulted in adaptation to the basil oil mixture, as well as cross protection against several antibiotics and better survival on harvested basil leaves. Collectively, this work highlights the hazard to public health while using plant essential oils without sufficient knowledge about their influence on pathogens at subinhibitory concentrations.

Mechanisms of resistance to linalool in Salmonella Senftenberg and their role in survival on basil.

Fresh produce contaminated with human pathogens raises vital and ecological questions about bacterial survival strategies. Such occurrence was basil harboring Salmonella enterica serovar Senftenberg that caused an outbreak in 2007. This host was unanticipated due to its production of antibacterial substances, including linalool. We show that linalool perforates bacterial membranes, resulting in increased permeability and leakage of vital molecules. It also inhibits cell motility and causes bacterial aggregation. Linalool-resistance was investigated by identification and characterization of S. Senftenberg mutants that perform altered resistance. Resistance mechanisms include selective permeability, regulated efflux/influx and chemotaxis-controlled motility. Moreover, survival of S. Senftenberg on basil leaves was substantially affected by McpL, a putative chemotaxis-related receptor, and RfaG, a component of the lipopolysaccharide production pathway, both have a role in resistance to linalool. Results reveal that adaptation to linalool occurs in nature by concurrent mechanisms. This adaption raises concerns about pathogens adaptation to new hosts including antimicrobial-compound-producing plants.

Preparation of single-cell suspensions of mouse glomeruli for high-throughput analysis.

The kidney glomerulus is essential for proper kidney function. Until recently, technical challenges associated with glomerular isolation and subsequent dissolution into single cells have limited the detailed characterization of cells in the glomerulus. Previous techniques of kidney dissociation result in low glomerular cell yield, which limits high-throughput analysis. The ability to efficiently purify glomeruli and digest the tissue into single cells is especially important for single-cell characterization methods. Here, we present a detailed and comprehensive technique for the extraction and preparation of mouse glomerular cells, with high yield and viability. The method includes direct renal perfusion of Dynabeads via the renal artery followed by kidney dissociation and isolation of glomeruli by magnet; these steps provide a high number and purity of isolated glomeruli, which are further dissociated into single cells. The balanced representation of podocytes, mesangial and endothelial cells in single-cell suspensions of mouse glomeruli, and the high cell viability observed, confirm the efficiency of our method. With some practice, the procedure can be done in <3 h (excluding equipment setup and data analysis). This protocol provides a valuable technique for advancing future single-cell-based studies of the glomerulus in health, injury and disease.

The Mesangial cell - the glomerular stromal cell.

Mesangial cells are stromal cells that are important for kidney glomerular homeostasis and the glomerular response to injury. A growing body of evidence demonstrates that mesenchymal stromal cells, such as stromal fibroblasts, pericytes and vascular smooth muscle cells, not only specify the architecture of tissues but also regulate developmental processes, vascularization and cell fate specification. In addition, through crosstalk with neighbouring cells and indirectly through the remodelling of the matrix, stromal cells can regulate a variety of processes such as immunity, inflammation, regeneration and in the context of maladaptive responses - fibrosis. Insights into the molecular phenotype of kidney mesangial cells suggest that they are a specialized stromal cell of the glomerulus. Here, we review our current understanding of mesenchymal stromal cells and discuss how it informs the function of mesangial cells and their role in disease. These new insights could lead to a better understanding of kidney disease pathogenesis and the development of new therapies for chronic kidney disease.

Progesterone Attenuates Brain Inflammatory Response and Inflammation-Induced Increase in Immature Myeloid Cells in a Mouse Model.

Progesterone has been shown to regulate immunity during pregnancy, and progesterone administration may reduce inflammation-induced preterm labor. We sought to determine the maternal brain immune response to LPS-induced inflammation in pregnant and non-pregnant mice and whether additional progesterone supplementation attenuates this response. Pregnant (P: n = 9) and non-pregnant mice (NP: n = 9) were randomized to pretreatment with vaginal progesterone/carrier (Replens), daily from days 13 to 16. On days 15 and 16, LPS/saline was administered by intraperitoneal injection (Replens + saline n = 3; Replens + LPS n = 3; progesterone + LPS n = 3). Mice were sacrificed on day 16 and maternal serum analyzed for IL-6 levels and brains analyzed for nNOS, NF-kB, IL-6 protein levels and for immature myeloid cells (IMCs) and microglial activity. LPS significantly increased brain nNOS, NF-kB, and IL-6 in both NP and P mice, with significantly greater responses in P mice. In both NP and P groups, progesterone significantly attenuated LPS-induced increase of nNOS and NF-kB, however with no effect on serum IL-6. In the NP brains, LPS significantly increased IMC population and progesterone reduced the IMC phenotype to levels similar to controls. In P mice, neither LPS nor LPS + progesterone altered the brain IMC population. LPS significantly increased the microglial activity in both NP and P groups, which was attenuated by progesterone. Progesterone attenuates brain inflammatory response to LPS in both NP and P mice although it has no effect on systemic inflammation. In NP mice, progesterone attenuated the increase in brain IMC following LPS administration. Our results suggest that endogenous progesterone during pregnancy may protect the brain from LPS-induced inflammation.

Short-term sleep deprivation in mice induces B cell migration to the brain compartment.

Increasing evidence highlight the involvement of immune cells in brain activity and its dysfunction. The brain's immune compartment is a dynamic ensemble of cells that can fluctuate even in naive animals. However, the dynamics and factors that can affect the composition of immune cells in the naive brain are largely unknown. Here, we examined whether acute sleep deprivation can affect the brain's immune compartment (parenchyma, meninges, and choroid plexus). Using high-dimensional mass cytometry analysis, we broadly characterized the effects of short-term sleep deprivation on the immune composition in the mouse brain. We found that after 6 h of sleep deprivation, there was a significant increase in the abundance of B cells in the brain compartment. This effect can be accounted for, at least in part, by the elevated expression of the migration-related receptor, CXCR5, on B cells and its ligand, cxcl13, in the meninges following sleep deprivation. Thus, our study reveals that short-term sleep deprivation affects the brain's immune compartment, offering a new insight into how sleep disorders can affect brain function and potentially contribute to neurodegeneration and neuroinflammation.

ATF3 and JDP2 deficiency in cancer associated fibroblasts promotes tumor growth via SDF-1 transcription.

The activating transcription factor 3 (ATF3) and the c-Jun dimerization protein 2 (JDP2) are members of the basic leucine zipper (bZIP) family of transcription factors. These proteins share a high degree of homology and both can activate or repress transcription. Deficiency of either one of them in the non-cancer host cells was shown to reduce metastases. As ATF3 and JDP2 compensate each other's function, we studied the double deficiency of ATF3 and JDP2 in the stromal tumor microenvironment. Here, we show that mice with ATF3 and JDP2 double deficiency (designated thereafter dKO) developed larger tumors with high vascular perfusion and increased cell proliferation rate compared to wild type (WT) mice. We further identify that the underlying mechanism involves tumor associated fibroblasts which secrete high levels of stromal cell-derived factor 1 (SDF-1) in dKO fibroblasts. SDF-1 depletion in dKO fibroblasts dampened tumor growth and blood vessel perfusion. Furthermore, ATF3 and JDP2 were found to regulate SDF-1 transcription and secretion in fibroblasts, a phenomenon that is potentiated in the presence of cancer cells. Collectively, our results suggest that ATF3 and JDP2 regulate the expression of essential tumor promoting factors expressed by fibroblasts within the tumor microenvironment, and thus restrain tumor growth.

Application of Chemogenetics and Optogenetics to Dissect Brain-Immune Interactions.

For many years, the complexity and multifactorial nature of brain-immune interactions limited our ability to dissect their underlying mechanisms. An especially challenging question was how the brain controls immunity, since the repertoire of techniques to control the brain's activity was extremely limited. New tools, such as optogenetics and chemogenetics (e.g., DREADDs), developed over the last decade, opened new frontiers in neuroscience with major implications for neuroimmunology. These tools enable mapping the causal effects of activating/attenuating defined neurons in the brain, on the immune system. Here, we present a detailed experimental protocol for the analysis of brain-immune interactions, based on chemogenetic or optogenetic manipulation of defined neuronal populations in the brain, and the subsequent analysis of immune cells. Such detailed and systematic dissection of brain-immune interactions has the potential to revolutionize our understanding of how mental and neurological states affect health and disease.

Mass cytometry analysis of immune cells in the brain.

Immune cells comprise a diverse and dynamic cell population that is responsible for a broad range of immunological activities. They act in concert with other immune and nonimmune cells via cytokine-mediated communication and direct cell-cell interactions. Understanding the complex immune network requires a broad characterization of its individual cellular components. This is especially relevant for the brain compartment, which is an active immunological site, composed of resident and infiltrating immune cells that affect brain development, tissue homeostasis and neuronal activity. Mass cytometry, or CyTOF (cytometry by time-of-flight), uses metal-conjugated antibodies to enable a high-dimensional description of tens of markers at the single-cell level, thereby providing a bird's-eye view of the immune system. This technique has been successfully applied to the discovery of novel immune populations in humans and rodents. Here, we provide a step-by-step description of a mass cytometry approach for the analysis of the mouse brain compartment. The different stages of the procedure include brain perfusion, extraction of the brain tissue and its dissociation into a single-cell suspension, followed by cell staining with metal-tagged antibodies, sample reading using a mass cytometer, and data analysis using SPADE and viSNE. This procedure takes <5 h (excluding data analysis) and can be applied to study modifications in the brain's immune populations under normal and pathological conditions.

Modulation of anti-tumor immunity by the brain's reward system.

Regulating immunity is a leading target for cancer therapy. Here, we show that the anti-tumor immune response can be modulated by the brain's reward system, a key circuitry in emotional processes. Activation of the reward system in tumor-bearing mice (Lewis lung carcinoma (LLC) and B16 melanoma) using chemogenetics (DREADDs), resulted in reduced tumor weight. This effect was mediated via the sympathetic nervous system (SNS), manifested by an attenuated noradrenergic input to a major immunological site, the bone marrow. Myeloid derived suppressor cells (MDSCs), which develop in the bone marrow, became less immunosuppressive following reward system activation. By depleting or adoptively transferring the MDSCs, we demonstrated that these cells are both necessary and sufficient to mediate reward system effects on tumor growth. Given the central role of the reward system in positive emotions, these findings introduce a physiological mechanism whereby the patient's psychological state can impact anti-tumor immunity and cancer progression.

High-dimensional, single-cell characterization of the brain's immune compartment.

The brain and its borders create a highly dynamic microenvironment populated with immune cells. Yet characterization of immune cells within the naive brain compartment remains limited. In this study, we used CyTOF mass cytometry to characterize the immune populations of the naive mouse brain using 44 cell surface markers. By comparing immune cell composition and cell profiles between the brain compartment and blood, we were able to characterize previously undescribed cell subsets of CD8 T cells, B cells, NK cells and dendritic cells in the naive brain. Using flow cytometry, we show differential distributions of immune populations between meninges, choroid plexus and parenchyma. We demonstrate the phenotypic ranges of resident myeloid cells and identify CD44 as a marker for infiltrating immune populations. This study provides an approach for a system-wide view of immune populations in the brain and is expected to serve as a resource for understanding brain immunity.

Activation of the reward system boosts innate and adaptive immunity.

Positive expectations contribute to the clinical benefits of the placebo effect. Such positive expectations are mediated by the brain's reward system; however, it remains unknown whether and how reward system activation affects the body's physiology and, specifically, immunity. Here we show that activation of the ventral tegmental area (VTA), a key component of the reward system, strengthens immunological host defense. We used 'designer receptors exclusively activated by designer drugs' (DREADDs) to directly activate dopaminergic neurons in the mouse VTA and characterized the subsequent immune response after exposure to bacteria (Escherichia coli), using time-of-flight mass cytometry (CyTOF) and functional assays. We found an increase in innate and adaptive immune responses that were manifested by enhanced antibacterial activity of monocytes and macrophages, reduced in vivo bacterial load and a heightened T cell response in the mouse model of delayed-type hypersensitivity. By chemically ablating the sympathetic nervous system (SNS), we showed that the reward system's effects on immunity are, at least partly, mediated by the SNS. Thus, our findings establish a causal relationship between the activity of the VTA and the immune response to bacterial infection.

Sleep disruption impairs haematopoietic stem cell transplantation in mice.

Many of the factors affecting the success of haematopoietic cell transplantation are still unknown. Here we show in mice that donor sleep deprivation reduces the ability of its haematopoietic stem cells (HSCs) to engraft and reconstitute the blood and bone marrow of an irradiated recipient by more than 50%. We demonstrate that sleep deprivation downregulates the expression of microRNA (miR)-19b, a negative regulator of the suppressor of cytokine signalling (SOCS) genes, which inhibit HSC migration and homing. Accordingly, HSCs from sleep-deprived mice have higher levels of SOCS genes expression, lower migration capacity in vitro and reduced homing to the bone marrow in vivo. Recovery of sleep after sleep deprivation restored the reconstitution potential of the HSCs. Taken together, this study provides insights into cellular and molecular mechanisms underlying the effects of sleep deprivation on HSCs, emphasizing the potentially critical role of donor sleep in the success of bone marrow transplantation.