A defined commensal consortium elicits CD8 T cells and anti-cancer immunity.

There is a growing appreciation for the importance of the gut microbiota as a therapeutic target in various diseases. However, there are only a handful of known commensal strains that can potentially be used to manipulate host physiological functions. Here we isolate a consortium of 11 bacterial strains from healthy human donor faeces that is capable of robustly inducing interferon-γ-producing CD8 T cells in the intestine. These 11 strains act together to mediate the induction without causing inflammation in a manner that is dependent on CD103+ dendritic cells and major histocompatibility (MHC) class Ia molecules. Colonization of mice with the 11-strain mixture enhances both host resistance against Listeria monocytogenes infection and the therapeutic efficacy of immune checkpoint inhibitors in syngeneic tumour models. The 11 strains primarily represent rare, low-abundance components of the human microbiome, and thus have great potential as broadly effective biotherapeutics.

Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation.

In mammals, the transcription factor SRY, encoded by the Y chromosome, is normally responsible for triggering the indifferent gonads to develop as testes rather than ovaries. However, testis differentiation can occur in its absence. Here we demonstrate in the mouse that a single factor, the forkhead transcriptional regulator FOXL2, is required to prevent transdifferentiation of an adult ovary to a testis. Inducible deletion of Foxl2 in adult ovarian follicles leads to immediate upregulation of testis-specific genes including the critical SRY target gene Sox9. Concordantly, reprogramming of granulosa and theca cell lineages into Sertoli-like and Leydig-like cell lineages occurs with testosterone levels comparable to those of normal XY male littermates. Our results show that maintenance of the ovarian phenotype is an active process throughout life. They might also have important medical implications for the understanding and treatment of some disorders of sexual development in children and premature menopause in women.

The effects of sildenafil citrate on intrauterine growth restriction: a systematic review and meta-analysis.

BACKGROUND: An increase in vascular resistance of uterine vessels is associated with intrauterine growth restriction (IUGR). Sildenafil citrate, a phosphodiesterase-5 inhibitor that stabilizes cyclic guanosine monophosphate (cGMP) and increases nitric oxide levels, improves placental perfusion by dilation of spiral arteries and is beneficial in managing IUGR. This study aims to determine the effectiveness of sildenafil citrate in improving perinatal outcomes in IUGR pregnancies. METHODS: Meta-analysis was performed on data extracted from all studies specific to sildenafil citrate in IUGR management, searching relevant articles on PubMed, Medline, Google Scholar, Embase, and Cochrane databases. Publications identified by the manual search, based on references in reviews, were also included. Dichotomous results were presented as risk ratio (95% confidence interval), while continuous results were expressed as mean difference (MD); samples represented by the random effects model. RESULTS: Nine trials were included where the sildenafil citrate effect was compared with a placebo or no intervention. A significant increase in birth weight [SMD (95% CI), 0.69 (0.31, 1.07)] was seen in IUGR pregnancies managed with sildenafil. However, gestational age (SMD (95% CI), 0.44 (-0.05, 0.94], fetal death rate [RR (95% CI), 0.56 (0.17, 1.79)] in IUGR pregnancies was not changed by sildenafil. Neonatal death [RR (95% CI), 0.93 (0.47, 1.86)] and neonatal intensive care unit (NICU) admissions [RR (95% CI), 0.76 (0.50, 1.17)] were not significantly different between sildenafil and control groups. CONCLUSION: Sildenafil citrate increases birth weight and prolonged pregnancies but did not affect stillbirth rate, neonatal death, and NICU admission. TRIAL REGISTRATION: The study was registered in PROSPERO on September 18, 2021 (CRD42021271992).

Dendritic cells ameliorate autoimmunity in the CNS by controlling the homeostasis of PD-1 receptor(+) regulatory T cells.

Mature dendritic cells (DCs) are established as unrivaled antigen-presenting cells (APCs) in the initiation of immune responses, whereas steady-state DCs induce peripheral T cell tolerance. Using various genetic approaches, we depleted CD11c(+) DCs in mice and induced autoimmune CNS inflammation. Unexpectedly, mice lacking DCs developed aggravated disease compared to control mice. Furthermore, when we engineered DCs to present a CNS-associated autoantigen in an induced manner, we found robust tolerance that prevented disease, which coincided with an upregulation of the PD-1 receptor on antigen-specific T cells. Additionally, we showed that PD-1 was necessary for DC-mediated induction of regulatory T cells. Our results show that a reduction of DCs interferes with tolerance, resulting in a stronger inflammatory response, and that other APC populations could compensate for the loss of immunogenic APC function in DC-depleted mice.

Ultraviolet B-Induced Maturation of CD11b-Type Langerin- Dendritic Cells Controls the Expansion of Foxp3+ Regulatory T Cells in the Skin.

Skin dendritic cells (DCs) are divided into several subsets with distinctive functions. This study shows a previously unappreciated role of dermal CD11b-type Langerin- DCs in maintaining immunological self-tolerance after UVB exposure. After UVB exposure, dermal CD11b-type Langerin- DCs upregulated surface CD86 expression, induced proliferation of Foxp3+ regulatory T (Treg) cells without exogenous Ags, and upregulated a set of genes associated with immunological tolerance. This Treg-expansion activity was significantly hampered by CD80/CD86 blockade in vivo. These results indicate that CD11b-type Langerin- DCs from the UVB-exposed skin are specialized to expand Treg cells in the skin, which suppress autoimmunity.

Sebaceous lipids are essential for water repulsion, protection against UVB-induced apoptosis and ocular integrity in mice.

Sebocytes, which are characterized by lipid accumulation that leads to cell disruption, can be found in hair follicle-associated sebaceous glands (SGs) or in free SGs such as the Meibomian glands in the eyelids. Because genetic tools that allow targeting of sebocytes while maintaining intact epidermal lipids are lacking, the relevance of sebaceous lipids in health and disease remains poorly understood. Using Scd3, which is expressed exclusively in mature sebocytes, we established a mouse line with sebocyte-specific expression of Cre recombinase. Both RT-PCR analysis and crossing into Rosa26-lacZ reporter mice and Kras(G12D) mice confirmed Cre activity specifically in SGs, with no activity in other skin compartments. Importantly, loss of SCD3 function did not cause detectable phenotypical alterations, endorsing the usefulness of Scd3-Cre mice for further functional studies. Scd3-Cre-induced, diphtheria chain A toxin-mediated depletion of sebaceous lipids resulted in impaired water repulsion and thermoregulation, increased rates of UVB-induced epidermal apoptosis and caused a severe pathology of the ocular surface resembling Meibomian gland dysfunction. This novel mouse line will be useful for further investigating the roles of sebaceous lipids in skin and eye integrity.

Promotion of periostin expression contributes to the migration of Schwann cells.

Neuregulin ligands and their ErbB receptors are important for the development of Schwann cells, the glial cells of the peripheral nervous system (PNS). ErbB3 deficiency is characterized by a complete loss of Schwann cells along axons of the peripheral nerves, impaired fasciculation and neuronal cell death. We performed comparative gene expression analysis of dorsal root ganglia (DRG) explant cultures from ErbB3-deficient and wild-type mice in order to identify genes that are involved in Schwann cell development and migration. The extracellular matrix (ECM) gene periostin was found to exhibit the most prominent down regulation in ErbB3-deficient DRG. Expression analysis revealed that the periostin-expressing cell population in the PNS corresponds to Schwann cell precursors and Schwann cells, and is particularly high in migratory Schwann cells. Furthermore, stimulation of Schwann cells with neuregulin-1 (NRG1) or transforming growth factor ß (TGFß-1) resulted in an upregulation of periostin expression. Interestingly, DRG explant cultures of periostin-deficient mice revealed a significant reduction of the number of migrating Schwann cells. These data demonstrate that the expression of periostin is stimulated by ErbB ligand NRG1 and influences the migration of Schwann cell precursors.

Sertoli cells control peritubular myoid cell fate and support adult Leydig cell development in the prepubertal testis.

Sertoli cells (SCs) regulate testicular fate in the differentiating gonad and are the main regulators of spermatogenesis in the adult testis; however, their role during the intervening period of testis development, in particular during adult Leydig cell (ALC) differentiation and function, remains largely unknown. To examine SC function during fetal and prepubertal development we generated two transgenic mouse models that permit controlled, cell-specific ablation of SCs in pre- and postnatal life. Results show that SCs are required: (1) to maintain the differentiated phenotype of peritubular myoid cells (PTMCs) in prepubertal life; (2) to maintain the ALC progenitor population in the postnatal testis; and (3) for development of normal ALC numbers. Furthermore, our data show that fetal LCs function independently from SC, germ cell or PTMC support in the prepubertal testis. Together, these findings reveal that SCs remain essential regulators of testis development long after the period of sex determination. These findings have significant implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

Lack of conventional dendritic cells is compatible with normal development and T cell homeostasis, but causes myeloid proliferative syndrome.

Dendritic cells are critically involved in the promotion and regulation of T cell responses. Here, we report a mouse strain that lacks conventional CD11c(hi) dendritic cells (cDCs) because of constitutive cell-type specific expression of a suicide gene. As expected, cDC-less mice failed to mount effective T cell responses resulting in impaired viral clearance. In contrast, neither thymic negative selection nor T regulatory cell generation or T cell homeostasis were markedly affected. Unexpectedly, cDC-less mice developed a progressive myeloproliferative disorder characterized by prominent extramedullary hematopoiesis and increased serum amounts of the cytokine Flt3 ligand. Our data identify a critical role of cDCs in the control of steady-state hematopoiesis, revealing a feedback loop that links peripheral cDCs to myelogenesis through soluble growth factors, such as Flt3 ligand.

Loss of caspase-8 in hepatocytes accelerates the onset of liver regeneration in mice through premature nuclear factor kappa B activation.

UNLABELLED: The cytokine tumor necrosis factor alpha (TNF-α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF-κB) activation, apoptosis signaling by way of caspase-8 (Casp8), and activation of cJun N-terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8(Δhepa) ) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8(Δhepa) mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF-dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor-interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF-κB and JNK/cJun related signals. In order to define the role of NF-κB in this setting we blocked NF-κB activation in Casp8(Δhepa) mice by concomitant inactivation of the NF-κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8(Δhepa) mice but resulted in incomplete termination of the regeneration process and hepatomegaly. CONCLUSION: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF-κB, and JNK activation. While loss of Casp8 triggers NF-κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly.

Crucial role of dendritic cells in the generation of anti-tumor T-cell responses and immunogenic tumor microenvironment to suppress tumor development.

Dendritic cells (DCs) are known as unique professional antigen (Ag)-presenting cells (APCs) to prime naïve T cells for the initiation of adaptive immunity. While DCs are believed to play a pivotal role in generating anti-tumor T-cell responses, the importance of DCs in the protection from the progression of tumors remains elusive. Here, we show how the constitutive deficiency of CD11chi DCs influences the progression of tumors with the use of binary transgenic mice with constitutive loss of CD11chi DCs. Constitutive loss of CD11chi DCs not only enhances the progression of tumors but also reduces the responses of Ag-specific T cells. Furthermore, the congenital deficiency of CD11chi DCs generates the immunosuppressive tumor microenvironment (TME) that correlates with the marked accumulation of myeloid-derived suppressor cells (MDSCs) and the prominent productions of immunosuppressive mediators. Thus, our findings suggest that CD11chi DCs are crucial for generating anti-tumor T-cell responses and immunogenic TME to suppress the development of tumors.

Neurofibromin modulates adult hippocampal neurogenesis and behavioral effects of antidepressants.

Neurogenesis persists in the rodent dentate gyrus (DG) throughout adulthood but declines with age and stress. Neural progenitor cells (NPCs) residing in the subgranular zone of the DG are regulated by an array of growth factors and respond to the microenvironment, adjusting their proliferation level to determine the rate of neurogenesis. Here we report that genetic deletion of neurofibromin (Nf1), a tumor suppressor with RAS-GAP activity, in adult NPCs enhanced DG proliferation and increased generation of new neurons in mice. Nf1 loss-associated neurogenesis had the functional effect of enhancing behavioral responses to subchronic antidepressants and, over time, led to spontaneous antidepressive-like behaviors. Thus, our findings establish an important role for the Nf1-Ras pathway in regulating adult hippocampal neurogenesis, and demonstrate that activation of adult NPCs is sufficient to modulate depression- and anxiety-like behaviors.

Functional characterization of bitter-taste receptors expressed in mammalian testis.

Mammalian spermatogenesis and sperm maturation are susceptible to the effects of internal and external factors. However, how male germ cells interact with and respond to these elements including those potentially toxic substances is poorly understood. Here, we show that many bitter-taste receptors (T2rs), which are believed to function as gatekeepers in the oral cavity to detect and innately prevent the ingestion of poisonous bitter-tasting compounds, are expressed in mouse seminiferous tubules. Our in situ hybridization results indicate that Tas2r transcripts are expressed postmeiotically. Functional analysis showed that mouse spermatids and spermatozoa responded to both naturally occurring and synthetic bitter-tasting compounds by increasing intracellular free calcium concentrations, and individual male germ cells exhibited different ligand-activation profiles, indicating that each cell may express a unique subset of T2r receptors. These calcium responses could be suppressed by a specific bitter-tastant blocker or abolished by the knockout of the gene for the G protein subunit α-gustducin. Taken together, our data strongly suggest that male germ cells, like taste bud cells in the oral cavity and solitary chemosensory cells in the airway, utilize T2r receptors to sense chemicals in the milieu that may affect sperm behavior and fertilization.

A stomatin-domain protein essential for touch sensation in the mouse.

Touch and mechanical pain are first detected at our largest sensory surface, the skin. The cell bodies of sensory neurons that detect such stimuli are located in the dorsal root ganglia, and subtypes of these neurons are specialized to detect specific modalities of mechanical stimuli. Molecules have been identified that are necessary for mechanosensation in invertebrates but so far not in mammals. In Caenorhabditis elegans, mec-2 is one of several genes identified in a screen for touch insensitivity and encodes an integral membrane protein with a stomatin homology domain. Here we show that about 35% of skin mechanoreceptors do not respond to mechanical stimuli in mice with a mutation in stomatin-like protein 3 (SLP3, also called Stoml3), a mammalian mec-2 homologue that is expressed in sensory neurons. In addition, mechanosensitive ion channels found in many sensory neurons do not function without SLP3. Tactile-driven behaviours are also impaired in SLP3 mutant mice, including touch-evoked pain caused by neuropathic injury. SLP3 is therefore indispensable for the function of a subset of cutaneous mechanoreceptors, and our data support the idea that this protein is an essential subunit of a mammalian mechanotransducer.

Epibranchial ganglia orchestrate the development of the cranial neurogenic crest.

The wiring of the nervous system arises from extensive directional migration of neuronal cell bodies and growth of processes that, somehow, end up forming functional circuits. Thus far, this feat of biological engineering appears to rely on sequences of pathfinding decisions upon local cues, each with little relationship to the anatomical and physiological outcome. Here, we uncover a straightforward cellular mechanism for circuit building whereby a neuronal type directs the development of its future partners. We show that visceral afferents of the head (that innervate taste buds) provide a scaffold for the establishment of visceral efferents (that innervate salivatory glands and blood vessels). In embryological terms, sensory neurons derived from an epibranchial placode--that we show to develop largely independently from the neural crest--guide the directional outgrowth of hindbrain visceral motoneurons and control the formation of neural crest-derived parasympathetic ganglia.

Genetically induced adult oligodendrocyte cell death is associated with poor myelin clearance, reduced remyelination, and axonal damage.

Loss of oligodendrocytes is a feature of many demyelinating diseases including multiple sclerosis. Here, we have established and characterized a novel model of genetically induced adult oligodendrocyte death. Specific primary loss of adult oligodendrocytes leads to a well defined and highly reproducible course of disease development that can be followed longitudinally by magnetic resonance imaging. Histological and ultrastructural analyses revealed progressive myelin vacuolation, in parallel to disease development that includes motor deficits, tremor, and ataxia. Myelin damage and clearance were associated with induction of oligodendrocyte precursor cell proliferation, albeit with some regional differences. Remyelination was present in the mildly affected corpus callosum. Consequences of acutely induced cell death of adult oligodendrocytes included secondary axonal damage. Microglia were activated in affected areas but without significant influx of B-cells, T-helper cells, or T-cytotoxic cells. Analysis of the model on a RAG-1 (recombination activating gene-1)-deficient background, lacking functional lymphocytes, did not change the observed disease and pathology compared with immune-competent mice. We conclude that this model provides the opportunity to study the consequences of adult oligodendrocyte death in the absence of primary axonal injury and reactive cells of the adaptive immune system. Our results indicate that if the blood-brain barrier is not disrupted, myelin debris is not removed efficiently, remyelination is impaired, and axonal integrity is compromised, likely as the result of myelin detachment. This model will allow the evaluation of strategies aimed at improving remyelination to foster axon protection.

MRI signature in a novel mouse model of genetically induced adult oligodendrocyte cell death.

Two general pathological processes contribute to multiple sclerosis (MS): acute inflammation and degeneration. While magnetic resonance imaging (MRI) is highly sensitive in detecting abnormalities related to acute inflammation both clinically and in animal models of experimental autoimmune encephalomyelitis (EAE), the correlation of these readouts with acute and future disabilities has been found rather weak. This illustrates the need for imaging techniques addressing neurodegenerative processes associated with MS. In the present work we evaluated the sensitivity of different MRI techniques (T(2) mapping, macrophage tracking based on labeling cells in vivo by ultrasmall particles of iron oxide (USPIO), diffusion tensor imaging (DTI) and magnetization transfer imaging (MTI)) to detect histopathological changes in a novel animal model making use of intrinsic, temporally and spatially controlled triggering of oligodendrocyte cell death. This mouse model allows studying the MRI signature associated to neurodegenerative processes of MS in the absence of adaptive inflammatory components that appear to be foremost in the EAE models. Our results revealed pronounced T(2) hyperintensities in brain stem and cerebellar structures, which we attribute to structural alteration of white matter by pronounced vacuolation. Brain areas were found devoid of significant macrophage infiltration in line with the absence of a peripheral inflammatory response. The significant decrease in diffusion anisotropy derived from DTI measures in these structures is mainly caused by a pronounced decrease in diffusivity parallel to the fiber indicative of axonal damage. Triggering of oligodendrocyte ablation did not translate into a significant increase in radial diffusivity. Only minor decreases in MT ratio have been observed, which is attributed to inefficient removal of myelin debris.

Loss of caspase-8 protects mice against inflammation-related hepatocarcinogenesis but induces non-apoptotic liver injury.

BACKGROUND & AIMS: Disruption of the nuclear factor-κB (NF-κB) essential modulator (NEMO) in hepatocytes of mice (NEMO(Δhepa) mice) results in spontaneous liver apoptosis and chronic liver disease involving inflammation, steatosis, fibrosis, and development of hepatocellular carcinoma. Activation of caspase-8 (Casp8) initiates death receptor-mediated apoptosis. We investigated the pathogenic role of this protease in NEMO(Δhepa) mice or after induction of acute liver injury. METHODS: We created mice with conditional deletion of Casp8 in hepatocytes (Casp8(Δhepa)) and Casp8(Δhepa)NEMO(Δhepa) double knockout mice. Acute liver injury was induced by Fas-activating antibodies, lipopolysaccharides, or concanavalin A. Spontaneous hepatocarcinogenesis was monitored by magnetic resonance imaging. RESULTS: Hepatocyte-specific deletion of Casp8 protected mice from induction of apoptosis and liver injury by Fas or lipopolysaccharides but increased necrotic damage and reduced survival times of mice given concanavalin A. Casp8(Δhepa)NEMO(Δhepa) mice were protected against steatosis and hepatocarcinogenesis but had a separate, spontaneous phenotype that included massive liver necrosis, cholestasis, and biliary lesions. The common mechanism by which inactivation of Casp8 induces liver necrosis in both injury models involves the formation of protein complexes that included the adaptor protein Fas-associated protein with death domain and the kinases receptor-interacting protein (RIP) 1 and RIP3-these have been shown to be required for programmed necrosis. We demonstrated that hepatic RIP1 was proteolytically cleaved by Casp8, whereas Casp8 inhibition resulted in accumulation of RIP complexes and subsequent liver necrosis. CONCLUSIONS: Inhibition of Casp8 protects mice from hepatocarcinogenesis following chronic liver injury mediated by apoptosis of hepatocytes but can activate RIP-mediated necrosis in an inflammatory environment.

Platelets play an essential role in separating the blood and lymphatic vasculatures during embryonic angiogenesis.

RATIONALE: Several mutations that impair the development of blood lineages in the mouse also impair the formation of the lymphatic vasculature and its separation from the blood vasculature. However, the basis for these defects has remained unknown because the mutations characterized affect more than one blood lineage. OBJECTIVE: We tested the hypothesis that megakaryocytes/platelets are required for the formation of the lymphatic vasculature and its separation from the blood vascular system. METHODS AND RESULTS: We characterized the vascular patterning defects of mice deficient for the homeodomain transcription factor Meis1 (myeloid ecotropic viral integration site 1), which completely lack megakaryocyte/platelets. Meis1 null embryos fail to separate the blood and lymphatic vasculature, showing blood-filled primary lymphatic sacs and superficial lymphatic vessels. To test the involvement of megakaryocytes/platelets in this phenotype, we generated megakaryocyte/platelet-specific deficient mice by targeted lineage ablation, without affecting other blood lineages. This model reproduces the lymphatic/blood vasculature separation defects observed in Meis1 mutants. A similar phenotype was induced by antibody-mediated ablation of circulating platelets in wild type mice. Strong association of platelets with vascular endothelium at regions of contact between lymphatic sacs and veins confirmed a direct role of platelets in the separation of the 2 vasculatures. CONCLUSIONS: In addition to their known protective function in the response accidental vascular injury, platelets are also required during embryonic lymphangiogenesis for the separation of the nascent lymphatic vasculature from blood vessels.

V1 spinal neurons regulate the speed of vertebrate locomotor outputs.

The neuronal networks that generate vertebrate movements such as walking and swimming are embedded in the spinal cord. These networks, which are referred to as central pattern generators (CPGs), are ideal systems for determining how ensembles of neurons generate simple behavioural outputs. In spite of efforts to address the organization of the locomotor CPG in walking animals, little is known about the identity and function of the spinal interneuron cell types that contribute to these locomotor networks. Here we use four complementary genetic approaches to directly address the function of mouse V1 neurons, a class of local circuit inhibitory interneurons that selectively express the transcription factor Engrailed1. Our results show that V1 neurons shape motor outputs during locomotion and are required for generating 'fast' motor bursting. These findings outline an important role for inhibition in regulating the frequency of the locomotor CPG rhythm, and also suggest that V1 neurons may have an evolutionarily conserved role in controlling the speed of vertebrate locomotor movements.