Gut-innervating nociceptors regulate the intestinal microbiota to promote tissue protection.

Nociceptive pain is a hallmark of many chronic inflammatory conditions including inflammatory bowel diseases (IBDs); however, whether pain-sensing neurons influence intestinal inflammation remains poorly defined. Employing chemogenetic silencing, adenoviral-mediated colon-specific silencing, and pharmacological ablation of TRPV1+ nociceptors, we observed more severe inflammation and defective tissue-protective reparative processes in a murine model of intestinal damage and inflammation. Disrupted nociception led to significant alterations in the intestinal microbiota and a transmissible dysbiosis, while mono-colonization of germ-free mice with Gram+Clostridium spp. promoted intestinal tissue protection through a nociceptor-dependent pathway. Mechanistically, disruption of nociception resulted in decreased levels of substance P, and therapeutic delivery of substance P promoted tissue-protective effects exerted by TRPV1+ nociceptors in a microbiota-dependent manner. Finally, dysregulated nociceptor gene expression was observed in intestinal biopsies from IBD patients. Collectively, these findings indicate an evolutionarily conserved functional link between nociception, the intestinal microbiota, and the restoration of intestinal homeostasis.

CTLA-4-expressing ILC3s restrain interleukin-23-mediated inflammation.

Interleukin (IL-)23 is a major mediator and therapeutic target in chronic inflammatory diseases that also elicits tissue protection in the intestine at homeostasis or following acute infection1-4. However, the mechanisms that shape these beneficial versus pathological outcomes remain poorly understood. To address this gap in knowledge, we performed single-cell RNA sequencing on all IL-23 receptor-expressing cells in the intestine and their acute response to IL-23, revealing a dominance of T cells and group 3 innate lymphoid cells (ILC3s). Unexpectedly, we identified potent upregulation of the immunoregulatory checkpoint molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on ILC3s. This pathway was activated by gut microbes and IL-23 in a FOXO1- and STAT3-dependent manner. Mice lacking CTLA-4 on ILC3s exhibited reduced regulatory T cells, elevated inflammatory T cells and more-severe intestinal inflammation. IL-23 induction of CTLA-4+ ILC3s was necessary and sufficient to reduce co-stimulatory molecules and increase PD-L1 bioavailability on intestinal myeloid cells. Finally, human ILC3s upregulated CTLA-4 in response to IL-23 or gut inflammation and correlated with immunoregulation in inflammatory bowel disease. These results reveal ILC3-intrinsic CTLA-4 as an essential checkpoint that restrains the pathological outcomes of IL-23, suggesting that disruption of these lymphocytes, which occurs in inflammatory bowel disease5-7, contributes to chronic inflammation.

ZBTB46 defines and regulates ILC3s that protect the intestine.

RORγt is a lineage-specifying transcription factor that is expressed by immune cells that are enriched in the gastrointestinal tract and promote immunity, inflammation and tissue homeostasis1-15. However, fundamental questions remain with regard to the cellular heterogeneity among these cell types, the mechanisms that control protective versus inflammatory properties and their functional redundancy. Here we define all RORγt+ immune cells in the intestine at single-cell resolution and identify a subset of group 3 innate lymphoid cells (ILC3s) that expresses ZBTB46, a transcription factor specifying conventional dendritic cells16-20. ZBTB46 is robustly expressed by CCR6+ lymphoid-tissue-inducer-like ILC3s that are developmentally and phenotypically distinct from conventional dendritic cells, and its expression is imprinted by RORγt, fine-tuned by microbiota-derived signals and increased by pro-inflammatory cytokines. ZBTB46 restrains the inflammatory properties of ILC3s, including the OX40L-dependent expansion of T helper 17 cells and the exacerbated intestinal inflammation that occurs after enteric infection. Finally, ZBTB46+ ILC3s are a major source of IL-22, and selective depletion of this population renders mice susceptible to enteric infection and associated intestinal inflammation. These results show that ZBTB46 is a transcription factor that is shared between conventional dendritic cells and ILC3s, and identify a cell-intrinsic function for ZBTB46 in restraining the pro-inflammatory properties of ILC3s and a non-redundant role for ZBTB46+ ILC3s in orchestrating intestinal health.

ILC3s select microbiota-specific regulatory T cells to establish tolerance in the gut.

Microbial colonization of the mammalian intestine elicits inflammatory or tolerogenic T cell responses, but the mechanisms controlling these distinct outcomes remain poorly understood, and accumulating evidence indicates that aberrant immunity to intestinal microbiota is causally associated with infectious, inflammatory and malignant diseases1-8. Here we define a critical pathway controlling the fate of inflammatory versus tolerogenic T cells that respond to the microbiota and express the transcription factor RORγt. We profiled all RORγt+ immune cells at single-cell resolution from the intestine-draining lymph nodes of mice and reveal a dominant presence of T regulatory (Treg) cells and lymphoid tissue inducer-like group 3 innate lymphoid cells (ILC3s), which co-localize at interfollicular regions. These ILC3s are distinct from extrathymic AIRE-expressing cells, abundantly express major histocompatibility complex class II, and are necessary and sufficient to promote microbiota-specific RORγt+ Treg cells and prevent their expansion as inflammatory T helper 17 cells. This occurs through ILC3-mediated antigen presentation, αV integrin and competition for interleukin-2. Finally, single-cell analyses suggest that interactions between ILC3s and RORγt+ Treg cells are impaired in inflammatory bowel disease. Our results define a paradigm whereby ILC3s select for antigen-specific RORγt+ Treg cells, and against T helper 17 cells, to establish immune tolerance to the microbiota and intestinal health.

Coordination of Mucosal Immunity by Innate Lymphoid Cells.

Mucosal barrier surfaces of the mammalian body are frequent sites of pathogen colonization or entry and are also densely colonized with trillions of normally beneficial microbes, termed the microbiota. Therefore, it is paramount that the host immune system recognizes these microbes and is capable of differentiating between them. To this end, a multitude of mechanisms have evolved to carefully balance the need for immune activation in the face of infections while maintaining an appropriate level of tolerance to protect both the host and the beneficial microbes from hyperactivation. These mechanisms include the deployment of an emerging class of tissue-resident innate immune cells, innate lymphoid cells (ILCs), that are enriched at mucosal barriers such as the lungs and intestines, and are critical mediators of tissue homeostasis, tolerance, repair, and innate immunity. Recent findings have provided insight into the regulation of these cells and their interactions, not only with microbes, both commensal and foreign, but also with other systems of the body to prevent disease and promote tissue health. Here, we discuss recent findings in the regulation and function of ILCs, including a focus on their interactions with bodily systems, such as the nervous system, and how these interactions affect their functionality in states of health, infection, and disease.

Immunology of the Uterine and Vaginal Mucosae.

Along with the maintenance of symbiotic mutualism with commensal microbes and protection against invasive infections common to all mucosal barrier tissues, female reproductive tissues have additional, unique tasks that include dynamic cyclic cellular turnover in menstruation and immunological tolerance to genetically foreign fetal antigens in pregnancy. Here we review current knowledge on distinct features of the immune cells in female reproductive tissue with regard to antimicrobial host defense and adaptations to accommodate the fetus during pregnancy. Outstanding areas for future research to obtain new functional insights on this enigmatic mucosal barrier are also highlighted.

A Germinal Center Checkpoint of AIRE in B Cells Limits Antibody Diversification.

In response to antigens, B cells undergo affinity maturation and class switching mediated by activation-induced cytidine deaminase (AID) in germinal centers (GCs) of secondary lymphoid organs, but uncontrolled AID activity can precipitate autoimmunity and cancer. The regulation of GC antibody diversification is of fundamental importance but not well understood. We found that autoimmune regulator (AIRE), the molecule essential for T cell tolerance, is expressed in GC B cells in a CD40-dependent manner, interacts with AID and negatively regulates antibody affinity maturation and class switching by inhibiting AID function. AIRE deficiency in B cells caused altered antibody repertoire, increased somatic hypermutations, elevated autoantibodies to T helper 17 effector cytokines and defective control of skin Candida albicans. These results define a GC B cell checkpoint of humoral immunity and illuminate new approaches of generating high-affinity neutralizing antibodies for immunotherapy.

Kaposi Sarcoma: A Rare Presentation of Elephantiasis Nostras Verrucosa.

Although a low-grade vascular tumor, Kaposi sarcoma (KS) can have mucosal, and visceral involvement. Additionally, disfiguring disseminated lesions can be seen in patients with human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS). KS may cause lymphatic obstruction leading to chronic lymphedema that further contributes to progressive cutaneous hypertrophy and severe disfigurement in the form of non-filarial elephantiasis nostras verrucosa (ENV). This report highlights a case of a 33-year-old male with AIDS who presented in acute respiratory distress with bilateral lower extremity nodular lesions. We confirmed a diagnosis of KS with overlying ENV via a multi-disciplinary approach. Collaboratively, we optimized our patient and observed adequate treatment response and overall improvement in clinical status. Our report emphasizes the importance of a multi-disciplinary approach in recognizing a rare presentation of ENV. Recognition of the disease and understanding the extent of the disease are crucial in preventing irreversible disease progression and allowing for maximum response.

The Role of the Automated Implantable Cardioverter Defibrillator (AICD) for Secondary Prevention in the COVID-19 Era.

Life-threatening arrhythmias have been variably reported among patients hospitalized for COVID-19 infection. Sudden cardiac arrest (SCA) in COVID-19 patients is an alarming concern for clinicians. Multiple factors play an important role in the development of SCA in patients with severe systemic illness. We describe a case of COVID-19 in a New York City hospital in Spring 2020 that rapidly developed SCA and, before discharge, received a single lead transvenous implantable cardioverter defibrillator for secondary prevention. This case highlights the use of an automated implantable cardioverter-defibrillator as a secondary preventive measure irrespective of left ventricular function as a means of preventing recurrence of SCA as a sequela of COVID-19.

Molecular mechanisms regulating lysophosphatidylcholine acyltransferase 1 (LPCAT1) in human pregnancy.

INTRODUCTION: Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) is necessary for surfactant production in fetal lungs. Mechanisms responsible for its regulation during gestation remain to be elucidated. Our goal is to evaluate molecular mechanisms regulating LPCAT1 expression during gestation and after glucocorticoid administration. METHODS: Placentas throughout gestation were assayed for LPCAT1 protein levels. A placental cell line, HTR-8/SVneo (HTR), was used as a model to test the effects of placental oxygen tension found during pregnancy as well as the effects of dexamethasone used therapeutically in the clinic. RESULTS: LPCAT1 protein levels are maximal in late third trimester placental samples and are expressed strongly on the basal plate. LPCAT1 was maximally upregulated at 4% O2 (P < 0.01), corresponding to oxygen tension found in placenta at term. Mitochondrial nuclear retrograde regulator 1 (MNRR1), a bi-organellar (mitochondria and nucleus) regulator, transcriptionally activates LPCAT1. Antenatal corticosteroids (ACS) upregulate LPCAT1, at least in part, by an MNRR1-dependent pathway. HTR cells treated with 25 nM dexamethasone for 24 h exhibited a 2-fold increase in LPCAT1 levels compared to controls. In MNRR1 knockout cells, the response to ACS is significantly blunted. DISCUSSION: LPCAT1 appears to be induced by MNRR1. Hypoxia and corticosteroids increase LPCAT1 expression through an MNRR1 dependent pathway. LPCAT1 protein levels can be measured in maternal plasma and rise throughout gestation and in response to ACS.

Stress Forces First Lineage Differentiation of Mouse Embryonic Stem Cells; Validation of a High-Throughput Screen for Toxicant Stress.

Mouse Embryonic Stem Cells (mESCs) are unique in their self-renewal and pluripotency. Hypothetically, mESCs model gestational stress effects or stresses of in vitro fertilization/assisted reproductive technologies or drug/environmental exposures that endanger embryos. Testing mESCs stress responses should diminish and expedite in vivo embryo screening. Transgenic mESCs for green fluorescent protein (GFP) reporters of differentiation use the promoter for platelet-derived growth factor receptor (Pdgfr)a driving GFP expression to monitor hyperosmotic stress-forced mESC proliferation decrease (stunting), and differentiation increase that further stunts mESC population growth. In differentiating mESCs Pdgfra marks the first-lineage extraembryonic primitive endoderm (ExEndo). Hyperosmotic stress forces mESC differentiation gain (Pdgfra-GFP) in monolayer or three-dimensional embryoid bodies. Despite culture with potency-maintaining leukemia inhibitory factor (LIF), stress forces ExEndo as assayed using microplate readers and validated by coexpression of Pdgfra-GFP, Disabled 2 (Dab2), and laminin by immunofluorescence and GFP protein and Dab2 by immunoblot. In agreement with previous reports, Rex1 and Oct4 loss was inversely proportional to increased Pdgfra-GFP mESC after treatment with high hyperosmotic sorbitol despite LIF. The increase in subpopulations of Pdgfra-GFP+ cells>background at ∼23% was similar to the previously reported ∼25% increase in Rex1-red fluorescent protein (RFP)-negative subpopulation at matched high sorbitol doses. By microplate reader, there is a ∼7-11-fold increase in GFP at a high nonmorbid and a morbid dose despite LIF, compared with LIF alone. By flow cytometry (FACS), the subpopulation of Pdgfra-GFP+ cells>background increases ∼8-16-fold at these doses. Taken together, the microplate, FACS, immunoblot, and immunofluorescence data suggest that retinoic acid or hyperosmotic stress forces dose-dependent differentiation whether LIF is present or not and this is negatively correlated with and possibly compensates for stress-forced diminished ESC population expansion and potency loss.

Interleukin-33-induced expression of PIBF1 by decidual B cells protects against preterm labor.

Preterm birth (PTB) is a leading cause of neonatal death worldwide. Intrauterine and systemic infection and inflammation cause 30-40% of spontaneous preterm labor (PTL), which precedes PTB. Although antibody production is a major immune defense mechanism against infection, and B cell dysfunction has been implicated in pregnancy complications associated with PTL, the functions of B cells in pregnancy are not well known. We found that choriodecidua of women undergoing spontaneous PTL harbored functionally altered B cell populations. B cell-deficient mice were markedly more susceptible than wild-type (WT) mice to PTL after inflammation, but B cells conferred interleukin (IL)-10-independent protection against PTL. B cell deficiency in mice resulted in a lower uterine level of active progesterone-induced blocking factor 1 (PIBF1), and therapeutic administration of PIBF1 mitigated PTL and uterine inflammation in B cell-deficient mice. B cells are a significant producer of PIBF1 in human choriodecidua and mouse uterus in late gestation. PIBF1 expression by B cells is induced by the mucosal alarmin IL-33 (ref. 9). Human PTL was associated with diminished expression of the α-chain of IL-33 receptor on choriodecidual B cells and a lower level of active PIBF1 in late gestation choriodecidua. These results define a vital regulatory cascade involving IL-33, decidual B cells and PIBF1 in safeguarding term pregnancy and suggest new therapeutic approaches based on IL-33 and PIBF1 to prevent human PTL.