Nmes1 is a novel regulator of mucosal response influencing intestinal healing potential.

The initiation of tissue remodeling following damage is a critical step in preventing the development of immune-mediated diseases. Several factors contribute to mucosal healing, leading to innovative therapeutic approaches for managing intestinal disorders. However, uncovering alternative targets and gaining mechanistic insights are imperative to enhance therapy efficacy and broaden its applicability across different intestinal diseases. Here we demonstrate that Nmes1, encoding for Normal Mucosa of Esophagus-Specific gene 1, also known as Aa467197, is a novel regulator of mucosal healing. Nmes1 influences the macrophage response to the tissue remodeling cytokine IL-4 in vitro. In addition, using two murine models of intestinal damage, each characterized by a type 2-dominated environment with contrasting functions, the ablation of Nmes1 results in decreased intestinal regeneration during the recovery phase of colitis, while enhancing parasitic egg clearance and reducing fibrosis during the advanced stages of Schistosoma mansoni infection. These outcomes are associated with alterations in CX3CR1+ macrophages, cells known for their wound-healing potential in the inflamed colon, hence promising candidates for cell therapies. All in all, our data indicate Nmes1 as a novel contributor to mucosal healing, setting the basis for further investigation into its potential as a new target for the treatment of colon-associated inflammation.

Increased activity of IRE1 improves the clinical presentation of EAE.

Activation of the endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme-1α (IRE1α) contributes to neuronal development and is known to induce neuronal remodeling in vitro and in vivo. On the contrary, excessive IRE1 activity is often detrimental and may contribute to neurodegeneration. To determine the consequences of increased activation of IRE1α, we used a mouse model expressing a C148S variant of IRE1α with increased and sustained activation. Surprisingly, the mutation did not affect the differentiation of highly secretory antibody-producing cells but exhibited a beneficial effect in a mouse model of experimental autoimmune encephalomyelitis (EAE). Although mechanical allodynia was unaffected, significant improvement in motor function was found in IRE1C148S mice with EAE relative to wild type (WT) mice. Coincident with this improvement, there was reduced microgliosis in the spinal cord of IRE1C148S mice, with reduced expression of proinflammatory cytokine genes. This was accompanied by reduced axonal degeneration and enhanced 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) levels, suggesting improved myelin integrity. Interestingly, while the IRE1C148S mutation is expressed in all cells, the reduction in proinflammatory cytokines and in the microglial activation marker ionized calcium-binding adapter molecule (IBA1), along with preservation of phagocytic gene expression, all point to microglia as the cell type contributing to the clinical improvement in IRE1C148S animals. Our data suggest that sustained increase in IRE1α activity can be beneficial in vivo, and that this protection is cell type and context dependent. Considering the overwhelming but conflicting evidence for the role of ER stress in neurological diseases, a better understanding of the function of ER stress sensors in physiological contexts is clearly needed.

Serotonin reduction in post-acute sequelae of viral infection.

Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.

The lncRNA HOXA11os regulates mitochondrial function in myeloid cells to maintain intestinal homeostasis.

This study reveals a previously uncharacterized mechanism to restrict intestinal inflammation via a regulatory RNA transcribed from a noncoding genomic locus. We identified a novel transcript of the lncRNA HOXA11os specifically expressed in the distal colon that is reduced to undetectable levels in colitis. HOXA11os is localized to mitochondria under basal conditions and interacts with a core subunit of complex 1 of the electron transport chain (ETC) to maintain its activity. Deficiency of HOXA11os in colonic myeloid cells results in complex I deficiency, dysfunctional oxidative phosphorylation (OXPHOS), and the production of mitochondrial reactive oxygen species (mtROS). As a result, HOXA11os-deficient mice develop spontaneous intestinal inflammation and are hypersusceptible to colitis. Collectively, these studies identify a new regulatory axis whereby a lncRNA maintains intestinal homeostasis and restricts inflammation in the colon through the regulation of complex I activity.

Quantitative control of Ets1 dosage by a multi-enhancer hub promotes Th1 cell differentiation and protects from allergic inflammation.

Multi-enhancer hubs are spatial clusters of enhancers present across numerous developmental programs. Here, we studied the functional relevance of these three-dimensional structures in T cell biology. Mathematical modeling identified a highly connected multi-enhancer hub at the Ets1 locus, comprising a noncoding regulatory element that was a hotspot for sequence variation associated with allergic disease in humans. Deletion of this regulatory element in mice revealed that the multi-enhancer connectivity was dispensable for T cell development but required for CD4+ T helper 1 (Th1) differentiation. These mice were protected from Th1-mediated colitis but exhibited overt allergic responses. Mechanistically, the multi-enhancer hub controlled the dosage of Ets1 that was required for CTCF recruitment and assembly of Th1-specific genome topology. Our findings establish a paradigm wherein multi-enhancer hubs control cellular competence to respond to an inductive cue through quantitative control of gene dosage and provide insight into how sequence variation within noncoding elements at the Ets1 locus predisposes individuals to allergic responses.

The enteric nervous system relays psychological stress to intestinal inflammation.

Mental health profoundly impacts inflammatory responses in the body. This is particularly apparent in inflammatory bowel disease (IBD), in which psychological stress is associated with exacerbated disease flares. Here, we discover a critical role for the enteric nervous system (ENS) in mediating the aggravating effect of chronic stress on intestinal inflammation. We find that chronically elevated levels of glucocorticoids drive the generation of an inflammatory subset of enteric glia that promotes monocyte- and TNF-mediated inflammation via CSF1. Additionally, glucocorticoids cause transcriptional immaturity in enteric neurons, acetylcholine deficiency, and dysmotility via TGF-ß2. We verify the connection between the psychological state, intestinal inflammation, and dysmotility in three cohorts of IBD patients. Together, these findings offer a mechanistic explanation for the impact of the brain on peripheral inflammation, define the ENS as a relay between psychological stress and gut inflammation, and suggest that stress management could serve as a valuable component of IBD care.

A miRNA/CXCR4 signaling axis impairs monopoiesis and angiogenesis in diabetic critical limb ischemia.

Patients with peripheral artery disease (PAD) and diabetes have the highest risk of critical limb ischemia (CLI) and amputation, yet the underlying mechanisms remain incompletely understood. MicroRNA (miRNA) sequencing of plasma from diabetic patients with or without CLI was compared to diabetic mice with acute or subacute limb ischemia to identify conserved miRNAs. miRNA-KO mice on high-fat diet were generated to explore the impact on CLI. Comparison of dysregulated miRNAs from diabetic individuals with PAD and diabetic mice with limb ischemia revealed conserved miR-181 family members. High-fat-fed, diabetic Mir181a2b2-KO mice had impaired revascularization in limbs due to abrogation of circulating Ly6Chi monocytes, with reduced accumulation in ischemic skeletal muscles. M2-like KO macrophages under diabetic conditions failed to produce proangiogenic cytokines. Single-cell transcriptomics of the bone marrow niche revealed that the reduced monocytosis in diabetic KO mice was a result of impaired hematopoiesis, with increased CXCR4 signaling in bone marrow Lineage-Sca1+Kit+ (LSK) cells. Exogenous Ly6Chi monocytes from nondiabetic KO mice rescued the impaired revascularization in ischemic limbs of diabetic KO mice. Increased Cxcr4 expression was mediated by the miR-181 target, Plac8. Taken together, our results show that MiR-181a/b is a putative mediator of diabetic CLI and contributes to changes in hematopoiesis, monocytosis, and macrophage polarization.

Multiscale 3D genome organization underlies ILC2 ontogenesis and allergic airway inflammation.

Innate lymphoid cells (ILCs) are well-characterized immune cells that play key roles in host defense and tissue homeostasis. Yet, how the three-dimensional (3D) genome organization underlies the development and functions of ILCs is unknown. Herein, we carried out an integrative analysis of the 3D genome structure, chromatin accessibility and gene expression in mature ILCs. Our results revealed that the local 3D configuration of the genome is rewired specifically at loci associated with ILC biology to promote their development and functional differentiation. Importantly, we demonstrated that the ontogenesis of ILC2s and the progression of allergic airway inflammation are determined by a unique local 3D configuration of the region containing the ILC-lineage-defining factor Id2, which is characterized by multiple interactions between the Id2 promoter and distal regulatory elements bound by the transcription factors GATA-3 and RORα, unveiling the mechanism whereby the Id2 expression is specifically controlled in group 2 ILCs.

The miR-181 family regulates colonic inflammation through its activity in the intestinal epithelium.

The intestinal epithelium is a key physical interface that integrates dietary and microbial signals to regulate nutrient uptake and mucosal immune cell function. The transcriptional programs that regulate intestinal epithelial cell (IEC) quiescence, proliferation, and differentiation have been well characterized. However, how gene expression networks critical for IECs are posttranscriptionally regulated during homeostasis or inflammatory disease remains poorly understood. Herein, we show that a conserved family of microRNAs, miR-181, is significantly downregulated in IECs from patients with inflammatory bowel disease and mice with chemical-induced colitis. Strikingly, we showed that miR-181 expression within IECs, but not the hematopoietic system, is required for protection against severe colonic inflammation in response to epithelial injury in mice. Mechanistically, we showed that miR-181 expression increases the proliferative capacity of IECs, likely through the regulation of Wnt signaling, independently of the gut microbiota composition. As epithelial reconstitution is crucial to restore intestinal homeostasis after injury, the miR-181 family represents a potential therapeutic target against severe intestinal inflammation.

The lncRNA ALPHA specifically targets chikungunya virus to control infection.

Arthropod-borne viruses, including the alphavirus chikungunya virus (CHIKV), cause acute disease in millions of people and utilize potent mechanisms to antagonize and circumvent innate immune pathways including the type I interferon (IFN) pathway. In response, hosts have evolved antiviral counterdefense strategies that remain incompletely understood. Recent studies have found that long noncoding RNAs (lncRNAs) regulate classical innate immune pathways; how lncRNAs contribute to additional antiviral counterdefenses remains unclear. Using high-throughput genetic screening, we identified a cytoplasmic antiviral lncRNA that we named antiviral lncRNA prohibiting human alphaviruses (ALPHA), which is transcriptionally induced by alphaviruses and functions independently of IFN to inhibit the replication of CHIKV and its closest relative, O'nyong'nyong virus (ONNV), but not other viruses. Furthermore, we showed that ALPHA interacts with CHIKV genomic RNA and restrains viral RNA replication. Together, our findings reveal that ALPHA and potentially other lncRNAs can mediate non-canonical antiviral immune responses against specific viruses.

Protocol to assess RNA-RNA interactions in situ using an RNA-proximity ligation assay.

Here, we describe a protocol to assess RNA-RNA interactions in situ using an adapted proximity ligation assay (PLA). We detail steps to perform RNA-probe hybridization, in situ rolling circle amplification, and immunofluorescence confocal microscopy. With these tools, it is possible to detect and characterize the intracellular localization of interacting RNA pairs using small cell numbers. This protocol provides a targeted approach to understanding RNA-RNA interactions in intact cells that can complement other established deep-sequencing-based approaches. For complete details on the use and execution of this protocol, please refer to Basavappa et al. (2022).1.

Evolving Views of Long Noncoding RNAs and Epigenomic Control of Lymphocyte State and Memory.

Not simply an attribute of the adaptive immune system, immunological memory can be viewed on multiple levels. Accordingly, the molecular basis of memory comprises multiple mechanisms. The advent of new sequencing technologies has greatly enhanced the understanding of gene regulation and lymphocyte specification, and improved measurement of chromatin states affords new insights into the epigenomic and transcriptomic programs that underlie memory. Beyond canonical genes, the involvement of long noncoding RNAs (lncRNAs) is becoming increasingly apparent, and it appears that there are more than two to three times as many lncRNAs as protein-coding genes. lncRNAs can directly interact with DNA, RNA, and proteins, and a single lncRNA can contain multiple modular domains and thus interact with different classes of molecules. Yet, most lncRNAs have not been tested for function, and even fewer knockout mice have been generated. It is therefore timely to consider new potential mechanisms that may contribute to immune memory.

Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection.

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1ß release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as an independently-generated CRISPR/Cas9 Card19 knockout mouse line (Card19Null) showed no defect in macrophage cell lysis. Notably, Card19 is located on chromosome 13, immediately adjacent to Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that Card19lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli. Card19lxcn mice exhibited increased susceptibility to Yersinia infection, whereas independently-generated Card19Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of Card19lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent gene Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.

Protein kinase R and the integrated stress response drive immunopathology caused by mutations in the RNA deaminase ADAR1.

The RNA deaminase ADAR1 is an essential negative regulator of the RNA sensor MDA5, and loss of ADAR1 function triggers inappropriate activation of MDA5 by self-RNAs. Mutations in ADAR, the gene that encodes ADAR1, cause human immune diseases, including Aicardi-Goutières syndrome (AGS). However, the mechanisms of MDA5-dependent disease pathogenesis in vivo remain unknown. Here we generated mice with a single amino acid change in ADAR1 that models the most common human ADAR AGS mutation. These Adar mutant mice developed lethal disease that required MDA5, the RIG-I-like receptor LGP2, type I interferons, and the eIF2α kinase PKR. A small-molecule inhibitor of the integrated stress response (ISR) that acts downstream of eIF2α phosphorylation prevented immunopathology and rescued the mice from mortality. These findings place PKR and the ISR as central components of immunopathology in vivo and identify therapeutic targets for treatment of human diseases associated with the ADAR1-MDA5 axis.

RIPK1 activates distinct gasdermins in macrophages and neutrophils upon pathogen blockade of innate immune signaling.

Injection of effector proteins to block host innate immune signaling is a common strategy used by many pathogenic organisms to establish an infection. For example, pathogenic Yersinia species inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signaling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1-caspase-8 death-inducing platform that confers antibacterial defense. While recent studies revealed that caspase-8 cleaves the pore-forming protein gasdermin D to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defense is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase-dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1ß release, which is critical for anti-Yersinia defense. During in vivo infection, IL-1ß neutralization increases bacterial burden in wild-type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signaling.

A new perspective on mesenchymal-immune interactions in adipose tissue.

The mammalian immune system has crucial homeostatic functions in different adipose depots. However, white adipose tissue (WAT) inflammation is a hallmark of obesity and can contribute to type 2 diabetes mellitus (T2DM). Recently, mesenchymal cells were identified as highly heterogenous populations displaying specialized immune functions in immune cell migration, activation, survival, and overall lymphoid tissue organization in several tissues. How they regulate the inflammatory milieu within different adipose depots remains unknown. Using recently published single-cell RNA-sequencing (scRNAseq) data sets, we analyze cytokine and chemokine expression of mouse WAT mesenchymal cell subpopulations to highlight potential immunological heterogeneity and specialization, hypothesizing on their immunological functions. This new perspective on immune-mesenchymal cell interactions in adipose tissue may promote studies that heighten our understanding of immune cell processes within WAT during health and obesity. We hope that these studies redefine our knowledge of the roles of mesenchymal cells in regulating adipose tissue inflammation and physiology.

Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1.

Mutations in KCNC3, which encodes the Kv3.3 potassium channel, cause degeneration of the cerebellum, but exactly how the activity of an ion channel is linked to the survival of cerebellar neurons is not understood. Here, we report that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies, and that this stimulation is greatly increased by a disease-causing Kv3.3 mutation. TBK1 activity is required for the binding of Kv3.3 to its auxiliary subunit Hax-1, which prevents channel inactivation with depolarization. Hax-1 is also an anti-apoptotic protein required for survival of cerebellar neurons. Overactivation of TBK1 by the mutant channel leads to the loss of Hax-1 by its accumulation in multivesicular bodies and lysosomes, and also stimulates exosome release from neurons. This process is coupled to activation of caspases and increased cell death. Our studies indicate that Kv3.3 channels are directly coupled to TBK1-dependent biochemical pathways that determine the trafficking of cellular constituents and neuronal survival.

Ontogeny and heterogeneity of innate lymphoid cells and the noncoding genome.

Since their discovery a decade ago, it has become evident that innate lymphoid cells (ILCs) play critical roles in protective immune responses against intracellular and extracellular pathogens but are also central regulators of epithelial barrier integrity and tissue homeostasis. ILCs populate almost every tissue in mammalian organisms; therefore, not surprisingly, dysregulation of their functions contributes to the development and progression of multiple inflammatory and metabolic diseases. Our knowledge of the transcriptional programs governing the development, differentiation, and functions of the different groups of ILCs has increased dramatically in the last ten years. However, with the advent of new technologies, an unprecedented level of heterogeneity, plasticity, and developmental complexity has started to be revealed. In this review, we highlight recent advances in our understanding of ILC development and their biological functions. In particular, we aim to emphasize how our increasing knowledge of the chromatin landscape and the noncoding genome of these innate lymphocytes is allowing us to better understand their development and functions in different contexts during homeostasis and inflammation. Moreover, we propose that the design of more refined genetic tools to study tissue-specific ILCs and their functions can be accomplished by leveraging our understanding of how specific noncoding elements of the genome regulate gene expression in ILCs.

Presynaptic Kv3 channels are required for fast and slow endocytosis of synaptic vesicles.

Since their discovery decades ago, the primary physiological and pathological effects of potassium channels have been attributed to their ion conductance, which sets membrane potential and repolarizes action potentials. For example, Kv3 family channels regulate neurotransmitter release by repolarizing action potentials. Here we report a surprising but crucial function independent of potassium conductance: by organizing the F-actin cytoskeleton in mouse nerve terminals, the Kv3.3 protein facilitates slow endocytosis, rapid endocytosis, vesicle mobilization to the readily releasable pool, and recovery of synaptic depression during repetitive firing. A channel mutation that causes spinocerebellar ataxia inhibits endocytosis, vesicle mobilization, and synaptic transmission during repetitive firing by disrupting the ability of the channel to nucleate F-actin. These results unmask novel functions of potassium channels in endocytosis and vesicle mobilization crucial for sustaining synaptic transmission during repetitive firing. Potassium channel mutations that impair these "non-conducting" functions may thus contribute to generation of diverse neurological disorders.