PD-L1+ and XCR1+ dendritic cells are region-specific regulators of gut homeostasis.

The intestinal mucosa constitutes an environment of closely regulated immune cells. Dendritic cells (DC) interact with the gut microbiome and antigens and are important in maintaining gut homeostasis. Here, we investigate DC transcriptome, phenotype and function in five anatomical locations of the gut lamina propria (LP) which constitute different antigenic environments. We show that DC from distinct gut LP compartments induce distinct T cell differentiation and cytokine secretion. We also find that PD-L1+ DC in the duodenal LP and XCR1+ DC in the colonic LP comprise distinct tolerogenic DC subsets that are crucial for gut homeostasis. Mice lacking PD-L1+ and XCR1+ DC have a proinflammatory gut milieu associated with an increase in Th1/Th17 cells and a decrease in Treg cells and have exacerbated disease in the models of 5-FU-induced mucositis and DSS-induced colitis. Our findings identify PD-L1+ and XCR1+ DC as region-specific physiologic regulators of intestinal homeostasis.

Regulation of splenic monocyte homeostasis and function by gut microbial products.

Splenic Ly6Chigh monocytes are innate immune cells involved in the regulation of central nervous system-related diseases. Recent studies have reported the shaping of peripheral immune responses by the gut microbiome via mostly unexplored pathways. In this study, we report that a 4-day antibiotic treatment eliminates certain families of the Bacteroidetes, Firmicutes, Tenericutes, and Actinobacteria phyla in the gut and reduces the levels of multiple pattern recognition receptor (PRR) ligands in the serum. Reduction of PRR ligands was associated with reduced numbers and perturbed function of splenic Ly6Chigh monocytes, which acquired an immature phenotype producing decreased levels of inflammatory cytokines and exhibiting increased phagocytic and anti-microbial abilities. Addition of PRR ligands in antibiotic-treated mice restored the number and functions of splenic Ly6Chigh monocytes. Our data identify circulating PRR ligands as critical regulators of the splenic Ly6Chigh monocyte behavior and suggest possible intervention pathways to manipulate this crucial immune cell subset.

Gut Microbiome in Progressive Multiple Sclerosis.

OBJECTIVE: This study was undertaken to investigate the gut microbiome in progressive multiple sclerosis (MS) and how it relates to clinical disease. METHODS: We sequenced the microbiota from healthy controls and relapsing-remitting MS (RRMS) and progressive MS patients and correlated the levels of bacteria with clinical features of disease, including Expanded Disability Status Scale (EDSS), quality of life, and brain magnetic resonance imaging lesions/atrophy. We colonized mice with MS-derived Akkermansia and induced experimental autoimmune encephalomyelitis (EAE). RESULTS: Microbiota ß-diversity differed between MS patients and controls but did not differ between RRMS and progressive MS or differ based on disease-modifying therapies. Disease status had the greatest effect on the microbiome ß-diversity, followed by body mass index, race, and sex. In both progressive MS and RRMS, we found increased Clostridium bolteae, Ruthenibacterium lactatiformans, and Akkermansia and decreased Blautia wexlerae, Dorea formicigenerans, and Erysipelotrichaceae CCMM. Unique to progressive MS, we found elevated Enterobacteriaceae and Clostridium g24 FCEY and decreased Blautia and Agathobaculum. Several Clostridium species were associated with higher EDSS and fatigue scores. Contrary to the view that elevated Akkermansia in MS has a detrimental role, we found that Akkermansia was linked to lower disability, suggesting a beneficial role. Consistent with this, we found that Akkermansia isolated from MS patients ameliorated EAE, which was linked to a reduction in RORγt+ and IL-17-producing γδ T cells. INTERPRETATION: Whereas some microbiota alterations are shared in relapsing and progressive MS, we identified unique bacteria associated with progressive MS and clinical measures of disease. Furthermore, elevated Akkermansia in MS may be a compensatory beneficial response in the MS microbiome. ANN NEUROL 2021;89:1195-1211.

Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses.

We present barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel insitu analyses (BOLORAMIS), a reverse transcription-free method for spatially-resolved, targeted, in situ RNA identification of single or multiple targets. BOLORAMIS was demonstrated on a range of cell types and human cerebral organoids. Singleplex experiments to detect coding and non-coding RNAs in human iPSCs showed a stem-cell signature pattern. Specificity of BOLORAMIS was found to be 92% as illustrated by a clear distinction between human and mouse housekeeping genes in a co-culture system, as well as by recapitulation of subcellular localization of lncRNA MALAT1. Sensitivity of BOLORAMIS was quantified by comparing with single molecule FISH experiments and found to be 11%, 12% and 35% for GAPDH, TFRC and POLR2A, respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglial cells. We used fluorescence in situ sequencing to detect error-robust 8-base barcodes associated with each of these genes. We then used this data to uncover the spatial relationship among cells and transcripts by performing single-cell clustering and gene-gene proximity analyses. We anticipate the BOLORAMIS technology for in situ RNA detection to find applications in basic and translational research.

Clostridium bolteae is elevated in neuromyelitis optica spectrum disorder in India and shares sequence similarity with AQP4.

OBJECTIVE: To understand the role of gut microbiome in influencing the pathogenesis of neuromyelitis optica spectrum disorders (NMOSDs) among patients of south Indian origin. METHODS: In this case-control study, stool and blood samples were collected from 39 patients with NMOSD, including 17 with aquaporin 4 IgG antibodies (AQP4+) and 36 matched controls. 16S ribosomal RNA (rRNA) sequencing was used to investigate the gut microbiome. Peripheral CD4+ T cells were sorted in 12 healthy controls, and in 12 patients with AQP4+ NMOSD, RNA was extracted and immune gene expression was analyzed using the NanoString nCounter human immunology kit code set. RESULTS: Microbiota community structure (beta diversity) differed between patients with AQP4+ NMOSD and healthy controls (p < 0.001, pairwise PERMANOVA test). Linear discriminatory analysis effect size identified several members of the microbiota that were altered in patients with NMOSD, including an increase in Clostridium bolteae (effect size 4.23, p 0.00007). C bolteae was significantly more prevalent (p = 0.02) among patients with AQP4-IgG+ NMOSD (n = 8/17 subjects) compared with seronegative patients (n = 3/22) and was absent among healthy stool samples. C bolteae has a highly conserved glycerol uptake facilitator and related aquaporin protein (p59-71) that shares sequence homology with AQP4 peptide (p92-104), positioned within an immunodominant (AQP4 specific) T-cell epitope (p91-110). Presence of C bolteae correlated with expression of inflammatory genes associated with both innate and adaptive immunities and particularly involved in plasma cell differentiation, B cell chemotaxis, and Th17 activation. CONCLUSION: Our study described elevated levels of C bolteae associated with AQP4+ NMOSD among Indian patients. It is possible that this organism may be causally related to the immunopathogenesis of this disease in susceptible individuals.

HCN2 Channel-Induced Rescue of Brain Teratogenesis via Local and Long-Range Bioelectric Repair.

Embryonic exposure to the teratogen nicotine results in brain defects, by disrupting endogenous spatial pre patterns necessary for normal brain size and patterning. Extending prior work in Xenopus laevis that showed that misexpression of ion channels can rescue morphogenesis, we demonstrate and characterize a novel aspect of developmental bioelectricity: channel-dependent repair signals propagate long-range across the embryo. We show that distal HCN2 channel misexpression and distal transplants of HCN2-expressing tissue, non-cell-autonomously reverse profound defects, rescuing brain anatomy, gene expression, and learning. Moreover, such rescue can be induced by small-molecule HCN2 channel activators, even with delayed treatment initiation. We present a simple, versatile computational model of bioelectrical signaling upstream of key patterning genes such as OTX2 and XBF1, which predicts long-range repair induced by ion channel activity, and experimentally validate the predictions of this model. Our results and quantitative model identify a powerful morphogenetic control mechanism that could be targeted by future regenerative medicine exploiting ion channel modulating drugs approved for human use.

HCN2 Rescues brain defects by enforcing endogenous voltage pre-patterns.

Endogenous bioelectrical signaling coordinates cell behaviors toward correct anatomical outcomes. Lack of a model explaining spatialized dynamics of bioelectric states has hindered the understanding of the etiology of some birth defects and the development of predictive interventions. Nicotine, a known neuroteratogen, induces serious defects in brain patterning and learning. Our bio-realistic computational model explains nicotine's effects via the disruption of endogenous bioelectrical gradients and predicts that exogenous HCN2 ion channels would restore the endogenous bioelectric prepatterns necessary for brain patterning. Voltage mapping in vivo confirms these predictions, and exogenous expression of the HCN2 ion channel rescues nicotine-exposed embryos, resulting in normal brain morphology and molecular marker expression, with near-normal learning capacity. By combining molecular embryology, electrophysiology, and computational modeling, we delineate a biophysical mechanism of developmental brain damage and its functional rescue.

HCN4 ion channel function is required for early events that regulate anatomical left-right patterning in a nodal and lefty asymmetric gene expression-independent manner.

Laterality is a basic characteristic of all life forms, from single cell organisms to complex plants and animals. For many metazoans, consistent left-right asymmetric patterning is essential for the correct anatomy of internal organs, such as the heart, gut, and brain; disruption of left-right asymmetry patterning leads to an important class of birth defects in human patients. Laterality functions across multiple scales, where early embryonic, subcellular and chiral cytoskeletal events are coupled with asymmetric amplification mechanisms and gene regulatory networks leading to asymmetric physical forces that ultimately result in distinct left and right anatomical organ patterning. Recent studies have suggested the existence of multiple parallel pathways regulating organ asymmetry. Here, we show that an isoform of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of ion channels (hyperpolarization-activated cyclic nucleotide-gated channel 4, HCN4) is important for correct left-right patterning. HCN4 channels are present very early in Xenopus embryos. Blocking HCN channels (Ih currents) with pharmacological inhibitors leads to errors in organ situs. This effect is only seen when HCN4 channels are blocked early (pre-stage 10) and not by a later block (post-stage 10). Injections of HCN4-DN (dominant-negative) mRNA induce left-right defects only when injected in both blastomeres no later than the 2-cell stage. Analysis of key asymmetric genes' expression showed that the sidedness of Nodal, Lefty, and Pitx2 expression is largely unchanged by HCN4 blockade, despite the randomization of subsequent organ situs, although the area of Pitx2 expression was significantly reduced. Together these data identify a novel, developmental role for HCN4 channels and reveal a new Nodal-Lefty-Pitx2 asymmetric gene expression-independent mechanism upstream of organ positioning during embryonic left-right patterning.