Biotin Supplementation Ameliorates Murine Colitis by Preventing NF-κB Activation.

Background & aimsBiotin is a water-soluble vitamin that is indispensable for human health. Biotin deficiency can cause failure-to-thrive, immunodeficiency, alopecia, dermatitis, and conjunctivitis. We previously reported that biotin deficiency also can lead to severe colitis in mice, which is completely reversed with supplementation. Our aim in this study was to determine if high-dose biotin supplementation can provide a therapeutic benefit in a preclinical model for inflammatory bowel disease (IBD) and to identify the molecular mechanism by which this occurs.MethodsMice were challenged with dextran sodium sulfate to induce colitis and were treated with 1 mmol/L biotin to induce or maintain remission. Clinical response was monitored by the Disease Activity Index and fecal calprotectin levels. The colon tissue was investigated for histology, length, as well as expression of inflammatory cytokines (interleukin 6, tumor necrosis factor-α, interleukin 1β), intestinal permeability, tight junctions (zonula occludens-1 and claudin-2), and the transcription factor nuclear factor-κB (NF-κB).ResultsBiotin therapy led to delayed onset and severity of colitis as well as accelerated healing. There was improvement in the Disease Activity Index, fecal calprotectin levels, colon length, and histology. In addition, biotin-treated mice had reduced expression of inflammatory cytokines, reduced intestinal permeability, and reduced activation of NF-κB.ConclusionsOral supplementation with biotin provides benefit for maintenance and induction of remission in the dextran sodium sulfate preclinical model for IBD. Biotin does this by reducing the activation of NF-κB, which prevents the production of inflammatory cytokines and helps maintain the integrity of the intestinal barrier. Clinically, the NF-κB pathway is important in the development of IBD and this finding suggests that biotin may have therapeutic potential for patients with IBD

Imaging regulatory T cell dynamics and CTLA4-mediated suppression of T cell priming

Foxp3(+) regulatory T cells (Tregs) maintain immune homoeostasis through mechanisms that remain incompletely defined. Here by two-photon (2P) imaging, we examine the cellular dynamics of endogenous Tregs. Tregs are identified as two non-overlapping populations in the T-zone and follicular regions of the lymph node (LN). In the T-zone, Tregs migrate more rapidly than conventional T cells (Tconv), extend longer processes and interact with resident dendritic cells (DC) and Tconv. Tregs intercept immigrant DCs and interact with antigen-induced DC: Tconv clusters, while continuing to form contacts with activated Tconv. During antigen-specific responses, blocking CTLA4-B7 interactions reduces Treg-Tconv interaction times, increases the volume of DC: Tconv clusters and enhances subsequent Tconv proliferation in vivo. Our results demonstrate a role for altered cellular choreography of Tregs through CTLA4-based interactions to limit T-cell priming

Imaging the Single Cell Dynamics of CD4+ T Cell Activation by Dendritic Cells in Lymph Nodes

The adaptive immune response is initiated in secondary lymphoid organs by contact between antigen-bearing dendritic cells (DCs) and antigen-specific CD4+ T cells. However, there is scant information regarding the single cell dynamics of this process in vivo. Using two-photon microscopy, we imaged the real-time behavior of naive CD4+ T cells and in vivo–labeled DCs in lymph nodes during a robust T cell response. In the first 2 h after entry into lymph nodes, T cells made short-lived contacts with antigen-bearing DCs, each contact lasting an average of 11–12 min and occurring mainly on dendrites. Altered patterns of T cell motility during this early stage of antigen recognition promoted serial engagement with several adjacent DCs. Subsequently, T cell behavior progressed through additional distinct stages, including long-lived clusters, dynamic swarms, and finally autonomous migration punctuated by cell division. These observations suggest that the immunological synapse in native tissues is remarkably fluid, and that stable synapses form only at specific stages of antigen presentation to T cells. Furthermore, the serial nature of these interactions implies that T cells activate by way of multiple antigen recognition events

Charge Screening by Internal pH and Polyvalent Cations as a Mechanism for Activation, Inhibition, and Rundown of TRPM7/MIC Channels

The Mg2+-inhibited cation (MIC) current, believed to represent activity of TRPM7 channels, is found in lymphocytes and mast cells, cardiac and smooth muscle, and several other eukaryotic cell types. MIC current is activated during whole-cell dialysis with divalent-free internal solutions. Millimolar concentrations of intracellular Mg2+ (or other divalent metal cations) inhibit the channels in a voltage-independent manner. The nature of divalent inhibition and the mechanism of channel activation in an intact cell remain unknown. We show that the polyamines (spermine, spermidine, and putrescine) inhibit the MIC current, also in a voltage-independent manner, with a potency that parallels the number of charges. Neomycin and poly-lysine also potently inhibited MIC current in the absence of Mg2+. These same positively charged ions inhibited IRK1 current in parallel with MIC current, suggesting that they probably act by screening the head group phosphates on PIP2 and other membrane phospholipids. In agreement with this hypothesis, internal protons also inhibited MIC current. By contrast, tetramethylammonium, tetraethylammonium, and hexamethonium produced voltage-dependent block but no inhibition. We show that inhibition by internal polyvalent cations can be relieved by alkalinizing the cytosol using externally applied ammonium or by increasing pH in inside-out patches. Furthermore, in perforated-patch and cell-attached recordings, when intracellular Mg2+ is not depleted, endogenous MIC or recombinant TRPM7 currents are activated by cytosolic alkalinization and inhibited by acidification; and they can be reactivated by PIP2 following rundown in inside-out patches. We propose that MIC (TRPM7) channels are regulated by a charge screening mechanism and may function as sensors of intracellular pH

A Store-operated Calcium Channel in Drosophila S2 Cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca2+-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca2+-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20–50 pA at −110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing ∼300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of ∼50 nM, and was also blocked by 20 μM SKF 96365 and by 20 μM 2-APB. At concentrations between 5 and 14 μM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells

The largest deep-ocean silicic volcanic eruption of the past century

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Science Advances 4 (2018): e1701121, doi:10.1126/sciadv.1701121.The 2012 submarine eruption of Havre volcano in the Kermadec arc, New Zealand, is the largest deep-ocean eruption in history and one of very few recorded submarine eruptions involving rhyolite magma. It was recognized from a gigantic 400-km2 pumice raft seen in satellite imagery, but the complexity of this event was concealed beneath the sea surface. Mapping, observations, and sampling by submersibles have provided an exceptionally high fidelity record of the seafloor products, which included lava sourced from 14 vents at water depths of 900 to 1220 m, and fragmental deposits including giant pumice clasts up to 9 m in diameter. Most (>75%) of the total erupted volume was partitioned into the pumice raft and transported far from the volcano. The geological record on submarine volcanic edifices in volcanic arcs does not faithfully archive eruption size or magma production.This research was funded by Australian Research Council Postdoctoral fellowships (DP110102196 and DE150101190 to R. Carey), a short-term postdoctoral fellowship grant from the Japan Society for the Promotion of Science (to R. Carey), National Science Foundation grants (OCE1357443 to B.H., OCE1357216 to S.A.S., and EAR1447559 to J.D.L.W.), and a New Zealand Marsden grant (U001616 to J.D.L.W.). J.D.L.W. and A.M. were supported by a research grant and PhD scholarship from the University of Otago. R.W. was supported by NIWA grant COPR1802. J.D.L.W. and F.C.-T. were supported by GNS Science grants CSA-GHZ and CSA-EEZ. M.J. was supported by the U.S. Department of Defense (DoD) through the National Defense Science and Engineering Graduate Fellowship (NDSEG) Program

Capacitative Calcium Entry Deficits and Elevated Luminal Calcium Content in Mutant Presenilin-1 Knockin Mice

Dysregulation of calcium signaling has been causally implicated in brain aging and Alzheimer's disease. Mutations in the presenilin genes (PS1, PS2), the leading cause of autosomal dominant familial Alzheimer's disease (FAD), cause highly specific alterations in intracellular calcium signaling pathways that may contribute to the neurodegenerative and pathological lesions of the disease. To elucidate the cellular mechanisms underlying these disturbances, we studied calcium signaling in fibroblasts isolated from mutant PS1 knockin mice. Mutant PS1 knockin cells exhibited a marked potentiation in the amplitude of calcium transients evoked by agonist stimulation. These cells also showed significant impairments in capacitative calcium entry (CCE, also known as store-operated calcium entry), an important cellular signaling pathway wherein depletion of intracellular calcium stores triggers influx of extracellular calcium into the cytosol. Notably, deficits in CCE were evident after agonist stimulation, but not if intracellular calcium stores were completely depleted with thapsigargin. Treatment with ionomycin and thapsigargin revealed that calcium levels within the ER were significantly increased in mutant PS1 knockin cells. Collectively, our findings suggest that the overfilling of calcium stores represents the fundamental cellular defect underlying the alterations in calcium signaling conferred by presenilin mutations

Mutations in Orai1 transmembrane segment 1 cause STIM1-independent activation of Orai1 channels at glycine 98 and channel closure at arginine 91

Stim and Orai proteins comprise the molecular machinery of Ca(2+) release-activated Ca(2+) (CRAC) channels. As an approach toward understanding the gating of Orai1 channels, we investigated effects of selected mutations at two conserved sites in the first transmembrane segment (TM1): arginine 91 located near the cytosolic end of TM1 and glycine 98 near the middle of TM1. Orai1 R91C, when coexpressed with STIM1, was activated normally by Ca(2+)-store depletion. Treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent Orai1 subunits and rapidly blocked STIM1-operated Ca(2+) current. Diamide-induced blocking was reversed by disulfide bond-reducing agents. These results indicate that R91 forms a very narrow part of the conducting pore at the cytosolic side. Alanine replacement at G98 prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) or proline (G98P) caused constitutive channel activation in a STIM1-independent manner. Both Orai1 G98 mutants formed a nonselective Ca(2+)-permeable conductance that was relatively resistant to block by Gd(3+). The double mutant R91W/G98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position found in some patients with severe combined immunodeficiency (SCID), and the double mutant R91C/G98D was resistant to diamide block. These data suggest that the channel pore is widened and ion selectivity is altered by mutations at the G98 site that may perturb α-helical structure. We propose distinct functional roles for G98 as a gating hinge and R91 as part of the physical gate at the narrow inner mouth of the channel

Human neural stem cells induce central nervous system specific regulatory T cells from the ex Treg pool and promote repair in models of multiple sclerosis

Abstract: Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease affecting the central nervous system (CNS) and for which there is no cure. Current treatments focus on suppression of the immune system but fail to repair the resulting damage to the CNS. Neural stem cell (NSC) transplantation is a promising therapeutic strategy for treating neurodegenerative diseases through cell replacement and repair however it is unclear how these cells would mediate repair in MS. We report that human NSCs promote CNS specific T regulatory cells (Tregs) which activate endogenous repair pathways and promote remyelination in a murine model of MS. We observed remyelination, decreased inflammation and an increase in (CNS)-infiltrating CD4+CD25+FoxP3+ Tregs in EAE mice receiving an intra-spinal transplant of NSCs. Recovery was not a result of cell replacement, as NSCs underwent xenograft rejection, and was Treg dependent, as ablation of Tregs abrogated histopathological improvement. RAG2−/−2D2 (R2D2) mice, which bear a TCR repertoire restricted to myelin oligodendrocyte glycoprotein (MOG) and neurofilament, lack CD25+FoxP3+ Tregs under homeostatic conditions; however, upon exposure to MOG, R2D2 mice developed CD25+FoxP3+ Tregs in cervical lymph nodes and the spinal cord. hNSCs also promoted Tregs in vitro in co-cultures with wild type B6 and R2D2 splenocytes, but not with RAG2−/− OT-II+ splenocytes. Additionally, hNSC-Tregs also appear to derive from the exTreg pool suggesting both antigen specific expansion and antigen dependent maintenance of FOXP3 in CNS-specific Tregs. hNSC Tregs also have a unique expression profile and express transglutimase-2 which is implicated in oligodendrocyte dependent repair in the CNS