Paré JF et al. (2017), Bioelectric regulation of innate immune system ...

XB-ART-54423

NPJ Regen Med 2017 Jan 01;2:15. doi: 10.1038/s41536-017-0019-y.

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Bioelectric regulation of innate immune system function in regenerating and intact Xenopus laevis.

Paré JF , Martyniuk CJ , Levin M .

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Two key inputs that regulate regeneration are the function of the immune system, and spatial gradients of transmembrane potential (Vmem). Endogenous bioelectric signaling in somatic tissues during regenerative patterning is beginning to be understood, but its role in the context of immune response has never been investigated. Here, we show that Vmem levels modulate innate immunity activity in Xenopus laevis embryos. We developed an assay in which X. laevis embryos are infected with a uropathogenic microorganism, in the presence or absence of reagents that modify Vmem, prior to the ontogenesis of the adaptive immune system. General depolarization of the organism's Vmem by pharmacological or molecular genetic (ion channel misexpression) methods increased resistance to infection, while hyperpolarization made the embryos more susceptible to death by infection. Hyperpolarized specimens harbored a higher load of infectious microorganisms when compared to controls. We identified two mechanisms by which Vmem mediates immune function: serotonergic signaling involving melanocytes and an increase in the number of primitive myeloid cells. Bioinformatics analysis of genes whose transcription is altered by depolarization revealed a number of immune system targets consistent with mammalian data. Remarkably, amputation of the tail bud potentiates systemic resistance to infection by increasing the number of peripheral myeloid cells, revealing an interplay of regenerative response, innate immunity, and bioelectric regulation. Our study identifies bioelectricity as a new mechanism by which innate immune response can be regulated in the context of infection or regeneration. Vmem modulation using drugs already approved for human use could be exploited to improve resistance to infections in clinical settings.

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Genes referenced: mmp7 spib

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	Fig. 1. Uropathogenic E. coli can produce systemic infection of gastrula-stage X. laevis embryos, whose V mem modulates immune response to infection. a Widely distributed uropathogenic E. coli-derived-GFP fluorescence is detected under fluorescence microscopy in embryos infected 24 h (panel i) or 48 h (panel ii) before the image was captured. In contrast, the non-pathogenic K12 E. coli lab strain harboring the same GFP reporter fails to colonize the embryos after injection (fluorescent imaging in panels i–iv, bright field imaging in panels v–vi). b Comparative survival curves of embryos injected with vehicle solution (no bacteria, green line), non-pathogenic E. coli (blue line), or uropathogenic E. coli (yellow line). Error bars represent the standard deviation from three biological replicates. Embryos showing no motion after poking or being in a state of lysis were counted as dead. c Embryo’s V mem modulates immune response to infection. General embryo depolarization increases the rate of survival following infection while general embryo hyperpolarization decreases post-infection survival. For chemical polarizations, embryos were kept in normal conditions until after infection (late gastrula stage), at which point the specified compounds were added to their media. For genetic modifications of polarity, one-cell embryos were injected with mRNAs coding for the specified transmembrane channels, incubated until they reach the gastrula stage, and infected with uropathogenic E. coli. Surviving embryos were counted 4 days after infection (chemical depolarization conditions in solid blue columns, chemical hyperpolarization condition in solid red column; genetic depolarization condition in dotted blue column, genetic hyperpolarization conditions in dotted red columns). Error bars represent the standard deviation from at least three biological replicates. *denotes p < 0.05. IVM ivermectin, NMDG N-methyl-d-glucamine. d Resistant embryos show peripheral mobilization of leukocytes. Fluorescence detection of leukocytes with the specific XL2 antibody in embryos surviving 4 days after infection. Panel i shows a non-infected embryo while panel ii shows an infected one
	Fig. 2. Increased resistance to infection following chemical depolarization is mediated through a serotonergic signaling pathway. a Following infection, embryos were immediately incubated with the specified compounds. b For genetic inhibition of serotonergic signaling, a dominant-negative rat serotonin transporter mRNA encoding a mutant (rSert-D98G) was injected into one-cell embryos, which were grown to the gastrula stage, and infected with uropathogenic E. coli. Surviving embryos were counted 4 days after infection. Error bars represent the standard deviation from two biological replicates. *denotes p < 0.05. IVM ivermectin, fluox fluoxetine
	Fig. 3. Melanocyte-derived factors increase resistance to infection. a Melanocytes migrate and fragment to injury sites. b Albino embryos’ resistance to infection keeps being increased by exposure to ivermectin. Infected embryos were exposed to ivermectin for 24 h following infection and the survival ratios were calculated 4 days post-infection. c Percentage increase in survival to infection following bacterial exposure to cell supernatants from proliferating or quiescent human neonatal melanocytes (HEMn). Error bars represent the standard deviation from three biological replicates. d Percentage increase in survival to infection following bacterial exposure to non-denaturing cell lysates from proliferating or quiescent human neonatal melanocytes (HEMn). Each column represents the compilation of three independent experiments within which each experiment used embryos from a single fertilization. *denotes p < 0.05
	Fig. 4. Human homologs of immune genes differentially expressed at stage 45 after depolarization with ivermectin (a) or glycine receptor overexpression (b). Proteins are red shapes, diseases are purple boxes, stimulatory regulatory events are indicated by an arrow and a plus sign on the relationship line, inhibitory regulatory events are indicated by a blunt line, and arrows without any sign indicate direct binding of proteins
	Fig. 5. Barium chloride-induced depolarization affects primitive myeloid cells distribution. a Visualization of spib-a and mmp7 expression by in situ hybridization at stage 26 in control (i, iii) and barium chloride-treated (ii, iv) conditions. b Quantification of spib-a and mmp7-positive cells in control and barium chloride-treated conditions reveals that depolarization by barium chloride significantly increases the number of mmp7-expressing cells at stage 26. Error bars represent the standard deviation from five biological replicates. *denotes p < 0.05. c Visualization of spib-a and mmp7 expression by in situ hybridization at stage 33 in control (i, iv), infected (ii, v), and resistant (iii, vi) embryos
	Fig. 6. Tail amputation increases resistance to infection. a Embryos at NF stage 12 (gastrula) were infected with uropathogenic E. coli and, after reaching NF stage 27 (post-hatching), were amputated from the posterior fifth of the body. The number of survivors was quantified 3 days later (4 days post-infection) and compared with non-amputated infected embryos. b Comparison of survival to infection percentages between non-amputated (plain column) and amputated (dotted column) embryos reveals an augmentation of survival by tail amputation and subsequent regeneration. Each column represents three independent assays. c Visualization of mmp7-expressing myeloid cells by in situ hybridization following amputation (top panel) reveals increased posterior concentration of primitive myeloid cells, and detection of depolarized cells by staining with DiBAC4(3) voltage dye (lower panel) reveals a cluster of highly depolarized cells at the posterior extremity of the embryo after tail amputation. d Quantification of mmp7-expressing cells following infection and/or amputation at NF stage 28 reveals significant increases in mmp7-expressing cells consequent to tail amputation. *denotes p < 0.05
	Fig. 7. Model integrating V mem signal into innate immune response. Unperturbed tadpoles harbor polarized cells, with specific native amounts and distributions of melanocytes and primitive myeloid cells. Following chemical/genetic treatments that depolarize V mem, pathways involving serotonin signaling induce proliferation and redistribution of melanocytes and primitive myeloid cells, leading to an increase in the efficiency of the immune response when stimulated with a pathogenic agent. Tail amputation induces a strong posterior V mem depolarization (at the site of injury), where melanocytes and primitive myeloid cells are recruited, resulting in a net increase of the latter in the embryo, leading to an enhanced innate immune response

References [+] :

Adams, Endogenous voltage gradients as mediators of cell-cell communication: strategies for investigating bioelectrical signals during pattern formation. 2013, Pubmed