Herrera-Rincon C et al. (2020), An in vivo brain-bacteria interface: the develo...

XB-ART-56679

NPJ Regen Med 2020 Feb 04;5:2. doi: 10.1038/s41536-020-0087-2.

Show Gene links Show Anatomy links

An in vivo brain-bacteria interface: the developing brain as a key regulator of innate immunity.

Herrera-Rincon C , Paré JF , Martyniuk CJ , Jannetty SK , Harrison C , Fischer A , Dinis A , Keshari V , Novak R , Levin M .

???displayArticle.abstract???
Infections have numerous effects on the brain. However, possible roles of the brain in protecting against infection, and the developmental origin and role of brain signaling in immune response, are largely unknown. We exploited a unique Xenopus embryonic model to reveal control of innate immune response to pathogenic E. coli by the developing brain. Using survival assays, morphological analysis of innate immune cells and apoptosis, and RNA-seq, we analyzed combinations of infection, brain removal, and tail-regenerative response. Without a brain, survival of embryos injected with bacteria decreased significantly. The protective effect of the developing brain was mediated by decrease of the infection-induced damage and of apoptosis, and increase of macrophage migration, as well as suppression of the transcriptional consequences of the infection, all of which decrease susceptibility to pathogen. Functional and pharmacological assays implicated dopamine signaling in the bacteria-brain-immune crosstalk. Our data establish a model that reveals the very early brain to be a central player in innate immunity, identify the developmental origins of brain-immune interactions, and suggest several targets for immune therapies.

???displayArticle.pubmedLink??? 32047653
???displayArticle.pmcLink??? PMC7000827
???displayArticle.link??? NPJ Regen Med
???displayArticle.grants??? [+]

Species referenced: Xenopus
Genes referenced: ctrl itih3 mmp7 nr2e1 slurp1l tub
GO keywords: immune response

???displayArticle.gses??? GSE119729: NCBI

???attribute.lit??? ???displayArticles.show???

	Fig. 1: The presence of a brain protects against systemic infection in Xenopus embryos. a) One day after fertilization (early- to mid-gastrula or stage 12), Xenopus embryos were microinjected with the pathogenic bacteria E. coli UTI89. The next day, surgeries were performed for removal of the brain (brainless or BR– embryos), a piece of cervical spinal cord (SC– embryos) or the tail bud (Tail– embryos). Embryos were collected for morphological and molecular analysis during the next three days post-surgery. The bacterial load is represented in red. b–e) Dorsal (left column) and lateral (right column) views of st. 48 embryos belonging Control or Intact (Ctrl; b, c) vs. Brainless (BR−; d, e) experimental groups. Green and red arrows point, respectively, the control or correct vs. aberrant morphologies after surgery removal. Eyes, gut and branchial arches (ba) are indicated for reference. Left: rostral is up. Right: rostral is left, dorsal is up. Rostral is left and dorsal is up. Scale bar = 500 μm. f, g) Survival rates (plotted as percentage, %) of each experimental group per each infection condition: without E. coli infection or not-infected animals (NI; f, one-way ANOVA P > 0.05) vs. E. coli UTI89 infected animals (UTI, evaluated four days after infection; g, one-way ANOVA P < 0.01). Data represent the mean and S.D. of, at least, five independent replicates. Each replicate is shown by one dot. h) Bacteria load measured and plotted as colony forming units per milliliter (cfu/ml) in independent embryos (dots) belonging each experimental group or surgery condition. Alive embryos were harvested for analysis 48 h after infection. Initial bacteria load or number of bacteria injected (average of three independent replicates) at t = 0 is plotted as a blue-dashed line. One-way ANOVA P > 0.05. i) Host-Pathogen Response Index (HPRI) = % survival/(1 + log10(CFU + 1)), for each experimental group, as a metric of tolerance. Data represent the mean and S.D. of five independent embryos. One-way ANOVA P < 0.01. g, i) P values after post hoc Bonferroni comparisons are indicated as *P < 0.01, P < 0.05, ns P > 0.05. See also Supplementary Fig. S1.
	Fig. 2: The presence of a brain protects against infection-induced death, which is more severe during the first 48 h post infection. a) Longitudinal assay, scoring survival rates after infection for Control (Ctrl, green), Brainless (BR–, red), Spinal Cord Resection (SC–, orange) and Tailless (Tail–, yellow) embryos at 2–4 h post-surgery (hps; corresponding to st. 26–27), 8 hps (st. 30), 24 hps (st. 35), 36 hps (st. 40), 48 hps (st. 42) and the end stage-48 time point (4 days ps). Data represent the mean and S.D. of three independent replicates. b–f) Number (b) and expression pattern (c–f) of apoptotic cells (positively reacted against Cleaved Caspase-3 Asp175 antibody; CC3) in Ctrl (green) and BR– (red) animals at the same longitudinal period than (a). Data represent the mean and S.D. of, at least, ten embryos coming from three different replicates. c–f) Lateral views after CC3 immunofluorescence of UTI-infected Ctrl (c, e) and BR– (d, f) embryos at st. 35 (c, d) and st. 40 (e, f). White arrows point CC3-expressing cells; red arrows indicate damage related to the infection along the gut area. Rostral is left and dorsal is up. Scale bar = 250 μm. a, b) Two-way ANOVA P < 0.01. P values after post hoc Bonferroni comparisons are indicated as **P < 0.01.
	Fig. 3: The absence of the brain during development affects the migration of macrophages in response to infection. a, b) Drawing of (a) and image after in situ hybridization (ISH) for mmp7+ (b) of one st. 35 Xenopus embryo indicating the landmarks or reference points (1–6) for the body subdivision in the four independent areas for quantification (see Methods for details). Dashed-purple line square indicates the area shown in m, n inserts. Rostral is left and dorsal is up. c–l) Quantification of number of mmp7+ cells in not-infected (NI, c–g) and infected (UTI, h–l), belonging each experimental group: Control (Ctrl, green), brainless (BR–; red), spinal cord resection (SC–; yellow) and Tailless (Tail–; orange). Values for normalized number of mmp7+ cells are plotted per each region and group: face (c, h), tail (d, i), dorsal (e, j), and ventral (f, k) to detect distribution and migration patterns. Total (g, l) is the sum of the four regions. One-way ANOVA P value showed significance for (d) (P < 0.01) and (k) (P < 0.05). m, n) High-magnification images showing detail of ventral region (purple square in (a)) with mmp7-positive cells in a Control (m) vs. Brainless (n) embryos. VBI: Ventral Blood Islands or primitive hematopoietic organ. o–r) Number of mmp7-positive myeloid cells (normalized to the area) in not-infected (green) vs. infected (red) embryos belonging Ctrl (o), BR– (p), SC– (q), and Tail– (r) groups. Counts were done on four independent regions of the whole animal body (plus the summation): face, tail, dorsal and ventral areas. Two-way ANOVA showed significant P values for all comparisons (P < 0.05). c–l, o–r) Data represent the mean and S.D. of, at least, ten different embryos, from three different replicates, per each group and condition. Significant P values after post hoc Bonferroni test are indicated as P < 0.05 and *P < 0.01. See also Supplementary Fig. S2.
	Fig. 4: The absence of early brain affects number and patterning of myeloid cells, distributed in proximity to the aberrant neural network. a–h) XL2 immunofluorescence to label leukocytes in uninfected (NI) and infected (UTI) embryos belonging Control (Ctrl, green), Brainless (BR–, red), Spinal Cord Resection (SC–), Tailless (Tail–), Simvastatin-treated, and Neural Crest ablation (NC–) groups. a) Drawing of a late-staged Xenopus embryo. Black rectangle indicates the tail area for counting. Magenta-dashed line outlines the region showed in (c)–(h). b) Number of XL2+ cells (normalized to the area) per each group and condition. c–h) Lateral views of Ctrl (c, d), NC– (e, f), and BR– (g, h); NI (c, e, g) vs. UTI (d, f, h) embryos. Red arrows point XL2+ cells. White arrows point the posterior region of the central blood vessels. i, j) Number of myeloid cells (i) and acetylated alpha-tubulin (Tub) optical density (OD; j) per each tail region for Ctrl (white), and BR– (gray) st. 46–48 embryos. k–o) Lateral views of xlurp::GFP Ctrl (k) and BR– (l) embryos after Tub immunofluorescence. GFP+ myeloid cells are green, Tub+ nerves are red, nuclei are blue after DAPI staining. Differences in tail morphology for BR– animals (deviating the normal body axis (l) with respect to Ctrl (k)) are due to developmental muscle mispatterning occurring when brain is absent. Two independent tail regions were counted: center (occupied by the myotomes; solid-white line) and peripheral (dorsal and ventral lateral fins; dashed-white line). Counts were made at the same region of the animals (e.g., at the same somite level). m–o) Details from (l), white-dashed square. White arrows indicate positive elements at the same point on the three images. Dashed-white line circle an area within scattered groups of immune cells lying in proximity to the highly disorganized neural networks. b, i, j) Data represent the mean and SD of, at least, ten different embryos, from different replicates, per each group and condition. Significant P values after post hoc tests are indicated as **P < 0.01, ns P > 0.05. Images: rostral is left, dorsal is up. Scale bar = 250 μm. See also Supplementary Fig. S3.
	Fig. 5: Transcriptional analysis of Control (Ctrl) and Brainless (BR–) datasets show quantitatively and qualitatively differences for regulated transcripts and cell processes after infection (UTI) or/and not-infection (NI) conditions. a, b) Venn diagram comparing genes (a) and sub-networks (b) differentially regulated for each experimental group-condition. c) Neural-related pathways unique to infection with a brain. d) Neural-related pathways unique to infection without a brain. e) Innate immunity response for brainless animals with infection. This network was significantly upregulated by 11%. f) Complement activation sub-network (classical pathway) exclusively present in infected embryos with absence of brain. Green = down gene, Red = up gene. Complete data are presented in Supplementary Data 1. All measured genes found in a pathway are located in Supplementary Data 2. See also Supplementary Figs S4 and S5.
	Fig. 6: Functional assays demonstrate a role for dopamine signaling in the protective effects of the brain on immune response. a) Dopamine concentration (in pg/μL) in infected Control (Ctrl, white) and Brainless (BR–, gray) embryos at early stage 35, measured by liquid chromatography–mass spectrometry (LC–MS/MS). Unpaired t-test P < 0.05. b) Survival rates (in percentage) of Ctrl (white) and BR– (gray) infected embryos after pharmacological treatment with drugs targeting the type-1 or type-2 family of dopamine (DA) receptors (D1R, D2R). Two-way ANOVA P < 0.01; P values after Bonferroni post hoc test are indicated as *P < 0.01, ns P > 0.05. a, b) Data represent the mean and S.D. of three independent replicates of 30 embryos. Each replicate is shown by one dot. See also Supplementary Fig. S6. c, d) Drawings represent the morphological and molecular events occurs in response to infection, in presence (c) or absence (d) of brain. Unique neural- and immune-related pathways are indicated in blue. Upregulation and downregulation of transcripts are indicated in black. E. coli is represented by red circles; apoptotic cells are represented by cyan circles; mmp7+ cells (early macrophages) are represented by pink stars; dopamine-activated macrophages are represented with a D on the pink star. Complete list of genes is presented in Supplementary Data 1. Green = down gene, Red = up gene. All measured genes found in a pathway are located in Supplementary Data 2.
	Supplementary Figure S1. Brain Removal, Spinal-Cord resection, Simvastatin-treated and Neural-Crest ablation. a,b) Photomicrographs of Control (Ctrl, a) and Brainless (BR–, b) stage-48 embryos from the transgenic line PNTub::GFP. To reveal potential differences in proliferative cells at the face region, we performed immunofluorescence on these embryos against the Histone H3 phosphorylated at serine 10 (H3P, red dots pointed by red arrow), a mitosis marker extensively used in Xenopus. Our results in PNTub::GFP brainless embryos show no growth of GFP-labeled tissue at the brain area, indicating absence of brain regeneration after the complete removal (compare white arrows in a indicating the brain and its extension vs. b with no regenerated tissue along the place where the brain should be). These embryos do not display changes in H3P-positive cells at this face area (157±39 for Ctrl and 168±32 for BR–; unpaired t-test P=0.76), suggesting there is no difference in the proliferative response targeted to regenerate the brain. c, d) Dorsal (c) and lateral (d) views of a late-staged embryo after resection of a constant portion of spinal cord (SC), cervical levels, at stage 25. Orange arrows point the place for surgery removal, showing complete discontinuity between brain and spinal cord. e-g) Neural Crest (NC) ablation assays performed on stage-17 embryos (e, red-dashed square indicates the removed NC domain) and the subsequent aberrant phenotype for the craniofacial elements (f), as defects in branchial arcs (ba) or cement gland (cg). g) Survival rates of Ctrl, BR– and NC– infected embryos. Data represent the mean and S.D. of three independent replicates (n=40, N=120). Each replicate is shown by one dot. One-way ANOVA P<0.05. Significant P values after Bonferroni post-hoc test are indicated as *P<0.05, ns P> 0.05. h-j. Dorsal (h) and lateral (i) views of a late-staged embryo after treatment with 0.4 uM Simvastatin for 18 hours, showing severe muscle and tail phenotype (red arrows) but normal brain morphology (white arrow). Photomicrographs after polarized light of the posterior region of the tail in an intact or control (j). a-c, h: rostral is up. d, f, i ,j: rostral is left, dorsal is up. Eyes, gut, and branchial arches (ba) are indicated for reference.

	Supplementary Figure S3. Number and distribution of myeloid precursors (xlurp+) respect to peripheral nerve network (Tub+ fibers) in Control (Ctrl), Brainless (BR–), Spinal-Cord resection (SC–) and Tailless (Tail–) embryos. a–d) Number of GFP+ cells (normalized to the area; a,b) and peripheral nerve intensity (measured as Tub OD; c, d) for st. 48 xlurp::GFP Control, BR–, SC–, Tail– and Simv groups. P<0.01 for Kruskal-Wallis test. P values after post-hoc Dunn test, when compared to Ctrl group, is indicated as ** P<0.01. Data represent the mean and S.D. of, at least, ten embryos from three independent replicates. e–m) High-magnification images of center (e-j) and peripheral (k–m) tail areas in Control (e–g, k–m) and BR– (h–j) xlurp::GFP embryos. Left column shows GFP+ myeloid cells, middle column shows neural network in red and right column shows co-location of immune cells and nerves. White arrows indicate positive elements at the same point on the three images of a row. n–q) Low-magnification lateral views of xlurp::GFP st. 48 Tail– (n) and SC– (o-q) embryos after whole-mount immunofluorescence with acetylated alpha-tubulin (Tub, red), showing the myeloid cells (GFP+, green), the neural network (Tub+, red), and the co-location for immune cells and nerves (nuclei are blue after DAPI staining in N). Yellow arrows in n indicate accumulation of immune cells at the bud or amputation plane. Orange arrows in o-q indicate the same point on the three images, noting an aberrant sprouted neural network (red) but without parallel ectopic pattern for macrophages (green; as detected in BR– animals, compare to Fig. 4m-o). All: Rostral is left, dorsal is up; scale bar = 250 μm.
	Supplementary Figure S4. Gene networks and cell processes unique to infection in presence or absence of a brain during development. a–d) Pie chart of the functional classification of the pathways exclusively regulated after infection in Control animals (a), after infection in BR– animals (b), after brain removal without infection (c), and between infected Ctrl and infected BR– embryos (d). e) Upregulation of bacteria-related genes induced by infection in brainless animals. These networks were up-regulated by 20%. f) Innate immune response after infection in intact (or developed with a brain) animals. This pathway responds 11% less in presence of a brain than it does in absence of a brain (see Fig. 6c). Green = down gene, Red = up gene. Complete data are presented in Supplementary Data 1. All measured genes found in a pathway are located in Supplementary Data 2.
	Supplementary Figure S5. Gene networks and cell processes unique to infection in presence of brain. a) Considering the role of the brain on macrophages, which in turn are required for regeneration1, we analyzed transcriptional sub-networks related to regeneration. Gene networks related to tissue regeneration/remodeling were exclusively regulated in intact animals (developing with brain) with presence of bacteria, suggesting a relationship between brain, bacteria, and regeneration. b) Membrane-Potential related genes. Likewise, genes related to membrane potential are exclusively up regulated by 12%, in the presence of brain, confirming our previous results on connection between electrical signaling from the brain and slow electrical flows in the long-distance somatic tissue2,3. c) Melanocyte-migration related genes are suppressed (12%) by infection in presence of a brain. Conversely, in absence of brain, this pathway is not affected. Green = down gene, Red = up gene. Complete data are presented in Supplementary Data 1. All measured genes found in a pathway are located in Supplementary Data 2.
	Supplementary Figure S6. Quantitative LC/MS/MS Analysis of Dopamine (DA) in Control (Ctrl) and Brainless (BR–) Xenopus embryos. a) Chromatogram of DA, its metabolite DA-d4 and labelled versions from wholemount embryos homogenate. b) Standard curve used for DA quantification, r2=0.99897605. X-axis is concentration (μM) and Y-axis if for relative response of DA corrected by its internal standard (DA-d4).

References [+] :

Agricola, Identification of genes expressed in the migrating primitive myeloid lineage of Xenopus laevis. 2016, Pubmed, Xenbase