Colonic goblet cell-associated antigen passages mediate physiologic and beneficial translocation of live gut bacteria in preweaning mice

Abstract

Gut-resident microorganisms have time-limited effects in distant tissues during early life. However, the reasons behind this phenomenon are largely unknown. Here, using bacterial culture techniques, we show that a subset of live gut-resident bacteria translocate and disseminate to extraintestinal tissues (mesenteric lymph nodes and spleen) in preweaning (day of life 17), but not adult (day of life 35), mice. Translocation and dissemination in preweaning mice appeared physiologic as it did not induce an inflammatory response and required host goblet cells, the formation of goblet cell-associated antigen passages, sphingosine-1-phosphate receptor-dependent leukocyte trafficking and phagocytic cells. One translocating strain, Lactobacillus animalis^WU, showed antimicrobial activity against the late-onset sepsis pathogen Escherichia coli ST69 in vitro, and its translocation was associated with protection from systemic sepsis in vivo. While limited in context, these findings challenge the idea that translocation of gut microbiota is pathological and show physiologic and beneficial translocation during early life.

Extended Data Figure 1:. Bionomial distribution of translocation, characterization of colon bacterial taxa at family level in preweaning and adult mice, luminal vs mucosal residence of L. animalis^WU, and changes in gut barrier with EGF:

A) Pattern of number of CFUs recovered from the MLN and spleen of DOL17 mice (relates to supplementary table 2. B) Relative frequency of bacterial taxa at family level compared by 16S v4 sequencing of specific pathogen free laboratory mice at DOL17 (n=3) and DOL35 (n=4). Lactobacillaceae family (denoted by red box in the legend and arrows on the graph) was one of the most common translocating taxa, is present at both ages. C) Culture of luminal vs mucosal scrapings from L. animalis^WU fed DOL17 mice (n=4) reveals that L. animalis^WU can be present in both compartments. D) Schematic of EGF treatment to inhibit GAPs and FTY720 treatment to inhibit trafficking. E) Assessment of gut barrier function in L. animalis^WU colonized mice in the presence and absence of EGF (n=3 per treatment group) using the 4kD FITC dextran leak assay. Graphs represent the mean +/− SEM. P value calculated using two-tailed Student’s t-test.

Extended Data Figure 2:. S1PR modulation alters immune cell trafficking to spleen in preweaning mice:

Percentage of immune cells expressing S1PR (%S1PR^GFP+CD45⁺) assessed in colon and spleen of S1PR-GFP reporter mice (B6.129P2-S1pr1^tm1Hrose/J) treated at DOL17 with pan-S1PR inhibitor (FTY720) (n=3) or not treated (control) (n=3). Statistical analyses were performed by two-tailed Student’s t test in GraphPad Prism. Data represented as mean with individual values. P values are as denoted.

Extended Figure 3:. CSF1R blockade, loss of cDC1 cells, and CX₃CR1 deletion does not impair L. animalis^WU translocation in preweaning mice:

(A) Flow cytometry plots and (B-E) graphs demonstrate that CSFR1 blockade in preweaning mice (n=3) reduces the CD45+ MHCII+ CD11c- colonic LP cellular population which can also express CX₃CR1 and F4/80. (F) CFU/organ of L. animalis^WU recovered from intestinal and extraintestinal tissues of L. animalis^WU fed preweaning mice that were nontreated controls (n=4) or treated with anti-CSF1R (n=3). (G) CFU/organ of L. animalis^WU recovered from intestinal and extraintestinal tissues of L. animalis^WU fed wildtype (n=6), cDC1 deficient (Irf8 delta 32) mice (n=6), or CX₃CR1 deficient preweaning mice (n=5). Graphs represent mean +/− SEM. Statistical analyses were performed by two- tailed Student’s t test for B - F, one-way ANOVA for intestinal tissues in G, and one-sided cumulative binomial distribution probability test for extraintestinal tissues in F and G.

Extended Figure 4:. Induction of Th1, Th2 and Th17 cytokines by MLN and splenic CD4⁺ T cells from preweaning mice given L. animalis^WU with and without EGF :

(A) Flow cytometry gating strategy for identifying Th1 cytokines (TNFα and IFNɣ) secreted by CD45⁺CD3⁺CD4⁺ T cells in MLNs and spleens. Frequency of TNFα+ and IFNɣ+ CD4+ T cells in (B-C) MLNs and (D-E) spleens of nontreated (control) (n=9), L. animalis^WU fed (n=5) and L. animalis^WU+EGF fed mice (n=6). (F) Flow cytometry gating strategy for identifying Th2 cytokines (IL4 and IL13) secreted by CD45⁺CD3⁺CD4⁺ T cells in MLNs and spleens. Frequency of IL13+ and IL4+ CD4+ T cells in (G-H) MLNs and (I-J) spleens of nontreated (control) (n=9), L. animalis^WU fed (n=5) and L. animalis^WU+EGF fed mice (n=6). (K) Flow cytometry gating strategy for identifying Th17 cytokines (IL17 and IL22) secreted by CD45⁺CD3⁺CD4⁺ T cells in MLNs and spleens. Frequency of IL17+ and IL22+ CD4+ T cells in (L-M) MLNs and (N-O) spleens of nontreated (control) (n=9), L. animalis^WU fed (n=5) and L. animalis^WU+EGF fed mice (n=6). Statistical analyses were performed by one-way ANOVA with Dunnett’s post test. Graphs represent mean+/− SEM. P values are as denoted.

Extended Data Figure 5:. Peripheral blood neutrophils increase in preweaning mice infected with E. coli ST69:

Neutrophil (CD45⁺Ly6G/C⁺ cells) numbers in non-infected mice (n=3) or mice infected with E. coli ST69 (n=5) gavaged orally at DOL17. Statistical analyses were performed by two-tailed Student’s t test in GraphPad Prism. Graph represents mean +/− SEM. P values are as denoted.

Extended Data Figure 6:. L. animalis^WU does not induce regulatory T cell subsets in preweaning mice:

A) Graphs and B) representative flow plots of Foxp3+ and RORγt+ Foxp3+ regulatory T cell populations in the MLN and spleen of unmanipulated mice (Control) (n=4 for MLNs and 3 for spleens), mice fed L. animalis^WU from DOL10–20 (L. animalis^WU) (n=4 for MLNs and 3 for spleens), and mice fed L. animalis^WU given EGF gavage from DOL10–20 (L. animalis^WU +EGF) (n=4 for MLNs and spleen). Graphs represent mean +/− SEM. Statistical analyses were performed by one way ANOVA with a Dunnett’s post test.

Extended Figure 7:. Characterization of MLN and splenic bacterial taxa at family level in preweaning mice with and without L. animalis^WU feeding.

Cellular populations from the A) MLN and B) spleen were isolated and treated with propidium monoazide and photoactivation, DNA was isolated and bacterial taxa were characterized by 16s rRNA v4 sequencing. The number of different taxa identified were not dramatically altered by L. animalis^WU feeding.

Extended Data Figure 8:. E. coli ST69 and L. animalis^WU are sensitive to vancomycin, neomycin, ampicillin and metronidazole:

Colony forming units (CFU) of (A-D) E. coli ST69 (n=3) and (E-H) L. animalis^WU (n=3) after plating bacteria treated with (A, E) Vancomycin (B, F) Neomycin (C, G) Ampicillin and (D, H) Metronidazole at the specified concentrations for 4 hours. Statistical analyses were performed by one way ANOVA with a Dunnett’s post test. Data represented as mean +/− SEM P values are as denoted.

Figure 1:. Live gut-resident bacteria spontaneously translocate to MLN and Spleen in preweaning mice:

(A) Schematic representation of the experimental set up used in the identification of translocating bacteria in mice. (B) CFU/organ (colony forming units per organ) of bacteria recovered from brain heart infusion (BHI) plates of spleen and MLN homogenates from DOL17 and DOL35 littermates (n=27 for DOL17 MLN, n=26 for DOL35 MLN, n=15 for DOL17 spleen and n=14 for DOL35 spleen) (C) Specific bacterial taxa identified from six DOL17 mice (M1 – M6) MLNs by full length 16s rRNA Sanger sequencing of isolates (D) Schematic representation of the experimental set up used to track L. animalis^WU colonization in intestinal tissues and translocation to MLN and spleen in DOL17 and DOL35 mice. (E) CFU/organ of L. animalis^WU recovered from intestinal and extraintestinal tissues of L. animalis^WU fed DOL17 (N=15) and DOL35 (N=15) mice. For B and E litters are color-coded, circle denotes females, and triangle denotes males to demonstrate that translocation was not litter or sex dependent but was age dependent. Graphs are presented as the mean +/− SEM. Statistical comparisons were performed using a one-sided cumulative binomial distribution probability for MLN and Spleen in panels 1B and 1E (see supplementary tables 2 and 3 for details) and two-tailed Students t-test for intestinal tissues, P values are as denoted.

Figure 2:. Early-life L. animalis^WU translocation requires goblet cells, GAPs, and host cells that express S1PR and are phagocytic :

(A) Representative images from the proximal colon of DOL17 mice with gut-resident bacteria (eubacterial FISH probe; red) identified within a goblet cell (UEA1+ green) and DAPI (in blue) visualized in XY, YZ and XZ plane. (B) CFU/organ of L. animalis^WU recovered from intestinal and extraintestinal tissues of Math1^f/f (n=9) and goblet cell deficient Math1^f/fvil-Cre-ER^T2 (n=13) DOL17 mice fed with L. animalis^WU. (C) CFU/organ of L. animalis^WU recovered from the intestine and extraintestinal tissues of nontreated (n=14), EGF treated (n=14) and pan-S1PR modulator (FTY720) treated (n=6) DOL17 mice fed with L. animalis^WU. (D) Flow cytometry plots and (E) graph showing depletion of F4/80+ MHCII+ CD45+ splenic cells in Clodrosome treated preweaning mice (n=5) when compared to nontreated mice (n=4). (F) CFU/organ of L. animalis^WU recovered intestinal and extraintestinal tissues of PBS (n=4) and Clodrosome (n=4) treated preweaning mice. (G) CFU of in vitro cultured L. animalis^WU recovered after treatment of 10³ L. animalis^WU with gentamicin at the denoted concentrations for 2 hours. CFU/organ of L. animalis^WU recovered from (H) MLNs (n=3) and (I) Spleens (n=4) of preweaning mice in presence or absence of gentamicin treatment before cell lysis. Image in panel A is representative of colon sections from unmanipulated DOL17 mice from 3 independent litters in which 13 of 125 goblet cells (UEA1+) and 1 of 553 (UEA1-) colonocytes imaged in 3 dimensions (z-stacks) contained bacteria (eubacterial FISH probe +). Bar graphs presented as mean+/− SEM. Data points in panels H and I represent paired observations of CFUs within one half of the same MLN or spleen. Two- tailed Student’s t-test was used in E, H and I and intestinal tissues in B and F. A one-sided cumulative binomial distribution probability test was used for extraintestinal tissues in B, C, and F. A one-way ANOVA with at Dunnett’s post test was used for intestinal tissues in C and G.

Figure 3:. L. animalis^WU translocation does not trigger a substantial systemic inflammatory response in preweaning mice:

(A) Schematic representation of experimental setup used in investigating transcriptomic changes associated with L. animalis^WU translocation in MLN of DOL17 mice. (B) Heatmap of 69 differentially expressed genes in sorted cellular populations defined as in (A) and confirmed by CFUs in the liver. (C) Peripheral blood neutrophils, and serum (D) IL6, (E) TNFα, (F) IL10, (G) CCL2, (H) CXCL9, (I) CXCL10 and (J) IFNα measured in DOL17 mice that are non-treated (n=6) or treated with L. animalis^WU fed orally (L. ani oral) (n=6) or administered by i.p. injection (L. ani i.p.) (n=6). Graphs presented as mean +/− SEM. Statistical analyses performed using DEseq2 with FDR<0.05 and >2 fold change for B in Partek^® FLOW^® and with one-way ANOVA with a Dunnett’s post test for C-J.

Figure 4:. L. animalis^WU translocation in early-life helps protect against systemic E. coli ST69 infection:

(A) Putative gramicidin-tyrocidine antibiotic coding sequences identified in L. animalis^WU by Prokka annotation and five other gene regions predicted by antiSMASH to encode secondary metabolites with potential antimicrobial activity (red and supplementary table 7). (B) Representative images of antibacterial activity of L. animalis^WU by agar diffusion in E. coli ST69 LB plates incubated with MRS broth (control), supernatant from overnight L. animalis^WU culture, L. animalis^WU culture supernatant treated with proteinase K (experiment was performed three times) (C). E. coli ST69 recovery from 6 hour cultured E. coli ST69 (n=3 per treatment group) grown in LB broth with no additional treatments (growth control), treated with MRS broth (vehicle control), L. animalis^WU culture supernatant or, L. animalis^WU culture supernatant treated with proteinase K, plated on LB agar. (D) Schematic representation of experimental setup used to assess the protective role of bacterial translocation in preweaning mice against systemic E. coli ST69 infection. Comparison of survival in (E) nontreated (n=9) and VNAM antibiotic treated (n=7) mice and (F) L. animalis^WU (n=5) and EGF + L. animalis^WU treated (n=5) mice that were infected with E. coli ST69 by i.p. injection. (G) Body weight, (H) percentage of segmented neutrophils in the peripheral blood (I) representative images of hepatic abscess (marked by black arrows in mice in both groups) and (J) quantification of hepatic abscesses per mm² in L. animalis^WU (n=4) and EGF + L. animalis^WU treated (n=4) mice infected 2.5 days post infection with E. coli ST69 by i.p injection. Graph in 4C represents mean+/−SD. Graphs in 4G, H, and J represent mean +/− SEM. Statistical analyses were performed by one-way ANOVA with a Dunnett’s post test for C and Log Rank (Mantel-Cox) test for E and F in GraphPad Prism, and a two-tailed Student’s t test for G, H and J; P values are as denoted.