(A) Cytokine production measured by enzymelinked immunosorbent assay (ELISA) in MLN four days after vehicle or antibiotic treatment (ABX; single dose of a combination of ampicillin, metronidazole, neomycin, and vancomycin). (B) Quantification of live bacteria by colony forming units (CFUs) cultured from the MLNs of mice treated with vehicle ABX four days earlier. (C) Principal component analysis preformed on bacterial species identified by 16s rRNA sequencing from MLN, colon contents, or SI contents of mice treated with ABX four days earlier. (D) Unsupervised hierarchical cluster of bacterial species present in the SI, colon, and MLN of four mice following ABX treatment. (E) Venn diagram showing overlap of species found on average at least once in each organ from four ABX treated mice. (F) Photomicrographs following subserosal injection of Chicago blue dye into the SI (left panel) or colon (right panel) identifying the MLNs (black boxes), blue dye within the MLN indicates the MLN drains the respective organ (white arrows). (G) Quantification of live bacteria by CFUs cultured from the SI or colon draining MLNs of mice treated with vehicle treated or ABX four days earlier. Data is presented as the mean +/− SEM, * = p
Figure 2. Live commensal bacteria translocation is…
Figure 2. Live commensal bacteria translocation is associated with the presence of colonic GCs and…
Figure 2. Live commensal bacteria translocation is associated with the presence of colonic GCs and GAPs (A) Confocal image of a single colonic crypt from CX3CR1gfp/+ mice given ABX demonstrating the lack of TEDs (epithelial border denoted by a white dotted line). (B) Quantification of live bacteria by CFUs cultured from the SI or colon draining MLNs of vehicle treated or ABX treated Math1iΔvil or littermate Math1f/f mice following tamoxifen administration and ABX treatment. (C) Confocal image of a colonic epithelial cells with GC morphology from ABX treated mice given luminal Enterococcus faecalis (green) and luminal dextran (red) four hours earlier demonstrating the presence of Enterococcus faecalis within the apical mucin compartment of a GC that has taken up dextran, or a GAP (D) Confocal image of colonic tissue from ABX treated Math1dtomato reporter mice given luminal Enterococcus faecalis (green) six hours earlier, demonstrating the presence of Enterococcus faecalis at the lateral border of a Math1 expressing cells with GC morphology. (E) Quantification of CFUs after plating sorted goblet cells (GCs) or non-GC intestinal epithelial cells (IECs) from ABX or vehicle treated mice. (F) Graphical representation of GAPs per GC and corresponding CFUs per MLN during continuous ABX therapy and withdrawal. (G) GAPs per GC in the SI and colon and (H) CFUs in the MLNs of wildtype, Myd88−/−, and Myd88iΔMath1 mice following treatment with RU486 to delete Myd88 in Math1 expressing cells. Scale bar in A = 50µm, scale bar in C and D = 20µm. Data is presented as the mean +/− SEM, * = p<0.05, ND = not detected, ns = not significant, n = 3 or more mice per group or time point panels A, and C-F. Each symbol represents one mouse panels B and H.
Figure 3. Myd88 dependent and independent microbial…
Figure 3. Myd88 dependent and independent microbial signals regulate trafficking of live bacteria to the…
Figure 3. Myd88 dependent and independent microbial signals regulate trafficking of live bacteria to the MLN by CX3CR1+ DCs following antibiotic therapy (A) Flow cytometry plots showing changes in the populations of CD103+ CD11b− CX3CR1− and CD103− CD11b+ CX3CR1+ DCs (CD45+ CD11c+ MHCII+) in the colonic MLN after vehicle or ABX treatment; histogram gated on the populations (right panel) demonstrates that CD11b+ CD103− DCs are CX3CR1+ and CD11b− CD103+ DCs are CX3CR1−. (B) Absolute number of CD103− CD11b+ CX3CR1+ DCs in the colonic MLN after vehicle or ABX treatment. (C) Quantification live bacteria in the MLN of ABX treated mice by CFUs demonstrating the total bacterial load, intracellular bacterial load, and antibiotic resistant bacteria determined as outlined in the methods. (D) Quantification of live bacteria by CFUs in the sorted MLN DCs from mice following vehicle or ABX treatment. (E) Quantification of live bacteria by CFUs in the SI and colonic MLNs from CX3CR1−/− and CX3CR1+/− mice following ABX treatment. (F) Graphical representation of GAPs per goblet cell in wildtype and CX3CR1−/− mice following vehicle or ABX treatment. (G) Confocal image of CX3CR1GFP+ cell interacting with a dextran (red) filled colonic GC following ABX therapy. (H) Absolute number of CD103−CD11b+ MLN DCs in wildtype mice, Math1iΔvil mice given tamoxifen to delete GC, or CX3CR1−/− mice following vehicle or ABX treatment. (I) Absolute number of CD103− CD11b+ MLN DCs in the MLN of wildtype mice, Myd88−/− mice, or Myd88iΔMath1 mice given RU486 to delete Myd88 given no antibiotics or continual ABX therapy in drinking water. Data is presented as the mean +/− SEM, * = p<0.05, ns = not significant, ABX= antibiotics, n=4 or more mice in each group for data.
Figure 4. Enhanced colitis following antibiotic treatment…
Figure 4. Enhanced colitis following antibiotic treatment is associated with GAPs and dependent upon GCs
Figure 4. Enhanced colitis following antibiotic treatment is associated with GAPs and dependent upon GCs (A) Weight loss in Myd88iΔMath1 mice given RU486 to delete Myd88 in GCs or littermate controls placed on 2% DSS in drinking water. (B) Quantification of GAPs per goblet cell and live bacteria in the MLNs by CFUs after a single gavage ABX on Day 0. (C) Weight loss in mice placed on 2% DSS in drinking water, after single dose of ABX or vehicle on Day 0. (D) Graphical representation of total histology scores and (E) representative images showing increased edema, ulceration (white arrows) and lymphocyte infiltration (black arrows) in DSS treated mice given vehicle or ABX as in C. (F) Weight loss and (G) graphical representation of total histology scores in Math1iΔvil mice given tamoxifen to delete GCs or littermate controls given ABX on day 0 and 1.5% DSS in drinking water. Data is presented as the mean +/− SEM, * = p<0.05, ns = not significant. Scale bar = 50 µm. n=5 mice per group panels A-E, n =3 mice per group panel F and G.
Figure 5. Single doses of antibiotics that…
Figure 5. Single doses of antibiotics that induce colonic GAPs and bacterial translocation worsen inflammatory…
Figure 5. Single doses of antibiotics that induce colonic GAPs and bacterial translocation worsen inflammatory responses and colitis in response to coincident epithelial injury (A) Quantification of live bacteria by CFUs cultured from the MLN and (B) quantification of GAPs per colonic crypt four days after single antibiotic treatment. (C) Quantification of MLN or serum cytokines by ELISA four days after single antibiotic treatment. (D) Weight loss in mice given single antibiotics on day 0 and placed on 2% DSS in drinking water. (E) Graphical representation of total histological scores in mice given single antibiotics on day 0 and given DSS in drinking water. Data is presented as the mean +/− SEM, * = p
Figure 6. Model of the antibiotic induced…
Figure 6. Model of the antibiotic induced bacterial translocation and inflammation
Figure 6. Model of the antibiotic induced bacterial translocation and inflammation (left panel) In the presence of a normal gut microbiota and intact GC microbial sensing, colonic GAPs and bacterial translocation are inhibited. (middle panel) Following antibiotic therapy the altered microbiota is no longer able to inhibit GAP formation and bacterial translocation across the epithelium resulting in low level inflammation. Coincident epithelial damage leads to increased inflammation and worsened colitis. (right panel) The microbiota has dual and opposite roles in bacterial translocation to distant sites. Myd88 dependent GC sensing of the microbiota inhibits GAP formation and bacterial translocation across the epithelium, while Myd88 independent microbial signals induce APCs to migrate to the MLNs.