Figure 6.
Removing ligatures and applying oral antibiotic ointment improved cGVHD by reducing allogeneic immune responses. (A) Improvement of CEJ-ABC measurement after removing ligatures without HCT. Day −14 is the day ligatures were inserted. Day 0 is the day ligatures were removed. Days 7, 14, and 21 indicate the time points after removing ligatures (day 0 vs each time point, n = 4-6 per group, ∗∗P < .01; and ∗∗∗P < .001 determined using a Mann-Whitney U test). Data represent means ± SDs. (B) The experiment protocol for ligature-removal experiments (C-H) is shown. “Ligature-removal” mice had ligatures inserted 28 days before HCT and removed 14 days before HCT. By contrast, ligatures were inserted 14 days before HCT in OLP mice and remained in place throughout HCT. (C-D) Representative cGVHD skin scores and OS of BALB/c recipients of B10.D2 donor grafts are shown. Statistical significance of cGVHD skin scores were analyzed using the Wilcoxon matched-pairs signed-rank test. Data represent means ± SEs (OLP allogeneic mice vs ligature-removal allogeneic mice, n = 5 per group). OS data were combined from 2 independent experiments. Statistical significance was determined using the log-rank test (OLP allogeneic mice vs ligature-removal allogeneic mice, n = 10 per group). (E) Relative abundance of oral microbiota (family level) in the allogeneic control, OLP, and ligature-removal mice on day 21 after HCT are shown (n = 4-5 per group). (F) PCoA by AMOVA of oral microbiota family composition of each mouse on day 21 after HCT is shown (n = 4-5 per group). (G) α-Diversity (Shannon index) of oral microbiota in the control, OLP, and ligature-removal groups on day 21 after HCT is shown (n = 4-5 per group, ∗P < .05; and ∗∗P < .01 determined using the Mann-Whitney U test). Data represent means ± SDs. (H) Total microbial burden per ligature in control, OLP, and ligature-removal group on day 21 after allo-HCT (BALB/c recipients from B10.D2 donor grafts) as determined by qPCR is shown. Data are mean ± SD (n = 4-5 per group, ∗P < .05; and ∗∗P < .01, estimated by Mann-Whitney U test). (I) Percentage CEJ-ABC lengths (relative to day 0) of untransplanted OLP mice on the day of ligature placement (day −14), on the day oral antibiotic therapy was started, on day 7 of oral antibiotics, on day 14 of oral antibiotics, and on day 21 of oral antibiotics are shown. Oral antibiotic ointment consisted of a combination of VCM, MINO, CLDM, metronidazole, and ciprofloxacin. Vehicle control mice received petroleum jelly (antibiotics treatment group vs vehicle group, each time point, n = 4 per group, ∗P < .05 determined using the Mann-Whitney U test). Data represent means ± SDs. (J-K) Representative cGVHD skin scores and OS of BALB/c recipients of B10.D2 donor grafts treated with or without oral antibiotic ointment from day 0 to day 35 after HCT are shown. Statistical significance of cGVHD skin scores were analyzed using a Wilcoxon matched-pairs signed-rank test. Data represent means ± SEs (OLP allogeneic mice without antibiotics vs OLP allogeneic mice with antibiotics, n = 5 per group). OS statistical significance was determined using the log-rank test (OLP allogeneic mice without antibiotics vs OLP allogeneic mice with antibiotics, n = 5 per group). (L) The relative abundance of oral microbiota (family level) in control mice, OLP mice treated with the antibiotic, and OLP mice not treated with antibiotics on day 21 after allo-HCT is shown (n = 3-5 per group). (M) PCoA by AMOVA of oral microbiota family composition of control mice, OLP mice treated with antibiotics, and OLP mice not treated with antibiotics on day 21 after allo-HCT is shown (n = 3-5 per group). (N) α-Diversity (Shannon index) of oral microbiota in control mice, OLP mice treated with antibiotics, and OLP mice not treated with antibiotics on day 21 after allo-HCT is shown (n = 4-5 per group, ∗P < .05; and ∗∗P < .01 determined using the Mann-Whitney U test). Data represent means ± SD. (O) Bacterial load per ligature in control mice, OLP mice treated with antibiotics, and OLP mice not treated with antibiotics on day 21 after allo-HCT (BALB/c recipients from B10.D2 donor grafts) measured by qPCR is shown. Data are mean ± SD (n = 3-5 per group, ∗P < .05; and ∗∗P < .01, estimated by Mann-Whitney U test). (P) DCs from the cervical LNs were isolated from BALB/c mice that had ligatures in place for 14 days before analysis; that had ligatures in place for 28 days before analysis and were treated with an oral antibiotic combination (VCM, CLDM, and MINO) 14 days before analysis; or that had ligatures placed 28 days before analysis but were removed 14 days before analysis. The DCs from each of these groups were used to stimulate T cells isolated from the spleens of C57BL/6 mice in MLRs for 48 hours. CD3, CD4, and CD8 T-cell proliferation are depicted as the percent that divided at least once determined by CellTrace violet dilution. Data represent means ± SDs (n = 4 per group, ∗P < .05; and ∗∗P < .01 determined using the Mann-Whitney U test). Panels C, J, and K show representative data of 3 independent experiments, and panel P shows representative data of 2 independent experiments.

Removing ligatures and applying oral antibiotic ointment improved cGVHD by reducing allogeneic immune responses. (A) Improvement of CEJ-ABC measurement after removing ligatures without HCT. Day −14 is the day ligatures were inserted. Day 0 is the day ligatures were removed. Days 7, 14, and 21 indicate the time points after removing ligatures (day 0 vs each time point, n = 4-6 per group, ∗∗P < .01; and ∗∗∗P < .001 determined using a Mann-Whitney U test). Data represent means ± SDs. (B) The experiment protocol for ligature-removal experiments (C-H) is shown. “Ligature-removal” mice had ligatures inserted 28 days before HCT and removed 14 days before HCT. By contrast, ligatures were inserted 14 days before HCT in OLP mice and remained in place throughout HCT. (C-D) Representative cGVHD skin scores and OS of BALB/c recipients of B10.D2 donor grafts are shown. Statistical significance of cGVHD skin scores were analyzed using the Wilcoxon matched-pairs signed-rank test. Data represent means ± SEs (OLP allogeneic mice vs ligature-removal allogeneic mice, n = 5 per group). OS data were combined from 2 independent experiments. Statistical significance was determined using the log-rank test (OLP allogeneic mice vs ligature-removal allogeneic mice, n = 10 per group). (E) Relative abundance of oral microbiota (family level) in the allogeneic control, OLP, and ligature-removal mice on day 21 after HCT are shown (n = 4-5 per group). (F) PCoA by AMOVA of oral microbiota family composition of each mouse on day 21 after HCT is shown (n = 4-5 per group). (G) α-Diversity (Shannon index) of oral microbiota in the control, OLP, and ligature-removal groups on day 21 after HCT is shown (n = 4-5 per group, ∗P < .05; and ∗∗P < .01 determined using the Mann-Whitney U test). Data represent means ± SDs. (H) Total microbial burden per ligature in control, OLP, and ligature-removal group on day 21 after allo-HCT (BALB/c recipients from B10.D2 donor grafts) as determined by qPCR is shown. Data are mean ± SD (n = 4-5 per group, ∗P < .05; and ∗∗P < .01, estimated by Mann-Whitney U test). (I) Percentage CEJ-ABC lengths (relative to day 0) of untransplanted OLP mice on the day of ligature placement (day −14), on the day oral antibiotic therapy was started, on day 7 of oral antibiotics, on day 14 of oral antibiotics, and on day 21 of oral antibiotics are shown. Oral antibiotic ointment consisted of a combination of VCM, MINO, CLDM, metronidazole, and ciprofloxacin. Vehicle control mice received petroleum jelly (antibiotics treatment group vs vehicle group, each time point, n = 4 per group, ∗P < .05 determined using the Mann-Whitney U test). Data represent means ± SDs. (J-K) Representative cGVHD skin scores and OS of BALB/c recipients of B10.D2 donor grafts treated with or without oral antibiotic ointment from day 0 to day 35 after HCT are shown. Statistical significance of cGVHD skin scores were analyzed using a Wilcoxon matched-pairs signed-rank test. Data represent means ± SEs (OLP allogeneic mice without antibiotics vs OLP allogeneic mice with antibiotics, n = 5 per group). OS statistical significance was determined using the log-rank test (OLP allogeneic mice without antibiotics vs OLP allogeneic mice with antibiotics, n = 5 per group). (L) The relative abundance of oral microbiota (family level) in control mice, OLP mice treated with the antibiotic, and OLP mice not treated with antibiotics on day 21 after allo-HCT is shown (n = 3-5 per group). (M) PCoA by AMOVA of oral microbiota family composition of control mice, OLP mice treated with antibiotics, and OLP mice not treated with antibiotics on day 21 after allo-HCT is shown (n = 3-5 per group). (N) α-Diversity (Shannon index) of oral microbiota in control mice, OLP mice treated with antibiotics, and OLP mice not treated with antibiotics on day 21 after allo-HCT is shown (n = 4-5 per group, ∗P < .05; and ∗∗P < .01 determined using the Mann-Whitney U test). Data represent means ± SD. (O) Bacterial load per ligature in control mice, OLP mice treated with antibiotics, and OLP mice not treated with antibiotics on day 21 after allo-HCT (BALB/c recipients from B10.D2 donor grafts) measured by qPCR is shown. Data are mean ± SD (n = 3-5 per group, ∗P < .05; and ∗∗P < .01, estimated by Mann-Whitney U test). (P) DCs from the cervical LNs were isolated from BALB/c mice that had ligatures in place for 14 days before analysis; that had ligatures in place for 28 days before analysis and were treated with an oral antibiotic combination (VCM, CLDM, and MINO) 14 days before analysis; or that had ligatures placed 28 days before analysis but were removed 14 days before analysis. The DCs from each of these groups were used to stimulate T cells isolated from the spleens of C57BL/6 mice in MLRs for 48 hours. CD3, CD4, and CD8 T-cell proliferation are depicted as the percent that divided at least once determined by CellTrace violet dilution. Data represent means ± SDs (n = 4 per group, ∗P < .05; and ∗∗P < .01 determined using the Mann-Whitney U test). Panels C, J, and K show representative data of 3 independent experiments, and panel P shows representative data of 2 independent experiments.

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