Microbiota control immune regulation in humanized mice

Abstract

The microbiome affects development and activity of the immune system, and may modulate immune therapies, but there is little direct information about this control in vivo. We studied how the microbiome affects regulation of human immune cells in humanized mice. When humanized mice were treated with a cocktail of 4 antibiotics, there was an increase in the frequency of effector T cells in the gut wall, circulating levels of IFN-γ, and appearance of anti-nuclear antibodies. Teplizumab, a non-FcR-binding anti-CD3ε antibody, no longer delayed xenograft rejection. An increase in CD8+ central memory cells and IL-10, markers of efficacy of teplizumab, were not induced. IL-10 levels were only decreased when the mice were treated with all 4 but not individual antibiotics. Antibiotic treatment affected CD11b+CD11c+ cells, which produced less IL-10 and IL-27, and showed increased expression of CD86 and activation of T cells when cocultured with T cells and teplizumab. Soluble products in the pellets appeared to be responsible for the reduced IL-27 expression in DCs. Similar changes in IL-10 induction were seen when human peripheral blood mononuclear cells were cultured with human stool samples. We conclude that changes in the microbiome may impact the efficacy of immunosuppressive medications by altering immune regulatory pathways.

Figure 1. Changes in microbiota and lymphocytes with antibiotics.

16S sequencing was performed on freshly isolated pellets from humanized mice (A) and humanized mice treated with the cocktail of 4 antibiotics for 2 weeks (B). Following antibiotic treatment there was a 24 ± 4.32-fold depletion of Eubacteria (n = 11, P = 0.0002) and a 34.7 ± 1.86-fold depletion of segmented filamentous bacteria (P = 4.33 × 10^–9). The flora was largely restricted to Ternicutes spp. after antibiotic treatment. (C) The number of CD2⁺ cells in the lamina propria of the mice treated with antibiotics (Abx) compared with mice that did not receive antibiotics is shown (each symbol represents an individual mouse, P = 0.0382, Student’s t test). (D) There was a significant interaction between antibiotic treatment and CD4⁺ T cell subsets in the gut (P = 0.0004, 2-way ANOVA). Naive and effector T cells were decreased and increased, respectively. *P < 0.05, **P < 0.01; n = 8 and 9/group. (E) Similar findings were seen within the CD8⁺ T cells but the differences were not statistically significant. (F) The number of CD19⁺ cells in the lamina propria of the mice treated with antibiotics compared with mice that did not receive antibiotics is shown (each symbol represents an individual mouse; P = 0.0261, Student’s t test) (G) The circulating levels of IFN-γ were elevated in the antibiotic-treated mice. Each symbol represents an individual mouse that was or was not treated with antibiotics. **P = 0.008, Student’s t test. CM, central memory T cell; EMRA, effector memory RA T cell.

Figure 2. Antinuclear antibodies (ANAs) in humanized mice treated with antibiotics.

After 2 weeks of treatment with antibiotics, serum was taken from humanized mice and ANAs were measured using HEp-2 slides. Representative staining is shown for serum from mice treated (A and B) or untreated (C and D) with antibiotics. Original magnification, ×200. ANAs were detected in 5 of 6 (83%) of the humanized mice that were treated with antibiotics but not (0 of 4) in the untreated mice. *P = 0.048, χ² test.

Figure 3. Effects of antibiotics on activity of teplizumab.

The rate of skin graft rejection was compared in humanized mice that were treated with teplizumab (Tep) (solid line, n = 9) or hIg (dotted line, n = 6) without antibiotics (Abx) (A) or with antibiotics (B) (hIg, dotted line, n = 9; teplizumab, solid line, n = 13, P = ns). (C) There was a significant difference in the rate of graft rejection following teplizumab treatment with (red) or without (blue) antibiotics. (D) The rates of rejection were similar in the hIg-treated mice whether or not (red or blue, respectively) they received antibiotics. *P = 0.026, **P = 0.001, log-rank Mantel-Cox test.

Figure 4. Effects of antibiotic treatment on cells in the gut wall after teplizumab treatment.

(A) The number of CD2⁺ T cells in the lamina propria was determined 18 hours after treatment with teplizumab in mice that had or had not received antibiotics (Abx). (B) Gene expression of RORγt was analyzed by RT-PCR in gut cells 18 hours after treatment with anti-CD3 mAb in mice that had or had not received antibiotics. There was a significant reduction in the expression of RORγc in the antibiotic-treated mice. *P = 0.035, Student’s t test; each symbol represents an individual mouse.

Figure 5. Effects of antibiotic treatment on peripheral cells.

(A) The expression of CD127 was measured on CD2⁺ cells in the mesenteric lymph nodes (MLNs) in mice treated with hIg or teplizumab in the presence or absence of antibiotics (Abx). In hIg- and antibiotic-treated mice, CD127 expression was reduced compared with mice that had not been treated with antibiotics (^†P = 0.016). The MFI was significantly reduced after teplizumab in mice that had not received antibiotics (*P = 0.02) but not in the mice treated with antibiotics (n = 3–4 mice/group). (B) There was an interactive effect of antibiotic treatment on the frequency of CD8CM in the mesenteric lymph nodes after teplizumab treatment (*P = 0.035). The frequency increased in mice that received teplizumab without antibiotics (^†P = 0.049) but not in mice that received antibiotics (n = 3–4 mice/group), and was higher than in mice treated with teplizumab and antibiotics (P = 0.035). (C and D) The frequency of IL-2–producing CD4⁺ and CD8⁺ T cells in the spleen was analyzed by flow cytometry following stimulation with PMA/ionomycin for 6 hours, 18 hours following treatment with teplizumab or hIg (n = 10 per group). There was a significant effect of antibiotic treatment on the frequency of IL-2⁺ CD4⁺ and CD8⁺ T cells. *P = 0.03, **P = 0.006, 2-way ANOVA. (E) p-STAT5 was increased in the CD8⁺ splenocytes from mice treated with antibiotics and anti-CD3 mAb compared with mice that received anti-CD3 mAb without antibiotics (*P = 0.02, 2-way ANOVA; each symbol represents 1 mouse). (F) Representative histograms from a mouse treated with teplizumab alone or teplizumab plus antibiotics are shown (red = isotype control, blue = p-STAT5 staining without IL-2, green = p-STAT5 staining with IL-2). Statistical significance in all panels was determined by multiple comparison from 2-way ANOVA.

Figure 6. CCR4⁺CD4⁺ and CCR4⁺CD8⁺ T cells in the gut wall and spleen.

The expression of CCR4 on CD4⁺ cells in the gut (A) and spleen (B) and on CD8⁺ cells in the gut (C) and spleen (D) was measured by flow cytometry in cells that were harvested 18 hours after mice that had (black bars) or had not (open bars) been treated with hIg or teplizumab (Tep). *P < 0.05, **P < 0.01 by 2-way ANOVA (n = 7–10 mice/group). Abx, antibiotics.

Figure 7. Effects of antibiotic treatment on IL-10 levels.

(A) Serum was harvested from the humanized mice with skin grafts that had (n = 12) or had not (n = 7) been treated with antibiotics (Abx) 18 hours after teplizumab (Tep) or control Ig (hIg) treatment. The levels of IL-10 were increased after teplizumab in the mice but not when they were treated with antibiotics. There was significantly less IL-10 after teplizumab in the mice that had versus had not received antibiotics. *P < 0.03, ***P = 0.0009, 2-way ANOVA. (B) The serum levels of IL-10 were compared in mice treated with all 4 or the individual antibiotics 18 hours after teplizumab (n = 4–18/group). The levels of IL-10 were reduced in the mice treated with all 4 antibiotics (All) but not in the mice treated with the individual antibiotics compared with the mice that did not receive antibiotics. *P = 0.03, ANOVA with Dunnett’s multiple comparison test. Amp, ampicillin; Metro, metronidazole; Neo, neomycin; Vanco, vancomycin; Untx, untreated.

Figure 8. Effects of antibiotic treatment on pellets and cells from humanized mice.

(A–C) Splenocytes from mice that had not been treated with antibiotics (Abx) were cultured with pools of pellets from mice that had not or had been treated with antibiotics. The levels of IFN-γ, IL-10, and TNF were measured in the supernatants after 5 days and compared by paired t test. *P = 0.04, n = 8 cell donors/group. (D) Subsets of human PBMCs were isolated by magnetic beads and cultured with pellets from mice that had or had not been treated with antibiotics (n = 5 each). There was increased IL-10 release when the DCs were cultured with pellets from both groups of mice but reduced release with pellets from the antibiotic-treated mice. *P = 0.03, ***P = 0.0008, ****P < 0.0001, 2-way ANOVA with paired comparisons. (E–I) DCs were isolated from the PBMCs from healthy donors and cytokine levels were measured after 5 days in culture with a pool of pellets from mice that had or had not been treated with antibiotics. Each symbol represents a DC cell donor. Culture with the pellets increased production of all cytokines (P < 0.0001) compared with the cultures without pellets (Ctrl) (P = 0.002 by ANOVA). (J) Human stool samples of antibiotic-treated patients and healthy controls were heat inactivated and cultured with PBMCs that were pooled from 4 healthy cell donors. The level of IL-10 in the supernatant after culture for 3 days is shown. Each symbol represents a stool sample donor. **P = 0.002, ****P < 0.0001 by Student’s t test.

Figure 9. DCs cultured with antibiotic-treated pellets enhance T cell activation with anti-CD3 mAb.

CD11c⁺ cells were isolated from PBMCs and cultured with pellets as described in the Methods. (A) The expression of CD86 on CD11b⁺CD11c⁺ DCs cultured in pellets from antibiotic-treated mice was increased. *P = 0.02 by multiple comparison from ANOVA. (B) Autologous T cells were then added to the cultures with teplizumab and the expression of CD69 was measured on the CD4⁺ and CD8⁺ T cells after overnight culture. There was increased expression of CD69 on CD8⁺ T cells that had been cultured with cells and antibiotic-treated pellets. *P < 0.05, ****P < 0.0001 by 2-way ANOVA with paired comparisons. Abx, antibiotics.

Figure 10. Effects of pellets on IL-27 production.

(A) IL-27 expression by intracellular cytokine staining in CD11b⁺ and CD11b⁺CD11c⁺ cells isolated from PBMCs cultured with pellets from mice that had or had not been treated with antibiotics (Abx). Ctrl (triangles) represents IL-27 expression in cells that had not been cultured with pellets. There was reduced IL-27 expression in cells exposed to pellets from mice that had been treated with antibiotics. ***P = 0.0007 by ANOVA with paired comparisons. (B) Pellets were harvested from 6 mice that had not (open circles) or had (closed circles) been treated with antibiotics and dissolved in PBS. The insoluble material or supernatants were cultured with CD11b⁺CD11c⁺ cells isolated from PBMCs by magnetic beads. Ctrl wells (triangles) represent cells without supernatants or pellets for each experiment. There was reduced IL-27 expression when the supernatants from mice that had been cultured with antibiotics were added to the CD11b⁺CD11c⁺ cells. *P = 0.01, ANOVA.