Change of Dendritic Cell Subsets Involved in Protection Against Listeria monocytogenes Infection in Short-Term-Fasted Mice

Abstract

The gastrointestinal tract is the first organ directly affected by fasting. However, little is known about how fasting influences the intestinal immune system. Intestinal dendritic cells (DCs) capture antigens, migrate to secondary lymphoid organs, and provoke adaptive immune responses. We evaluated the changes of intestinal DCs in mice with short-term fasting and their effects on protective immunity against Listeria monocytogenes (LM). Fasting induced an increased number of CD103⁺CD11b^- DCs in both small intestinal lamina propria (SILP) and mesenteric lymph nodes (mLN). The SILP CD103⁺CD11b^- DCs showed proliferation and migration, coincident with increased levels of GM-CSF and C-C chemokine receptor type 7, respectively. At 24 h post-infection with LM, there was a significant reduction in the bacterial burden in the spleen, liver, and mLN of the short-term-fasted mice compared to those fed ad libitum. Also, short-term-fasted mice showed increased survival after LM infection compared with ad libitum-fed mice. It could be that significantly high TGF-β2 and Aldh1a2 expression in CD103⁺CD11b^- DCs in mice infected with LM might affect to increase of Foxp3⁺ regulatory T cells. Changes of major subset of DCs from CD103⁺ to CD103^- may induce the increase of IFN-γ-producing cells with forming Th1-biased environment. Therefore, the short-term fasting affects protection against LM infection by changing major subset of intestinal DCs from tolerogenic to Th1 immunogenic.

Keywords: Dendritic cells; Fasting; Listeria monocytogenes.

Figure 1. Change of CD11c^hi cells in the SILP and mLN after short-term fasting. Mice were fasted for 24 h. (A) Absolute number of CD45⁺ leukocytes in the mLN and SILP. (B) Percentage and absolute number of CD45⁺F4/80⁻CD11c^hi DCs in the mLN and SILP. Unpaired Student’s t-test. Data are representative of two or three independent experiments, n=3–6 mice.

^*p<0.05; ^**p<0.01.

Figure 2. Subtypes of CD11c^hi DCs based on CD103 and CD11b expression in mLN and SILP after short-term fasting. Mice were fasted for 24 h. (A, C) Contour plots of CD45⁺F4/80⁻CD11c^hi DCs based on CD103 and CD11b expression in the (A) mLN and (C) SILP. (B and D) Absolute number of CD45⁺F4/80⁻CD11c^hi DC subsets in the (B) mLN and (D) SILP. Unpaired Student’s t-test. Data are representative of two or three independent experiments, n=3–5 mice.

^**p<0.01; ^***p<0.001.

Figure 3. Increase of CD11c^hi DC subsets in the SILP after short-term fasting. Mice were fasted for 24 h. (A) BrdU uptake by CD45⁺F4/80⁻CD11c^hi DCs subsets. (B) mRNA level of GM-CSF in total SILP cells. (C) CCR7 expression in CD45⁺F4/80⁻CD11c^hiCD103⁺ DCs. Unpaired Student’s t-test. Results are representative of two or three independent experiments, n=3–4 mice.

^*p<0.05; ^**p<0.01.

Figure 4. Bacterial burden and survival rate in short-term-fasted mice infected with LM. Mice were fasted for 24 h and infected with LM. (A, B) Number of CFUs in (A) peripheral organs (spleen, mLN, and liver) and (B) serum at 0, 3, 9, 24, 48, and 72 hpi with Lm-OVA. (C) Survival rate over 8 days; log-rank (Mantel–Cox) test. Other analyses by unpaired Student’s t-test. Results are representative of two or three independent experiments, n=5–6 mice.

^*p<0.05; ^***p<0.001.

Figure 5. Induction of CD103⁺CD11b⁻ DCs and Foxp3⁺ Tregs in short-term-fasted mice infected with LM. Mice were fasted for 24 h and infected with LM. (A, B) Percentage (left panel) and absolute number (right panel) of CD45⁺F4/80⁻CD11c^hi DC subsets in (A) mLN and (B) SILP of mice infected with LM. (C, D) Foxp3⁺ Tregs in the mLN at 1 dpi: (C) dot plot and (D) percentage of CD3⁺CD4⁺ cells. (E) Percentage of Foxp3⁺CD103⁺ Tregs among CD3⁺CD4⁺ cells. (F) mRNA levels of foxp3, gata3, and t-bet in CD3⁺CD4⁺ cells in the mLN. Unpaired Student’s t-test. Results are representative of two or three independent experiments, n=4–6 mice.

^*p<0.05; ^**p<0.01.

Figure 6. Tolerogenic characteristics of CD103⁺ DCs in short-term-fasted mice infected with LM. Mice were fasted for 24 h and infected with LM. (A) Expression of CCR7, PD-L1, CD205, CD86, MHC II, and CD62L in CD45⁺F4/80⁻CD11c^hiCD103⁺ DCs. (B) mRNA levels of TGF-β1, TGF-β2, and aldehyde dehydrogenase 2 (aldh1a2) in CD45⁺F4/80⁻CD11c^hiCD103⁺ DCs. Unpaired Student’s t-test. Results are representative of two or three independent experiments, n=4–5 mice.

^*p<0.05; ^**p<0.01.

Figure 7. Composition of IFN-γ⁺ cells among CD4⁺ T cells and CD8⁺ T cells in short-term-fasted mice infected with LM. Mice were fasted for 24 h and infected with LM. (A) Percentage and absolute number of CD45⁺F4/80⁻CD11c^hi DC subsets in the mLN at 3 dpi. (B, C) Percentage and absolute number of IFN-γ⁺ cells among (B) CD4⁺ T lymphocytes (CD3⁺CD4⁺), and (C) CD8⁺ T lymphocytes (CD3⁺CD8⁺) in the mLN at 1, 2, and 3 dpi. Unpaired Student’s t-test. Results are representative of two or three independent experiments, n=4–6 mice.

^*p<0.05; ^**p<0.01.