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. 2022 Mar 30;22(2):e16. doi: 10.4110/in.2022.22.e16

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

Young-Jun Ju 1,†, Kyung-Min Lee 1,†, Girak Kim 1,†, Yoon-Chul Kye 1, Han Wool Kim 1, Hyuk Chu 2, Byung-Chul Park 3, Jae-Ho Cho 4, Pahn-Shick Chang 1,5,6, Seung Hyun Han 7, Cheol-Heui Yun 1,3,6,✉
PMCID: PMC9066004  PMID: 35573152

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: Fasting, Dendritic cells, Listeria monocytogenes

INTRODUCTION

Periodic fasting extends the lifespan of bacteria, yeast, worms, and mice compared to an ad libitum diet (1). Intermittent fasting protects mice from infectious and non-infectious diseases such as diabetes, cancer, and neurodegeneration (2). For instance, mice fasting for 24–72 h before Listeria monocytogenes (LM) infection showed a reduced bacterial burden and prolonged survival (3). Furthermore, fasting increased the survival rate after kidney and liver transplantation and ischemia-reperfusion injury in mice (4).

Even short-term nutritional depletion (i.e., 24 h) reduces the total number of cells in the bone marrow and thymus (5). Because the gastrointestinal tract is the first organ directly affected by fasting, proteins related to metabolism are decreased, and protein synthesis is reduced after fasting for 24 h. Interestingly, however, proteins involved in cellular protection such as preservation of intestinal integrity were significantly increased (6). Also, nutritional depletion alters hormone levels and immune cell function (7). For example, leptin promoted expansion of naïve T cells in an IL-2–dependent manner and switched from Th2 to Th1 responses (8,9). Leptin also promoted dendritic cell (DC) maturation by inducing co-stimulatory molecules, proinflammatory cytokines (10), and migration to inflamed tissues (11).

To provoke an adaptive immune response, activation of and antigen presentation by professional antigen-presenting cells (APCs) is required. DCs survey and capture antigen at the local site and deliver it to the draining secondary lymphoid organ for naïve T-cell priming (12). DCs have heterogenic subsets depending on their state of activation, tissue, and differentiation lineage (13). In the gastrointestinal tract, especially in the small intestinal lamina propria (SILP), DCs can be classified based on the expression of CD103 (14). CD103+ DCs in SILP capture antigens, including apoptotic epithelial cells (15) and bacterial antigens (16), and migrate to the mesenteric lymph node (mLN) in a C-C chemokine receptor type 7 (CCR7)-dependent manner (17). Moreover, together with TGF-β (18,19) and retinoic acid (RA) (20), they induce Treg differentiation. CD103+ DCs can be divided into several subtypes depending on their CD11b or CD8α expression. CD103+CD11b−CD8α+ DCs are specialized for cross-presentation of cell-associated antigens and priming of CD8+ T cells (21). Also, in Batf3-/- mice which lack intestinal CD103+CD11b- DCs, there was no evidence of spontaneous gastrointestinal inflammation (22). So, CD103+CD11b+ DCs have been postulated to play a role for immune tolerance in the intestine; however, paradoxically, it has been reported that those cells induce a Th1 and Th17 response under inflammatory conditions (23,24). Likewise, a minor population of intestinal CD103−CD11b+ DCs prime naïve CD4+ T cells and induce differentiation to IL-17– or IFN-γ–producing effector CD4+ T cells (25).

The primary function of the small intestine is digestion and absorption of nutrients. Upon fasting, there are structural and functional changes and reduced metabolic activities (26). Although the effect of fasting on intestinal epithelial cells has been documented, its impact on intestinal immune cells, DCs in particular, is unclear. We evaluated the changes in CD103+ DCs in gut-associated lymphoid organs (such as mLN and SILP) caused by short-term fasting and investigated the immune context induced by these changes in mice infected with LM.

MATERIALS AND METHODS

Animals and short-term fasting

Female BALB/c mice, 6 weeks old, were purchased from Orient Bio Inc., South Korea. Mice were divided into two groups, one fed ad libitum and the other fasted for 24 h with water provided. To prevent the mice from eating their own feces during starvation, they were transferred to new bedding cages when fasting started. The experimental procedures were approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-130510-4-1). Animal experiments were carried out in compliance with the ARRIVE guidelines.

Bacteria preparation and infection

Recombinant LM expressing ovalbumin (LM-OVA) and the parental 1043S strain were provided by Dr. Hao Shen (University of Pennsylvania, Philadelphia, PA, USA). Bacteria cultured in brain heart infusion medium for 8 h at 140 rpm on a shaking incubator at 30°C were harvested by centrifugation and thoroughly washed twice with PBS. Bacteria were enumerated by measuring the optical density at 600 nm (27). The number of bacteria administered to the mice was validated by colony forming units (CFUs) counting by serial dilution and plating. For infection in vivo, 1×108 CFU of LM-OVA in 200 µL of PBS were administered intragastrically after fasting for 24 h (28).

Enumeration of bacteria

The spleen, liver, and mLNs were removed after perfusion with PBS. Each organ was homogenized in PBS with 0.1% Triton x-100. To enumerate Listeria, at least 200 µl of blood were collected by eye-bleeding and centrifuged at 6,300×g for 10 min to separate serum. Serial dilutions were plated on brain heart infusion agar for 12 to 16 h at 37°C and CFUs were counted.

SILP cell isolation

The small intestine, fat, connective tissues, and Peyer’s patches were removed, cut longitudinally, and washed in cold PBS. The organs were cut into 1-cm pieces and transferred to flasks containing 20 ml of digestion solution comprising 1× Hank’s balanced salt solution without Ca2+ or Mg2+ (Sigma-Aldrich, St. Louis, MO, USA), 5% fetal bovine serum (FBS; Gendepot, Barker, TX, USA), 1 mM DL-dithiothreitol (Sigma-Aldrich), and 2 mM EDTA (Sigma-Aldrich). Tissues were dissociated by gentle stirring for 20 min at 37°C and the supernatant was discarded. The SILP fractions were chopped using scissors and digested by stirring in RPMI-1640 medium containing 2% FBS, 0.5 mg/mL collagenase VIII (Sigma-Aldrich), and 40 µg/ml DNase I (Roche, Indianapolis, IN, USA) for 30 min at 37°C. Lamina propria suspensions were passed through a 70-µm filter and washed with RPMI-1640.

In vivo proliferation assay

During short-term fasting, mice were injected intraperitoneally with 1 mg of BrdU in distilled PBS. After 12 h, single cells were prepared from the SILP and mLN. After surface staining with fluorochrome-conjugated antibodies, the cells were washed thoroughly. Then, the cells were fixed and permeabilized in 100 µl of Cytofix/Cytoperm buffer (BD Biosciences, San Jose, CA, USA) for 20 min at room temperature (RT) and washed with BD perm/wash buffer (BD Biosciences). Cells were suspended in 100 µl of BD perm/wash buffer plus (BD Biosciences) and incubated for 10 min at 4°C in the dark. Next, the cells were washed with BD perm/wash buffer and centrifuged. After fixation in 100 µl of buffer, 1×106 cells were incubated in DNase reaction solution for 1 h at 37°C. The cells were suspended in 50 µl of BD perm/wash buffer containing fluorescent anti-BrdU-FITC. The cells were incubated for 20 min at RT, washed with BD perm/wash buffer, and subjected to flow cytometry analysis.

Flow cytometry, intracellular staining, and Foxp3 staining

For cell surface staining, anti-CD11c FITC (HL3), -CD11b PE-Cy7 (M1/70), -CD103 BV421 (M290) or APC (2E7), -CD8a BV421 (53-6.7) or V450 (53-6.7), -I-Ad APC (AM5-32.1), -CD25 PE-Cy7 (PC61), -CD62L BV605 or APC-Cy7 (MEL-14), -CD3e FITC (145-2C11), -PD-L1 PE (J43), -CD80 PE (16-10A1), -CD86 PE (GL1), -NK1.1 (PK136) -CD44 APC-Cy7 (1M7), and -CD45 APC (30-F11) fluorochrome-conjugated antibodies were purchased from BD Biosciences. Mouse anti-CD69 PerCP-Cy5.5 (H1.2F3), -F4/80 APC (BM8), -Ly6G BV421 (1A8), -CD11b BV605 (M1/70), -CD4 BV605 (RM 4-5), and -CCR7 Alexa647 (4B12) monoclonal antibodies were purchased from BioLegend (San Diego, CA, USA). The cells were stained with the appropriate antibodies and incubated for 20 min at 4°C in the dark.

For intracellular staining, isolated single cells were stimulated with phorbol 12-myristate 13-acetate (20 ng/ml) and ionomycin (200 ng/ml) in the presence of 3 µl/ml Brefeldin A (BD Biosciences) and incubated for 5 h at 37°C. Next, cells were stained with anti-IFNγ-PE (XMG1.2) and -IL-17A-PerCP-Cy5.5 (TC11-18H10) antibodies (BD Biosciences) for 20 min at 4°C in the dark.

For Foxp3 intracellular staining, cells were incubated with anti-Foxp3 Alexa647 antibody (MF23) (BD Biosciences) for 20 min at RT in the dark. Next, flow cytometry was performed using a FACS Canto II (BD Biosciences) and analyzed by FlowJo software (Ashland, OR, USA). Cell sorting was performed using a FACS Aria (BD Biosciences). For all staining protocols, cells were analyzed by staining with live/dead discriminating dye (Tonbo Biosciences, CA, USA); dead cells were excluded.

Real-time quantitative PCR

cDNA was subjected to real-time quantitative PCR using Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK). The primers were gm-csf: forward 5′-CTG CCT TAA AGG GAC CAA GAG A-3′, reverse 5′-TTC CGC TGT CCA AGC TGA GT-3′; foxp3: forward 5′-GGA TGA GCT GAC TGC AAT TCT G-3′, reverse 5′-GTA CCT AGC TGC CCT GCA TGA-3′; gata3: forward 5′-GCC TCG GCC ATT CGT ACA T-3′, reverse 5′-GTA GCC CTG ACG GAG TTT C-3′; t-bet: forward 5′-TCG TGG AGG TGA ATG ATG GA-3′, reverse 5′-GA GTG ATC TCT GCG TTC TGG TA-3′; aldh1a2: forward 5′-TTG GCT TAC GGG AGT ATT CAG AA-3′, reverse 5′-GCC TCG GCC TCT TAG GAG TT-3′; tgfb1: forward 5′-TCG ACA TGG AGC TGG TGA AA-3′, reverse 5′-GAG CCT TAG TTT GGA CAG GAT CTG-3′; and tgfb2: forward 5′-GCC CCT GCT GTA CCT TCG T-3′, reverse 5′-TGC CAT CAA TAC CTG CAA ATC T-3′. Thirty PCR cycles were performed in duplicate for each primer. Relative quantification was performed using the ΔΔCt method and normalized to expression of the housekeeping gene gapdh: forward 5′-CTC CAC TCA CGG CAA ATT CA-3′, reverse 5′-GCC TCA CCC CAT TTG ATG TT-3′.

Statistical analysis

The mean value ± standard deviation was determined for each group. For comparison of means, the two-tailed unpaired Student’s t-test was used. Differences were considered significant at p<0.05 unless otherwise specified.

RESULTS

CD11chi DCs were increased in the mLN and SILP of short-term-fasted mice

The gastrointestinal tract consumes much energy and so is directly affected by fasting (6), but little is known about the influence of fasting on gastrointestinal immunity. Therefore, we focused on changes in the intestinal immune system caused by short-term fasting. After 24 h of fasting, a significantly increased number of CD45+ leukocytes was observed in the mLN and SILP of fasted mice compared to ad libitum-fed mice (Fig. 1A). Next, we investigated the numbers of CD45+ DCs, neutrophils, macrophages, B cells, natural killer (NK) cells, and T cells. A significant increase in the number of CD11chi DCs was observed in the mLN and SILP compared to ad libitum-fed mice (Fig. 1B and Supplementary Fig. 1). In contrast, neutrophils, macrophages, NK cells, and lymphocyte populations (including B cells, CD4, CD8, and γδ T cells) in the mLN and SILP were not significantly changed (Supplementary Fig. 1 and Supplementary Fig. 2).

Figure 1. Change of CD11chi 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−CD11chi DCs in the mLN and SILP. Unpaired Student’s t-test. Data are representative of two or three independent experiments, n=3–6 mice.

Figure 1

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*p<0.05; **p<0.01.

Taken together, the results showed that short-term fasting leads to a significant increase in the population of intestinal CD11chi DCs, but not of other immune cell types.

CD103+ DCs were dramatically increased in mLN and SILP from short-term-fasted mice

Intestinal CD11chi DCs can be categorized into several subsets based on their CD103 and CD11b expression (29). The majority of CD11chi DCs in the small intestine expresses the integrin αE referred to as CD103 paired with β7 (30). Furthermore, intestinal CD103+ DCs play an important role in maintaining tolerance to food antigens and commensal bacteria. In addition, immunological tolerance maintains intestinal homeostasis and suppresses unnecessary intestinal hyper-inflammation, which can occur even in normal individuals (14). To investigate the DC subsets increased by short-term fasting, we examined CD11chi DCs based on CD103 and CD11b expression. Intriguingly, the number of CD103+CD11b− DCs was significantly increased in the mLN of short-term-fasted mice (Fig. 2A and B). Furthermore, CD103+ DCs, but not CD103− DCs, in the SILP were significantly increased in short-term-fasted mice compared to ad libitum-fed mice (Fig. 2C and D).

Figure 2. Subtypes of CD11chi 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−CD11chi DCs based on CD103 and CD11b expression in the (A) mLN and (C) SILP. (B and D) Absolute number of CD45+F4/80−CD11chi 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.

Figure 2

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

Collectively, these results indicate that the increased CD11chi DCs in short-term-fasted mice were mainly CD103+CD11b− DCs, but not CD103− DCs, in the mLN and SILP.

CD103+ DCs proliferate in the SILP by GM-CSF

We hypothesized that the increased number of CD103+ DCs in the SILP was caused by local cell proliferation or migration or both. To investigate CD103+ DC proliferation, we performed a bromodeoxyuridine (BrdU) incorporation assay. Interestingly, BrdU uptake significantly increased in CD103+CD11b− and CD103+CD11b+ DCs in the SILP of short-term-fasted mice compared to ad libitum-fed mice (Fig. 3A). Furthermore, BrdU uptake increased in CD103+CD11b− DCs in the mLN of short-term-fasted mice (Supplementary Fig. 3A). These results were correlated with the increased numbers of cell subsets in the SILP and mLN (Fig. 2).

Figure 3. Increase of CD11chi DC subsets in the SILP after short-term fasting. Mice were fasted for 24 h. (A) BrdU uptake by CD45+F4/80−CD11chi DCs subsets. (B) mRNA level of GM-CSF in total SILP cells. (C) CCR7 expression in CD45+F4/80−CD11chiCD103+ DCs. Unpaired Student’s t-test. Results are representative of two or three independent experiments, n=3–4 mice.

Figure 3

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*p<0.05; **p<0.01.

GM-CSF is required for the development of DCs under steady-state and inflammatory conditions (31). GM-CSF also induces the development and expansion of conventional DCs (32) and facilitates the recruitment of intestinal DCs (33). Therefore, we evaluated the mRNA level of GM-CSF in the SILP. The expression of GM-CSF was significantly increased in the SILP after short-term fasting (Fig. 3B). To assess their migration capacity, expression of CCR7 on CD103+ DCs in the SILP was examined (34). CCR7 expression was significantly increased in intestinal CD103+ DCs after short-term fasting compared to ad libitum-fed mice (Fig. 3C). In addition, other functional markers of DCs, such as major histocompatibility complex class II (MHC II), CD205, and PD-L1, were increased (Supplementary Fig. 3B).

Therefore, short-term fasting increased the number of DC subsets, largely CD103+CD11b− and CD103+CD11b+ DCs, in the SILP in correlation with increase of GM-CSF and CCR7 expression, respectively.

Short-term fasting protects mice against LM infection

Next, we postulated that the increase in CD103+ DCs caused by short-term fasting modulates intestinal immunity because CD103+ DCs may be tolerogenic (14). To elucidate the role of CD103+ DCs in infection, mice were infected with LM, which induces Th1 and Th17 responses (35) and the bacterial burden was measured. The number of CFUs was significantly decreased in the spleen, mLN, and liver at 48 h post-infection (hpi) in short-term-fasted mice (Fig. 4A). In addition, high bacteremia was detected in mice fed ad libitum but not those on short-term fasting (Fig. 4B). Furthermore, and ruling out the possibility that short-term-fasted mice consumed food more rapidly after re-feeding, the number of CFUs in the stomach at 3 hpi was not different (Supplementary Fig. 4A).

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.

Figure 4

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

Next, we evaluated the effect of short-term fasting on survival. Consistent with the bacterial burden, the survival of short-term-fasted mice was increased compared to ad libitum-fed mice (Fig. 4C). Also, the body weight change of short-term-fasted mice infected with LM-OVA was comparable to that of the PBS group (Supplementary Fig. 4B).

Therefore, short-term fasting protects against gastrointestinal LM infection.

CD103+ DCs and Foxp3+ Tregs were increased in the mLN during early Listeria infection after short-term fasting

Next, we examined the role of the increased intestinal CD103+ DCs in LM-OVA infection in short-term fasting mice. CD103+CD11b− DCs are tolerogenic and mediate the differentiation of Foxp3+ Tregs by expressing anti-inflammatory cytokines and inhibitory surface molecules (14). Therefore, to investigate whether intestinal CD103+CD11b− DCs contribute to the induction of Foxp3+ Tregs during LM infection, CD103+CD11b− DCs and Foxp3+ Tregs were examined in LM-infected mice with/without fasting. CD103+CD11b− DCs were significantly increased in the mLN (Fig. 5A) and SILP (Fig. 5B) of short-term-fasted mice compared to ad libitum-fed mice. In the mLN, LM infection induced an increase in number of Foxp3+ Tregs in short-term-fasted and ad libitum-fed mice at 1 dpi; the magnitude of the increase was greater in the short-term-fasted mice (Fig. 5C top and D). CD103 is a marker of in vivo-activated Foxp3+ Tregs (36,37,38). Therefore, to determine whether the increased Foxp3+ Tregs in short-term-fasted mice were functionally active, we examined their CD103 expression. The number of in vivo-activated CD103+Foxp3+ Tregs among Foxp3+ Tregs was higher in short-term-fasted mice compared to ad libitum-fed mice (Fig. 5C bottom and E). By contrast, in the spleen, Foxp3+ Tregs were comparable in the two groups and their composition did not differ (Supplementary Fig. 5). Also, the foxp3 mRNA level was threefold higher in short-term-fasted mice than in ad libitum-fed mice at 1 dpi (Fig. 5F left). The t-bet mRNA level was significantly upregulated in short-term-fasted mice compared to ad libitum-fed mice at 3 dpi (Fig. 5F right).

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−CD11chi 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.

Figure 5

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*p<0.05; **p<0.01.

Collectively, these results suggest that the increased CD103+CD11b− DCs in short-term-fasted mice induced functional Foxp3+ Tregs upon LM infection, especially at the early stage.

Increased TGF-β and RA levels contributed to the tolerogenicity of CD103+ DCs

Next, to elucidate the factors responsible for the increase of Foxp3+ Tregs, we examined the cell surface molecules of CD103+ DCs. Increased PD-L1 and decreased CD86 and MHC II expression are phenotypic characteristics of tolerogenic DCs (39), and CD205+CD8α+ DCs producing TGF-β increase Foxp3+ Tregs (40). At 1 dpi, PD-L1, CD205, and CCR7 expression was significantly increased in CD103+ DCs of short-term-fasted mice compared to ad libitum-fed mice, but CD86 and MHC II expression was unchanged (Fig. 6A).

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−CD11chiCD103+ DCs. (B) mRNA levels of TGF-β1, TGF-β2, and aldehyde dehydrogenase 2 (aldh1a2) in CD45+F4/80−CD11chiCD103+ DCs. Unpaired Student’s t-test. Results are representative of two or three independent experiments, n=4–5 mice.

Figure 6

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*p<0.05; **p<0.01.

CD103+ DCs producing TGF-β, RA, and aldehyde dehydrogenase A2 (Aldh1a2) induce Foxp3+ Tregs (18,19). Therefore, we examined their expression in purified intestinal CD103+ DCs. Interestingly, the TGF-β2 and Aldh1a2 mRNA levels in intestinal CD103+ DCs were significantly increased in short-term-fasted mice compared to ad libitum-fed mice (Fig. 6B).

Taken together, the results suggested that increased TGF-β2, Aldh1a2, PD-L1, and CD205 expression may contribute to the tolerogenicity of CD103+ DCs. Such tolerogenic CD103+ DCs may have a correlation with an increase of Foxp3+ Tregs in short-term-fasted mice infected with LM.

Short-term fasting upregulated the Th1 response in mice infected with LM

Next, we investigated the LM burden after 48 hpi. CD103− DCs preferentially induce the differentiation of naïve CD4+ T cells to IFN-γ–producing Th1 cells (25). Therefore, we expected that the number of CD103− DCs would increase after 2 dpi in short-term-fasted mice. At 3 dpi, the percentage and absolute number of CD103−CD11b+ DCs were significantly increased in short-term-fasted mice compared to ad libitum-fed mice; by contrast, the percentage and absolute number of CD103+CD11b− DCs were reduced significantly (Fig. 7A). We further investigated whether CD103− DCs promote a Th1 environment (25). The percentage and absolute number of IFN-γ+ cells among CD4+CD3+ (Fig. 7B), CD8+CD3+ (Fig. 7C), and NK1.1+CD3− (Supplementary Fig. 6) cells were increased at 2 and 3 dpi in short-term-fasted mice, in agreement with the increased t-bet expression at 3 dpi (Fig. 5F).

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−CD11chi 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.

Figure 7

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*p<0.05; **p<0.01.

In summary, increased IFN-γ+ cells in short-term-fasted mice at the later phase of infection may be correlated with an increase of CD103- DCs in accordance with reduction of the bacterial burden by enhancing T cell–mediated immune responses.

DISCUSSION

We investigated the functional alterations of intestinal immune cells, especially CD11chi DCs, caused by short-term fasting with/without LM infection. The findings were as follows: short-term fasting altered the composition of intestinal innate immune cells, including an increase in CD11chi DCs; among CD11chi cells, CD103+ DCs in the mLN and SILP from short-term-fasted mice proliferated more than those from ad libitum-fed mice; the expansion and migration of CD103+ DCs in the SILP after fasting was linked to upregulation of GM-CSF and CCR7, respectively; and short-term fasting significantly contributed for the protection in mice infected with LM through induction of Foxp3+ Tregs for regulating excessive immunopathology at the early phase and IFN-γ+ cells to deal with the infected cells at the later phase.

Unexpected immune responses may be provoked by fasting. For instance, fasting during anorexia enhances survival in mice with experimental autoimmune encephalomyelitis (41) or LM infection (42). By contrast, intermittent fasting suppresses antigen-specific antibody production after immunization with ovalbumin and cholera toxin (43). Fasting for 24 h altered the properties of intestinal immune cells, particularly DCs, protecting against LM infection. A fasting strategy to induce CD11chi DC subset alterations and the duration of protection against LM infection warrant further studies.

CD103+ DCs were the most increased DC subset in the mLN and SILP upon short-term fasting. The increase of CD103+CD11b− DCs in the SILP was a result of cell proliferation and migration. Also, the rate of proliferation was higher in the SILP than the mLN, as reported previously (44), showing that mLN CD103+ DCs proliferate more slowly than SILP CD103+ DCs. In addition, GM-CSF is essential for the development of CD103+ DCs, but not CD103− DCs, in non-lymphoid tissues, including the SILP (45). Indeed, the GM-CSF in the SILP was increased in short-term-fasted mice compared to ad libitum-fed mice, which may be linked to the increased number of intestinal CD103+ DCs. Flt3 ligand also plays an important role in the differentiation of hematopoietic stem cells into conventional DCs (cDCs) (46). In fact, Flt3 ligand maintains a normal number of cDCs by directly regulating their proliferation in the periphery (47). It was reported that absence of GM-CSFR in Csf2r-/- mice affects mostly the development of CD103+CD11b+ DCs in SILP, but not of CD103-CD11b+ DCs. Also, in Flt3-/- mice, the development of CD103+CD11b+ DCs in SILP was impaired, and CD103+CD11b- and CD103-CD11b+ DCs were significantly diminished in SILP (48). In addition, in the same context, it was reported that CX3CR1hi (CD103-CD11b+) DCs in SILP have poor responsiveness to Flt3 and GM-CSF (49), whereas CD103+ DCs in SILP responded stronger to Flt3, coincident with strong antigen-presentation ability (16). Therefore, although our results suggested a role for GM-CSF in the increased proliferation of CD103+ DC subsets in short-term-fasted mice compared to ad libitum-fed mice, an investigation of the direct effect of Flt3 ligand with/without GM-CSF on proliferation is needed.

It has been well reported that CCR7 plays an important role for migration of DCs (34). We showed CD103+ DCs in fasted mice express increased CCR7 compared to ad libitum-fed mice (Fig. 3C). So, we could have inferred that CD103+ DCs in fasted mice could have migrated better into mLN than those of ad libitum-fed mice, thus likely proliferated by increased GM-CSF.

Foxp3+ Tregs not only prevent autoimmune diseases (50,51), but also curb vigorous antimicrobial immune responses by restricting inflammation (52,53). We report a correlation between CD103+ DCs and Foxp3+ Tregs, and that short-term fasting protects mice against LM. During the early stage of infection, CD103+CD11b− DCs and Foxp3+ Tregs were significantly increased in short-term-fasted mice. Foxp3+ Tregs induced by CD103+ DCs prevent excessive immune responses to pathogens (18). It is important to note that CD103+ DCs can be classified into two distinct subsets based on the expression of CD11b. It has been demonstrated that lack of intestinal CD103+CD11b- DCs in Batf3-/- mice have no symptoms of spontaneous inflammation in the intestine. The authors postulated that CD103+CD11b+ DCs would play a role for maintaining intestinal homeostasis via regulating Tregs induction (22). Although this is a report contrary to what we propose in the present study, it is not yet known how short-term fasting affects the relationship between intestinal CD103+CD11b+ DC and induction of Tregs. It appears to be necessary, in future, to examine the cause and consequence of changes in the intestinal DC subsets when Batf3-/- mice are applied to the short-term fasting model.

We have suggested that the increase of Foxp3+ Tregs was a result of increased TGF-β2 expression in short-term-fasted mice. TGF-β promotes expansion of Foxp3+ Tregs in vivo (54). Therefore, the increase of Foxp3+ Tregs during early infection may be mediated by TGF-β. This should be verified by transferring Foxp3+ Tregs into TGF-β-deficient or DC-specific IRF8-deficient mice. Furthermore, Aldh1a2 expression was higher in CD103+ DCs from short-term-fasted mice, which might have caused the increase in Foxp3+ Tregs. RA is mainly produced by intestinal DCs and epithelial cells, and inhibition of RA receptor reduced the induction of Foxp3+ Tregs (55). Therefore, tolerogenic conditions in short-term-fasted mice might restrain the immune response and prevent tissue damage during the early phase of LM infection.

PD-L1 expression, together with CCR7 and CD205, was also significantly increased in short-term-fasted mice infected with LM. PD-L1-expressing DCs function as tolerogenic DCs by inducing Foxp3+ Tregs (39,40). Therefore, we investigated the roles of CCR7 and CD205 in PD-L1-expressing DCs from short-term-fasted mice. CCR7 regulates migration of DCs from tissues to draining lymph nodes (34). CD205 is directly associated with antigen uptake and enhances antigen presentation by the MHC I and II pathways (56,57). It has been suggested that intestinal CD103+ DCs have better potential and ability to uptake non-invasive bacteria efficiently using intraepithelial dendrites (16). In addition, CD103+CD11b+ DCs have been reported to be the first DC subset to transport bacteria, Salmonella Typhimurium, to the mLN after oral infection (48). Collectively, our results suggested that the increased CD103+ DCs in short-term-fasted mice are migratory DCs maintaining intestinal tolerance, and can transport bacterial antigen the most firstly when the host infected.

CD103− DCs are more immunogenic than CD103+ DCs under steady state and infectious conditions and induce differentiation of naïve T cells into IFN-γ–producing Th1 cells (25) and production of proinflammatory cytokines (18). However, the number of CD103− DCs was lower than that of CD103+ DCs, indicating maintenance of tolerance. Indeed, at the early stage (1 or 2 days) after bacterial infection, Foxp3+ Tregs are important for inducing a protective and non-pathogenic Th17 response and later maximizing the gut Th1 response (58). At 1 dpi, CD103+CD11b− DCs and Foxp3+ Tregs were increased in number in short-term-fasted mice compared to ad libitum-fed mice. By contrast, the Th1 response at 3 dpi was increased in short-term-fasted mice infected with LM. Although it is still a controversy, CD103-CD11b+ DCs in mLN are known to be a subset derived from blood, not from intestine (25). So, we have thought reason that increase of CD103-CD11b+ DCs at 3 dpi is causing by the migration, not by subset switching from CD103+ into CD103-. However, the exact mechanisms on how short-term-fasted mice alter the major subset of DCs in intestine during bacterial infection should be followed up by further investigation.

Our results suggest that the changes of intestinal CD11chi DC subsets in short-term-fasted mice are critical for maintaining intestinal tolerance during early LM infection and later for forming a Th1-biased environment by increasing the number of CD103− DCs. It has been suggested that intestinal CD103-CD11b+ DCs induce differentiation of naïve CD4+ T cells into IFN-γ–producing Th1 cells (25). On the other hand, there is another intestinal DC subset, CD103+CD11b+XCR1+ DCs of which to differentiation of naive T cells into Th1 cells (59). The authors demonstrated that there was a significant decrease of intestinal T cell population, especially potential to produce IFN-γ, in mice when XCR1+ DCs were depleted. In addition, the mice lacking XCR1+ DCs showed more susceptible to DSS-induced colitis. Therefore, further investigation on the contribution of CD103+CD11b-XCR1+ DCs to the increase of IFN-γ producing cells would be meaningful.

In the context with induction of IFN-γ+ cells at 3 dpi, the present study showed that neutrophils were increased in short-term fasting mice infected with LM when compared to the ad libitum-fed mice (Supplementary Fig. 7). It could be that the increase of neutrophils may enhance bacterial clearance in LM infection. This is in agreement with the previous report that the infiltration of Ly6G+ neutrophils is critical for bacterial clearance and host survival (60). Based on these findings, we could suggest that establishment of Th1 environment in mice infected with LM by increased CD11b+ DCs is important for the protective immunity.

To examine the antigen-specific CD4+ and CD8+ T cells upon LM infection, it is usual to check IFN-γ production in intestinal CD4+ or CD8+ T cells at 7-8 dpi by re-stimulating with cognate epitope in vitro (61,62,63). However, unfortunately, in experimental condition of current study, we could not have checked antigen-specific IFN-γ producing CD4+ or CD8+ T cells because infected mice with ad libitum-fed (as the control group) were severely affected by infection and many are almost dead (Fig. 4B). Therefore, further works would be required to overcome the limitation of current study through finding even more perfect infection condition verifiable both unique effect of short-term fasting to intestinal DCs and surviving the mice for investigating antigen-specific T cell response.

Although we have suggested the effect of fasting to intestinal DC subsets only in the present study, it has been demonstrated that effect of mild or transient restriction of dietary intake is not limited to DCs, but can affect various immune cells, such as T cells, B cells, neutrophils, macrophages, and monocytes, to modulate immune responses (64). Furthermore, fasting could increase the resistance to colonization of Salmonella Typhimurium, thus reduce host inflammatory responses through suppressing NF-κB expression and downstream inflammatory mediators in whole cecal tissue lysates (65). Conversely, it has been reported that fasting suppresses antigen-specific antibody production in the Ova-vaccination model and Ova-induced diarrhea model by inducing the migration of naïve B cells to the bone-marrow in addition to the immune-enhancing effect (43). Therefore, further study that examines the comprehensive effect of fasting to modulate host immune responses from the perspective of changes in immune cells together with microbiome would be required.

In summary, short-term fasting influenced the characteristics of intestinal CD11chi DCs to balance tolerance and the immune response to LM infection. The balance was regulated by induction of intestinal CD103+CD11b− DCs and Foxp3+ Tregs during the early phase of infection, followed by induction of CD103−CD11b+ DCs with IFN-γ+ cells. These results provide insight into the influence of fasting on the innate immune system and could inform the development of strategies for oral prophylactic vaccination and treatment.

ACKNOWLEDGEMENTS

This work was carried out with the support of Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ016201 and Project No. PJ016613), Rural Development Administration, Republic of Korea. Y-JJ, K-ML, GK, Y-CK, and HWK were supported by the BK21 Plus Program of the Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.

Abbreviations

APC

antigen-presenting cell

CCR7

C-C chemokine receptor type 7

DC

dendritic cell

hpi

hours post-infection

LM

Listeria monocytogenes

mLN

mesenteric lymph nodes

RT

room temperature

SILP

small intestinal lamina propria

Footnotes

Conflicts of interest: The authors declare no potential conflicts of interest.

Author Contributions:
  • Conceptualization: Lee KM, Yun CH.
  • Data curation: Ju YJ, Lee KM, Kim G, Yun CH.
  • Funding acquisition: Yun CH.
  • Investigation: Lee KM, Yun CH.
  • Project administration: Ju YJ, Lee KM, Yun CH.
  • Supervision: Yun CH.
  • Validation: Ju YJ, Yun CH.
  • Writing - original draft: Ju YJ, Lee KM, Yun CH.
  • Writing - review & editing: Ju YJ, Lee KM, Kim G, Kye YC, Kim HW, Chu H, Park BC, Cho JH, Chang PS, Han SH, Yun CH.

SUPPLEMENTARY MATERIALS

Supplementary Figure 1

Gating strategy for flow cytometry analysis of immune cells. Single cells were prepared and pre-gated for single live CD45+ cells, and then specifically further gated for each cell type as displayed, respectively.

in-22-e16-s001.ppt (360KB, ppt)
Supplementary Figure 2

Changes on the composition of CD45+ cells in mLN and SI LP from mice with short-term fasting. Mice were fasted for 24 h. Changes of neutrophils and macrophages (A), B cells and NK cells (B), and T cells (C) were examined. Statistical significance was examined by using unpaired Student’s t-test. The representative results from 2–3 independent experiments, n=3–6 mice.

in-22-e16-s002.ppt (1,019.5KB, ppt)
Supplementary Figure 3

Increase of the CD11chi DC subsets in mice with short-term fasting. Mice were fasted for 24 h. (A) BrdU uptake among CD45+F4/80-CD11chi DCs subsets were examined in mLN. (B) Expression of MHC II, CD86, CD205, and PD-L1 was examined in CD45+F4/80-CD11chiCD103+ DCs of SILP. Statistical significance was examined by using unpaired Student’s t-test. The representative results from 2–3 independent experiments, n=3–4 mice.

in-22-e16-s003.ppt (247.5KB, ppt)
Supplementary Figure 4

Bacterial burden in stomach and body weight change after short-term fasting in mice infected with LM. Mice were fasted for 24 h and then infected with LM. (A) CFU was measured in stomach at 3 hpi. (B) Body weight was monitored for 8 days. To note that the body weight of short-term fasting group was measured from day-1 because of the fasting. The statistics for body weight result was analyzed by log-rank (Mantel-Cox) test and all other statistical significance by unpaired Student’s t-test. The representative results from 2–3 independent experiments, n=5–6 mice.

in-22-e16-s004.ppt (193.5KB, ppt)
Supplementary Figure 5

Induction of splenic Foxp3+ Tregs in mice with short-term fasting followed by LM infection. Mice were fasted for 24 h and then infected with LM. (A, B) Foxp3+ Tregs were analyzed in spleen at 1 dpi and shown in (A) dot plot and (B) percentage among CD3+CD4+ cells. Statistical significance was examined by using unpaired Student’s t-test. The representative results from 2–3 independent experiments, n=3-4 mice.

in-22-e16-s005.ppt (232KB, ppt)
Supplementary Figure 6

Composition of IFN-γ+ cells among NK cells in short-term fasting mice infected with LM. Mice were fasted for 24 h and then infected with LM. The percentage and absolute number of IFN-γ+ cells among NK cells (NK1.1+CD3-NKp46+) in mLN at 1, 2 and 3 dpi. Statistical significance was examined by using unpaired Student’s t-test. The representative results from 2–3 independent experiments, n=4–6 mice.

in-22-e16-s006.ppt (199KB, ppt)
Supplementary Figure 7

Changes of neutrophils and macrophages in mice infected with LM. Mice were fasted for 24 h and then infected with LM. Percentage and absolute number of neutrophils and macrophages from SILP was examined at 3 dpi. Statistical significance was examined by using unpaired Student’s t-test. Results are representative of two or three independent experiments, n=4–6 mice.

in-22-e16-s007.ppt (215.5KB, ppt)

References

  • 1.Fontana L, Partridge L, Longo VD. Extending healthy life span--from yeast to humans. Science. 2010;328:321–326. doi: 10.1126/science.1172539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Longo VD, Mattson MP. Fasting: molecular mechanisms and clinical applications. Cell Metab. 2014;19:181–192. doi: 10.1016/j.cmet.2013.12.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Wing EJ, Young JB. Acute starvation protects mice against Listeria monocytogenes . Infect Immun. 1980;28:771–776. doi: 10.1128/iai.28.3.771-776.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Mitchell JR, Verweij M, Brand K, van de Ven M, Goemaere N, van den Engel S, Chu T, Forrer F, Müller C, de Jong M, et al. Short-term dietary restriction and fasting precondition against ischemia reperfusion injury in mice. Aging Cell. 2010;9:40–53. doi: 10.1111/j.1474-9726.2009.00532.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Shushimita S, de Bruijn MJ, de Bruin RW, IJzermans JN, Hendriks RW, Dor FJ. Dietary restriction and fasting arrest B and T cell development and increase mature B and T cell numbers in bone marrow. PLoS One. 2014;9:e87772. doi: 10.1371/journal.pone.0087772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Lenaerts K, Sokolović M, Bouwman FG, Lamers WH, Mariman EC, Renes J. Starvation induces phase-specific changes in the proteome of mouse small intestine. J Proteome Res. 2006;5:2113–2122. doi: 10.1021/pr060183+. [DOI] [PubMed] [Google Scholar]
  • 7.Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, Flier JS. Role of leptin in the neuroendocrine response to fasting. Nature. 1996;382:250–252. doi: 10.1038/382250a0. [DOI] [PubMed] [Google Scholar]
  • 8.Saucillo DC, Gerriets VA, Sheng J, Rathmell JC, Maciver NJ. Leptin metabolically licenses T cells for activation to link nutrition and immunity. J Immunol. 2014;192:136–144. doi: 10.4049/jimmunol.1301158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.La Cava A, Matarese G. The weight of leptin in immunity. Nat Rev Immunol. 2004;4:371–379. doi: 10.1038/nri1350. [DOI] [PubMed] [Google Scholar]
  • 10.Moraes-Vieira PM, Larocca RA, Bassi EJ, Peron JP, Andrade-Oliveira V, Wasinski F, Araujo R, Thornley T, Quintana FJ, Basso AS, et al. Leptin deficiency impairs maturation of dendritic cells and enhances induction of regulatory T and Th17 cells. Eur J Immunol. 2014;44:794–806. doi: 10.1002/eji.201343592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Al-Hassi HO, Bernardo D, Murugananthan AU, Mann ER, English NR, Jones A, Kamm MA, Arebi N, Hart AL, Blakemore AI, et al. A mechanistic role for leptin in human dendritic cell migration: differences between ileum and colon in health and Crohn’s disease. Mucosal Immunol. 2013;6:751–761. doi: 10.1038/mi.2012.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18:767–811. doi: 10.1146/annurev.immunol.18.1.767. [DOI] [PubMed] [Google Scholar]
  • 13.Eisenbarth SC. Dendritic cell subsets in T cell programming: location dictates function. Nat Rev Immunol. 2019;19:89–103. doi: 10.1038/s41577-018-0088-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Scott CL, Aumeunier AM, Mowat AM. Intestinal CD103+ dendritic cells: master regulators of tolerance? Trends Immunol. 2011;32:412–419. doi: 10.1016/j.it.2011.06.003. [DOI] [PubMed] [Google Scholar]
  • 15.Huang FP, Platt N, Wykes M, Major JR, Powell TJ, Jenkins CD, MacPherson GG. A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J Exp Med. 2000;191:435–444. doi: 10.1084/jem.191.3.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Farache J, Koren I, Milo I, Gurevich I, Kim KW, Zigmond E, Furtado GC, Lira SA, Shakhar G. Luminal bacteria recruit CD103+ dendritic cells into the intestinal epithelium to sample bacterial antigens for presentation. Immunity. 2013;38:581–595. doi: 10.1016/j.immuni.2013.01.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Jang MH, Sougawa N, Tanaka T, Hirata T, Hiroi T, Tohya K, Guo Z, Umemoto E, Ebisuno Y, Yang BG, et al. CCR7 is critically important for migration of dendritic cells in intestinal lamina propria to mesenteric lymph nodes. J Immunol. 2006;176:803–810. doi: 10.4049/jimmunol.176.2.803. [DOI] [PubMed] [Google Scholar]
  • 18.Coombes JL, Siddiqui KR, Arancibia-Cárcamo CV, Hall J, Sun CM, Belkaid Y, Powrie F. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. J Exp Med. 2007;204:1757–1764. doi: 10.1084/jem.20070590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Sun CM, Hall JA, Blank RB, Bouladoux N, Oukka M, Mora JR, Belkaid Y. Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. J Exp Med. 2007;204:1775–1785. doi: 10.1084/jem.20070602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Hill JA, Hall JA, Sun CM, Cai Q, Ghyselinck N, Chambon P, Belkaid Y, Mathis D, Benoist C. Retinoic acid enhances Foxp3 induction indirectly by relieving inhibition from CD4+CD44hi cells. Immunity. 2008;29:758–770. doi: 10.1016/j.immuni.2008.09.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Cerovic V, Houston SA, Westlund J, Utriainen L, Davison ES, Scott CL, Bain CC, Joeris T, Agace WW, Kroczek RA, et al. Lymph-borne cd8alpha dendritic cells are uniquely able to cross-prime CD8 T cells with antigen acquired from intestinal epithelial cells. Mucosal Immunol. 2015;8:38–48. doi: 10.1038/mi.2014.40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Edelson BT, Kc W, Juang R, Kohyama M, Benoit LA, Klekotka PA, Moon C, Albring JC, Ise W, Michael DG, et al. Peripheral CD103+ dendritic cells form a unified subset developmentally related to CD8alpha+ conventional dendritic cells. J Exp Med. 2010;207:823–836. doi: 10.1084/jem.20091627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Liang J, Huang HI, Benzatti FP, Karlsson AB, Zhang JJ, Youssef N, Ma A, Hale LP, Hammer GE. Inflammatory Th1 and Th17 in the intestine are each driven by functionally specialized dendritic cells with distinct requirements for myd88. Cell Reports. 2016;17:1330–1343. doi: 10.1016/j.celrep.2016.09.091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Welty NE, Staley C, Ghilardi N, Sadowsky MJ, Igyártó BZ, Kaplan DH. Intestinal lamina propria dendritic cells maintain T cell homeostasis but do not affect commensalism. J Exp Med. 2013;210:2011–2024. doi: 10.1084/jem.20130728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Cerovic V, Houston SA, Scott CL, Aumeunier A, Yrlid U, Mowat AM, Milling SW. Intestinal CD103(-) dendritic cells migrate in lymph and prime effector T cells. Mucosal Immunol. 2013;6:104–113. doi: 10.1038/mi.2012.53. [DOI] [PubMed] [Google Scholar]
  • 26.Wang T, Hung CC, Randall DJ. The comparative physiology of food deprivation: from feast to famine. Annu Rev Physiol. 2006;68:223–251. doi: 10.1146/annurev.physiol.68.040104.105739. [DOI] [PubMed] [Google Scholar]
  • 27.Koch AL. Turbidity measurements of bacterial cultures in some available commercial instruments. Anal Biochem. 1970;38:252–259. doi: 10.1016/0003-2697(70)90174-0. [DOI] [PubMed] [Google Scholar]
  • 28.Foulds KE, Zenewicz LA, Shedlock DJ, Jiang J, Troy AE, Shen H. Cutting edge: CD4 and CD8 T cells are intrinsically different in their proliferative responses. J Immunol. 2002;168:1528–1532. doi: 10.4049/jimmunol.168.4.1528. [DOI] [PubMed] [Google Scholar]
  • 29.Joeris T, Müller-Luda K, Agace WW, Mowat AM. Diversity and functions of intestinal mononuclear phagocytes. Mucosal Immunol. 2017;10:845–864. doi: 10.1038/mi.2017.22. [DOI] [PubMed] [Google Scholar]
  • 30.Cepek KL, Shaw SK, Parker CM, Russell GJ, Morrow JS, Rimm DL, Brenner MB. Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin. Nature. 1994;372:190–193. doi: 10.1038/372190a0. [DOI] [PubMed] [Google Scholar]
  • 31.Becher B, Tugues S, Greter M. GM-CSF: from growth factor to central mediator of tissue inflammation. Immunity. 2016;45:963–973. doi: 10.1016/j.immuni.2016.10.026. [DOI] [PubMed] [Google Scholar]
  • 32.van de Laar L, Coffer PJ, Woltman AM. Regulation of dendritic cell development by GM-CSF: molecular control and implications for immune homeostasis and therapy. Blood. 2012;119:3383–3393. doi: 10.1182/blood-2011-11-370130. [DOI] [PubMed] [Google Scholar]
  • 33.Hirata Y, Egea L, Dann SM, Eckmann L, Kagnoff MF. GM-CSF-facilitated dendritic cell recruitment and survival govern the intestinal mucosal response to a mouse enteric bacterial pathogen. Cell Host Microbe. 2010;7:151–163. doi: 10.1016/j.chom.2010.01.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Clatworthy MR, Aronin CE, Mathews RJ, Morgan NY, Smith KG, Germain RN. Immune complexes stimulate CCR7-dependent dendritic cell migration to lymph nodes. Nat Med. 2014;20:1458–1463. doi: 10.1038/nm.3709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Orgun NN, Mathis MA, Wilson CB, Way SS. Deviation from a strong Th1-dominated to a modest Th17-dominated CD4 T cell response in the absence of IL-12p40 and type I IFNs sustains protective CD8 T cells. J Immunol. 2008;180:4109–4115. doi: 10.4049/jimmunol.180.6.4109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Zhao D, Zhang C, Yi T, Lin CL, Todorov I, Kandeel F, Forman S, Zeng D. In vivo-activated CD103+CD4+ regulatory T cells ameliorate ongoing chronic graft-versus-host disease. Blood. 2008;112:2129–2138. doi: 10.1182/blood-2008-02-140277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Suffia I, Reckling SK, Salay G, Belkaid Y. A role for CD103 in the retention of CD4+CD25+ Treg and control of Leishmania major infection. J Immunol. 2005;174:5444–5455. doi: 10.4049/jimmunol.174.9.5444. [DOI] [PubMed] [Google Scholar]
  • 38.Zhu J, Davidson TS, Wei G, Jankovic D, Cui K, Schones DE, Guo L, Zhao K, Shevach EM, Paul WE. Down-regulation of Gfi-1 expression by TGF-beta is important for differentiation of Th17 and CD103+ inducible regulatory T cells. J Exp Med. 2009;206:329–341. doi: 10.1084/jem.20081666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Yoo S, Ha SJ. Generation of tolerogenic dendritic cells and their therapeutic applications. Immune Netw. 2016;16:52–60. doi: 10.4110/in.2016.16.1.52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Yamazaki S, Dudziak D, Heidkamp GF, Fiorese C, Bonito AJ, Inaba K, Nussenzweig MC, Steinman RM. CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells. J Immunol. 2008;181:6923–6933. doi: 10.4049/jimmunol.181.10.6923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Cignarella F, Cantoni C, Ghezzi L, Salter A, Dorsett Y, Chen L, Phillips D, Weinstock GM, Fontana L, Cross AH, et al. Intermittent fasting confers protection in CNS autoimmunity by altering the gut microbiota. Cell Metab. 2018;27:1222–1235.e6. doi: 10.1016/j.cmet.2018.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Wang A, Huen SC, Luan HH, Yu S, Zhang C, Gallezot JD, Booth CJ, Medzhitov R. Opposing effects of fasting metabolism on tissue tolerance in bacterial and viral inflammation. Cell. 2016;166:1512–1525.e12. doi: 10.1016/j.cell.2016.07.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Nagai M, Noguchi R, Takahashi D, Morikawa T, Koshida K, Komiyama S, Ishihara N, Yamada T, Kawamura YI, Muroi K, et al. Fasting-refeeding impacts immune cell dynamics and mucosal immune responses. Cell. 2019;178:1072–1087.e14. doi: 10.1016/j.cell.2019.07.047. [DOI] [PubMed] [Google Scholar]
  • 44.Jaensson E, Uronen-Hansson H, Pabst O, Eksteen B, Tian J, Coombes JL, Berg PL, Davidsson T, Powrie F, Johansson-Lindbom B, et al. Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans. J Exp Med. 2008;205:2139–2149. doi: 10.1084/jem.20080414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Greter M, Helft J, Chow A, Hashimoto D, Mortha A, Agudo-Cantero J, Bogunovic M, Gautier EL, Miller J, Leboeuf M, et al. GM-CSF controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells. Immunity. 2012;36:1031–1046. doi: 10.1016/j.immuni.2012.03.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Karsunky H, Merad M, Cozzio A, Weissman IL, Manz MG. Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo . J Exp Med. 2003;198:305–313. doi: 10.1084/jem.20030323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Waskow C, Liu K, Darrasse-Jèze G, Guermonprez P, Ginhoux F, Merad M, Shengelia T, Yao K, Nussenzweig M. The receptor tyrosine kinase Flt3 is required for dendritic cell development in peripheral lymphoid tissues. Nat Immunol. 2008;9:676–683. doi: 10.1038/ni.1615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Bogunovic M, Ginhoux F, Helft J, Shang L, Hashimoto D, Greter M, Liu K, Jakubzick C, Ingersoll MA, Leboeuf M, et al. Origin of the lamina propria dendritic cell network. Immunity. 2009;31:513–525. doi: 10.1016/j.immuni.2009.08.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Schulz O, Jaensson E, Persson EK, Liu X, Worbs T, Agace WW, Pabst O. Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions. J Exp Med. 2009;206:3101–3114. doi: 10.1084/jem.20091925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell. 2008;133:775–787. doi: 10.1016/j.cell.2008.05.009. [DOI] [PubMed] [Google Scholar]
  • 51.Levings MK, Allan S, d’Hennezel E, Piccirillo CA. Functional dynamics of naturally occurring regulatory T cells in health and autoimmunity. Adv Immunol. 2006;92:119–155. doi: 10.1016/S0065-2776(06)92003-3. [DOI] [PubMed] [Google Scholar]
  • 52.Belkaid Y, Tarbell K. Regulatory T cells in the control of host-microorganism interactions. Annu Rev Immunol. 2009;27:551–589. doi: 10.1146/annurev.immunol.021908.132723. [DOI] [PubMed] [Google Scholar]
  • 53.Belkaid Y, Rouse BT. Natural regulatory T cells in infectious disease. Nat Immunol. 2005;6:353–360. doi: 10.1038/ni1181. [DOI] [PubMed] [Google Scholar]
  • 54.Peng Y, Laouar Y, Li MO, Green EA, Flavell RA. TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes. Proc Natl Acad Sci U S A. 2004;101:4572–4577. doi: 10.1073/pnas.0400810101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Mucida D, Park Y, Kim G, Turovskaya O, Scott I, Kronenberg M, Cheroutre H. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science. 2007;317:256–260. doi: 10.1126/science.1145697. [DOI] [PubMed] [Google Scholar]
  • 56.Bonifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, Steinman RM. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. J Exp Med. 2002;196:1627–1638. doi: 10.1084/jem.20021598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Mahnke K, Guo M, Lee S, Sepulveda H, Swain SL, Nussenzweig M, Steinman RM. The dendritic cell receptor for endocytosis, DEC-205, can recycle and enhance antigen presentation via major histocompatibility complex class II-positive lysosomal compartments. J Cell Biol. 2000;151:673–684. doi: 10.1083/jcb.151.3.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Clay SL, Bravo-Blas A, Wall DM, MacLeod MK, Milling SW. Regulatory T cells control the dynamic and site-specific polarization of total CD4 T cells following Salmonella infection. Mucosal Immunol. 2020;13:946–957. doi: 10.1038/s41385-020-0299-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Ohta T, Sugiyama M, Hemmi H, Yamazaki C, Okura S, Sasaki I, Fukuda Y, Orimo T, Ishii KJ, Hoshino K, et al. Crucial roles of XCR1-expressing dendritic cells and the XCR1-XCL1 chemokine axis in intestinal immune homeostasis. Sci Rep. 2016;6:23505. doi: 10.1038/srep23505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Shi C, Hohl TM, Leiner I, Equinda MJ, Fan X, Pamer EG. Ly6G+ neutrophils are dispensable for defense against systemic Listeria monocytogenes infection. J Immunol. 2011;187:5293–5298. doi: 10.4049/jimmunol.1101721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Huleatt JW, Pilip I, Kerksiek K, Pamer EG. Intestinal and splenic T cell responses to enteric Listeria monocytogenes infection: distinct repertoires of responding CD8 T lymphocytes. J Immunol. 2001;166:4065–4073. doi: 10.4049/jimmunol.166.6.4065. [DOI] [PubMed] [Google Scholar]
  • 62.Kursar M, Bonhagen K, Köhler A, Kamradt T, Kaufmann SH, Mittrücker HW. Organ-specific CD4+ T cell response during Listeria monocytogenes infection. J Immunol. 2002;168:6382–6387. doi: 10.4049/jimmunol.168.12.6382. [DOI] [PubMed] [Google Scholar]
  • 63.Pope C, Kim SK, Marzo A, Masopust D, Williams K, Jiang J, Shen H, Lefrançois L. Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection. J Immunol. 2001;166:3402–3409. doi: 10.4049/jimmunol.166.5.3402. [DOI] [PubMed] [Google Scholar]
  • 64.Collins N, Belkaid Y. Control of immunity via nutritional interventions. Immunity. 2022;55:210–223. doi: 10.1016/j.immuni.2022.01.004. [DOI] [PubMed] [Google Scholar]
  • 65.Graef FA, Celiberto LS, Allaire JM, Kuan MT, Bosman ES, Crowley SM, Yang H, Chan JH, Stahl M, Yu H, et al. Fasting increases microbiome-based colonization resistance and reduces host inflammatory responses during an enteric bacterial infection. PLoS Pathog. 2021;17:e1009719. doi: 10.1371/journal.ppat.1009719. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure 1

Gating strategy for flow cytometry analysis of immune cells. Single cells were prepared and pre-gated for single live CD45+ cells, and then specifically further gated for each cell type as displayed, respectively.

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Supplementary Figure 2

Changes on the composition of CD45+ cells in mLN and SI LP from mice with short-term fasting. Mice were fasted for 24 h. Changes of neutrophils and macrophages (A), B cells and NK cells (B), and T cells (C) were examined. Statistical significance was examined by using unpaired Student’s t-test. The representative results from 2–3 independent experiments, n=3–6 mice.

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Supplementary Figure 3

Increase of the CD11chi DC subsets in mice with short-term fasting. Mice were fasted for 24 h. (A) BrdU uptake among CD45+F4/80-CD11chi DCs subsets were examined in mLN. (B) Expression of MHC II, CD86, CD205, and PD-L1 was examined in CD45+F4/80-CD11chiCD103+ DCs of SILP. Statistical significance was examined by using unpaired Student’s t-test. The representative results from 2–3 independent experiments, n=3–4 mice.

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Supplementary Figure 4

Bacterial burden in stomach and body weight change after short-term fasting in mice infected with LM. Mice were fasted for 24 h and then infected with LM. (A) CFU was measured in stomach at 3 hpi. (B) Body weight was monitored for 8 days. To note that the body weight of short-term fasting group was measured from day-1 because of the fasting. The statistics for body weight result was analyzed by log-rank (Mantel-Cox) test and all other statistical significance by unpaired Student’s t-test. The representative results from 2–3 independent experiments, n=5–6 mice.

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Supplementary Figure 5

Induction of splenic Foxp3+ Tregs in mice with short-term fasting followed by LM infection. Mice were fasted for 24 h and then infected with LM. (A, B) Foxp3+ Tregs were analyzed in spleen at 1 dpi and shown in (A) dot plot and (B) percentage among CD3+CD4+ cells. Statistical significance was examined by using unpaired Student’s t-test. The representative results from 2–3 independent experiments, n=3-4 mice.

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Supplementary Figure 6

Composition of IFN-γ+ cells among NK cells in short-term fasting mice infected with LM. Mice were fasted for 24 h and then infected with LM. The percentage and absolute number of IFN-γ+ cells among NK cells (NK1.1+CD3-NKp46+) in mLN at 1, 2 and 3 dpi. Statistical significance was examined by using unpaired Student’s t-test. The representative results from 2–3 independent experiments, n=4–6 mice.

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Supplementary Figure 7

Changes of neutrophils and macrophages in mice infected with LM. Mice were fasted for 24 h and then infected with LM. Percentage and absolute number of neutrophils and macrophages from SILP was examined at 3 dpi. Statistical significance was examined by using unpaired Student’s t-test. Results are representative of two or three independent experiments, n=4–6 mice.

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