Short-term Fasting Enhances the Protection Against Listeria Monocytogenes Infection Through Changes of Intestinal CD103+ Dendritic Cells

Gastrointestinal tract is the rst organ to be directly affected upon fasting. However, little is known about how the fasting inuences intestinal immune system. In the present study, we focused on the changes of intestinal dendritic cells (DCs) in mice upon short-term fasting and how the changes inuence protective immunity against Listeria monocytogenes (LM) infection. We found that the fasting induces an increased number of CD103 + CD11b - DCs in both small intestinal lamina propria (SI LP) and mesenteric lymph nodes (mLN) and the SI LP CD103 + CD11b - DCs undergo an active proliferation and migration by increased levels of GM-CSF and CCR7, respectively. At 24 hours post-infection (hpi) of LM, there was a signicant reduction of bacterial burden from the spleen, liver, and mLN of the short-term fasting mice compared to those of ad libitum mice. Accordingly, short-term fasting mice showed enhanced survival against LM infection when compared with ad libitum mice. Furthermore, signicantly high amount of TGF-β2 and Aldh1a2 expression from CD103 + CD11b - DCs in mouse infected with LM sequentially caused the following events: the increase of Foxp3 + Tregs, preferential change in the composition of CD103 + to CD103 - DCs, and the induction of IFN-γ producing cells. Collectively, increase of intestinal CD103 + DCs by short-term fasting is a key player for protection against LM infection through the changes of functional features from tolerogenic to Th1 immunogenic. -IL-17A-PerCP-Cy5.5 (TC11-18H10) (BD For Foxp3 intracellular staining, the incubated with anti-Foxp3 Alexa647 antibody (MF23) (BD Biosciences) for 20 min at RT in the After the staining, ow cytometric analysis was performed using FACS Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star, CA, USA). Cell sorting was performed using FACS Aria (BD Biosciences).


Introduction
Periodic fasting could extend lifespan of bacteria, yeast, worms and mice compared to ad libitum diet 1 . It has been suggested that intermittent fasting protects mice from various infectious and non-infectious diseases such as diabetes, cancers and neurodegeneration 2 . For instance, the mice which had been fasting for 24-72 h before L. monocytogenes infection reduced bacterial burden and prolonged host survival 3 . Furthermore, fasting appeared to have a positive impact on survival rate after the transplantation of kidney and liver, and the ischemia-reperfusion injury in mice 4 .
The nutritional depletion even for a short time period (i.e., 24 h) reduced total number of cells in the bone marrow and thymus 5 . As gastrointestinal tract is the rst organ to be directly affected after the fasting, proteins related to metabolism including glycolysis are decreased, and overall protein synthesis is reduced after the fasting for 24 h. Interestingly, however, proteins involved in cellular protection such as preservation of intestinal integrity were signi cantly increased at the same time 6 . And, it has been suggested that nutritional depletion could induce the changes in hormones as well as the function of immune cells 7 . For example, leptin can promote expansion of naïve T cells in an IL-2-dependent manner and switch toward more Th1 responses than Th2 responses 8,9 . Leptin also promotes the maturation of dendritic cells (DCs) through increasing co-stimulatory molecules, producing pro-in ammatory cytokines 10 , and the migration to in amed tissues 11 . CD103 + DCs in small intestinal lamina propria (SI LP) serve to capture antigens, including apoptotic epithelial cells 12 and bacterial antigens 13 , and migrate into mesenteric lymph node (mLN) in a CCR7-dependent manner 14 . Moreover, they together with TGF-β 15,16 and retinoic acid 17 induce regulatory T cell (Treg) differentiation. CD103 + DCs can be divided into several subtypes depending on their CD11b or CD8α expression. CD103 + CD11b − CD8α + DCs are known to be specialized for the cross-presentation of cell-associated antigens and priming of CD8 + T cells 18 . By contrast, CD103 + CD11b + DCs appear to induce immune tolerance at the steady-state, particularly in intestine; however, paradoxically, these cells were prone to induce Th1 response if exposed to in ammatory condition 19 . Likewise, a minor population of intestinal CD103 − CD11b + DCs appear to e ciently prime naïve CD4 + T cells and induce differentiation to either IL-17 or IFN-γ producing effector CD4 + T cells 20 .
The primary function of small intestine is to perform digestion and absorption of incoming nutrients, while upon the fasting, there are structural and functional changes with reduction of metabolic activities 21 . Although effects of fasting on the intestinal epithelial cells have been previously documented, its impact on the intestinal immune cells, DCs in particular, has not yet been fully understood. In this study, changes of CD103 + DCs in gut-associated lymphoid organs such as mLN and SI LP by short-term fasting, and immune context by these changes were investigated using a mouse model infected with L. monocytogenes.

Results
CD11c hi DCs were increased in mLN and SI LP of shortterm fasting mice Gastrointestinal tract consumes enormous amount of energy and, therefore, could be directly and greatly affected upon the fasting 6 but little is known about how it in uences the gastrointestinal immunity. Thus, we focused on intestinal immune cells under the short-term fasting condition. After 24 h (short-term) of fasting 5 , signi cantly increased number of CD45 + leukocytes in mLN and SI LP was observed from fasting mice when compared to those of ad libitum mice (Fig. 1A). Next, to investigate which cell types have been increased among CD45 + leukocytes after short-term fasting, various immune cell types including dendritic cells (DCs), neutrophils, macrophages, B cells, natural killer (NK) cells, and T cells were analyzed. Signi cant increase in a number of CD11c hi DCs in mLN as well as in SI LP were observed when compared to ad libitum group (Fig. 1B). On the other hand, neutrophils, macrophages, NK cells, and lymphocyte populations including B cells, CD4, CD8 and γδ T cells, were not signi cantly changed in both mLN and SI LP (Supplementary Fig. 1A-C).
Taken together, the results showed that short-term fasting leads to the signi cant increase of intestinal CD11c hi DCs, not of other major immune cells.
CD103 + DCs were dramatically increased in mLN and SI LP from short-term fasting mice It has been reported that intestinal CD11c hi DCs could be further categorized to several subsets based on the expression of CD103 and CD11b molecules 22 . The majority of CD11c hi DCs in small intestine expresses the integrin α E referred as CD103 paired with β 7 23 . Furthermore, intestinal CD103 + DCs play an important role in the maintenance of tolerance against food antigens or commensal bacteria. In addition, this immunological tolerance maintains intestinal homeostasis while suppressing unnecessary intestinal hyper-in ammation that may occur even in the normal individuals 24 . To investigate which subsets of DCs are increased by short-term fasting, we further examined the change of CD11c hi DCs based on its expression of CD103 and CD11b. Intriguingly, the results showed that the number of CD103 + CD11b − DCs was signi cantly increased in mLN from mice with short-term fasting ( Fig. 2A and B). Furthermore, CD103 + DCs, but not CD103 − DCs, in SI LP were signi cantly increased from mice with short-term fasting when compared with those of ad libitum ( Fig. 2C and D).
Collectively, these results indicate that increase of CD11c hi DCs in short-term fasting mice mainly arose from the increased number of CD103 + CD11b − DCs, but not from CD103 − DCs, in both mLN and SI LP.
CD103 + DCs were actively proliferated in SI LP by GM-CSF that increased after the short-term fasting We hypothesized that increased number of CD103 + DCs in the SI LP was caused by either active cell proliferation at local site or migration. To investigate whether CD103 + DCs were proliferating more upon short-term fasting than ad libitum, BrdU incoporation assay was perfomed and the proliferation of intestinal DCs was monitored. Interestingly, there was a signi cant increase of BrdU uptake in CD103 + DCs, both CD103 + CD11b − and CD103 + CD11b + subsets, in the SI LP of short-term fasting mice compared to those of ad libitum mice, which is consistent with increased cellularity of these subsets in the SI LP ( Fig. 3A). However, no signi cant increase was seen in any subpopulations of CD11 hi DCs in mLN (Supplementary Fig. 2A).
It is well known that GM-CSF is required for the development of DCs at steady-state as well as in ammatory situation 25 . GM-CSF also induces the development and expansion of conventional DCs 26 , and facilitates the recruitment of intestinal DCs 27 . Thus, to investigate whether the increasd number of CD103 + DCs in short-term fasting mice was affected by GM-CSF increasement, mRNA expression of GM-CSF in SI LP was examined. As expected, the expression of GM-CSF was signi cantly increased in the SI LP after the short-term fasting (Fig. 3B). In order to con rm the possibility of migration mentioned above along with proliferation, expression of the surface proteins on CD103 + DCs in SI LP. The results showed that signi cant increase of CCR7, MHC II, CD205, and PD-L1 on the intestinal CD103 + DCs from shortterm fasting mice compared to ad libitum ( Fig. 3C and Supplementary Fig. 2B).
These results suggest that the short-term fasting increases the number of DC subsets, largely CD103 + CD11b − and CD103 + CD11b + DCs in the SI LP, through augmenting cell expansion and migration due to the increase of GM-CSF and CCR7 expression, respectively.
Short-term fasting protects mice against L. monocytogenes infection We postulated that increase of CD103 + DCs by short-term fasting can affect intestinal immunity against pathogenic infection because CD103 + DCs were suggested to act as tolerogenic DCs 24 . In order to elucidate the role of CD103 + DCs increased at short-term fasting in infectious condition, the mice were infected with L. monocytogenes that is known to preferentially induce Th1 and Th17 responses 28 and bacterial burden was measured. The result showed that colony forming unit (CFU) was signi cantly decreased in spleen, mLN, and liver at 48 hours post-infection (hpi) in short-term fasting group (Fig. 4A).
Together with bacterial burden, to measure the degree of systemic bacteremia, CFU in serum was examined. With consistency, high bacteremia was observed from the sera of mice fed with ad libitum but not with a short-term fasting group (Fig. 4B). Furthermore, in order to rule out the possibility that shortterm fasting mice consumed the feed much faster after re-feeding during the infection than ad libitum mice which could in uence severeness of the invasion, CFU in the stomach at 3 hpi was examined and found no differences ( Supplementary Fig. 3A).
Next, we questioned whether the reduced bacterial burden in both tissues and serum from short-term fasting mice has a correlation of protection based on host survival. Interestingly, consistent with bacterial burden, the survival of mice which had been short-term fasting was increased compared to that of ad libitum mice (Fig. 4C). In the context with survival rate, body weight change of short-term fasting group in Lm-OVA infected group was comparable to that of PBS group ( Supplementary Fig. 3B).
Collectively, these data suggest that short-term fasting has bene cial effect on the protection against gastrointestinal L. monocytogenes infection. CD103 is a marker for identifying in vivo-activated Foxp3 + Tregs 29 . Thus, to know whether the increased Foxp3 + Tregs in short-term fasting mice are activated functionally, expression of CD103 on Foxp3 + Tregs was examined. The results showed that increase of in vivo-activated CD103 + Foxp3 + Tregs was observed within the group of increased Foxp3 + Tregs in short-term fasting mice compared to those of ad libitum mice (Fig. bottom of 5C and E). In contrast to mLN, in spleen, Foxp3 + Tregs were comparable in both groups with no difference in the composition of CD103 + Foxp3 + Tregs ( Supplementary Fig. 4). Consistent with the increased Foxp3 + Tregs, mRNA level of foxp3 was 3-fold higher in short-term fasting mice than in that of ad libitum mice at 1 dpi (Fig. 5F left). Contrary to foxp3 level, it was observed that the mRNA levels of t-bet were upregulated at 3 days after the infection (Fig. 5F right).
Collectively, it suggests that increased CD103 + CD11b − DCs in short-term fasting mice induced functional Foxp3 + Tregs upon L. monocytogenes infection, especially at early stage.
Increased level of TGF-β and RA contributed to CD103 + DC with the feature of tolerogenic DCs We suggested intestinal CD103 + DCs to promote increase of Foxp3 + Tregs upon L. monocytogenes infection in short-term fasting mice. Thus, to elucidate the factors to promote the increase of Foxp3 + Tregs, rst, changes of the surface molecules on CD103 + DCs were examined. It has been reported that increased PD-L1 and decreased CD86 and MHC class II are the phenotypic characteristics of tolerogenic DCs 30 , and CD205-expressing CD8α + DCs also can induce Foxp3 + Tregs through producing TGF-β 31 .
Consistent with the previous reports, at 1 dpi, signi cant increase of PD-L1, CD205 and CCR7 was observed on CD103 + DCs from short-term fasting mice compared to ad libitum, while there were no changes on CD86 and MHC II (Fig. 6A).
With same context, it has been reported that CD103 + DCs producing TGF-β, RA and aldehyde dehydrogenase A2, which is the key enzyme for converting vitamin A to RA, are able to induce Foxp3 + Tregs 15,16 . So, we analyzed the expression of those soluble factors in puri ed intestinal CD103 + DCs of ad libitum mice and short-term fasting mice. interestingly, the mRNA levels of TGF-β2 and Aldh1a2 from the puri ed intestinal CD103 + DCs were signi cantly increased in short-term fasting mice compared to those from ad libitum mice (Fig. 6B).
Taken together, the results suggested that increased TGF-β2 and Aldh1a2 could contribute to tolerogenic feature of CD103 + DCs together with signi cant increase of PD-L1 and CD205, and those tolerogenic features of CD103 + DCs would contribute to increase of Foxp3 + Tregs in short-term fasting mice during L. monocytogenes infection.
Short-term fasting induced up-regulation of Th1 response in mice infected with L. monocytogenes Even though the results so far suggested increase of CD103 + tolerogenic DCs together with Foxp3 + Tregs in short-term fasting mice could be involved in the host survival, we went step further to investigate how the bacterial burden was signi cantly reduced during later time point, after 48 hpi (Fig. 4). It has been well reported that CD103 − DCs preferentially induce differentiation of naïve CD4 + T cells to IFN-γ-producing Th1 cells 20 . Thus, we hypothesized that there should be likely the change of CD103 + to CD103 − DC subsets after 2 dpi in short-term fasting mice that may re ect to reducing bacterial load. So, we tried to look at changes of DC subsets at 3 dpi. Interestingly, the results showed that the percentage and absolute number of CD103 − CD11b + DCs are signi cantly increased in short-term fasting mice compared to ad libitum mice at 3 dpi while, reversely, the percentage and absolute number of CD103 + CD11b − DCs reduced signi cantly (Fig. 7A). We further investigated whether CD103 − DCs have helped forming Th1 environment as reported in the previous study 20 . Both percentage and absolute number of IFN-γ + cells among CD4 + CD3e + (Fig. 7B), CD8 + CD3e + cells (Fig. 7C), and NK1.1 + CD3e − (Supplementary Fig. 5A), were increased at 2 and 3 dpi in short-term fasting mice, in agreement with the increased level of t-bet expression at 3 dpi (Fig. 5F).
Collectively, in short-term fasting mice, increase of IFN-γ + cells at the later phase of infection is highly affected by increase of CD103 − DCs that contributes to the reduction of bacterial burden by enhancing T cell immune response.

Discussion
In the present study, functional alteration of intestinal immune cells, especially CD11c hi DCs, caused by short-term fasting with/without L. monocytogenes infection was investigated. Major ndings are as follow: (1)  CD103 + DCs, in the present study, were the most increased DC subset in mLN and SI LP upon fasting. The increase of CD103 + CD11b − DCs in SI LP was due to the active proliferation and migration of the cell. It should be noted that the rate of proliferation was much higher in SI LP than mLN, which is consistent with previous report 35 showing that mLN CD103 + DCs have slower kinetics of proliferation than SI LP CD103 + DCs. On the other hand, increased GM-CSF could in uence the rapid recruitment of CD11c hi DCs 27 . Concordant with this report, we also observed increased GM-CSF in SI LP from short-term fasting mice, suggesting that local increase of GM-CSF induced by fasting might affect the expansion and migration of intestinal CD11c hi CD103 + DCs.
In addition to the GM-CSF we have proposed, there are other factors that can affect changing the number of DCs. One of the best known is the t3 ligand, which plays an important role in the differentiation of hematopoietic stem cells into cDCs 36 . In addition, t3 ligand can maintain the normal number of cDCs by directly regulating their proliferation in the periphery 37 . Therefore, although the results in the present study suggested a role of GM-CSF for the proliferation of CD11c hi cells, it is seemingly necessary to conduct future study for investigating a direct effect of t3 ligand with/without GM-CSF on proliferation of CD11 hi DC subset.
It has been well reported that Foxp3 + Tregs not only prevent autoimmune diseases 38,39 , but also curb vigorous antimicrobial immune responses by restricting excessive in ammation 40,41 . In the present study, we are proposing a correlation between CD103 + DCs and Foxp3 + Tregs that short-term fasting is bene cial for the protection of the mice infected with L. monocytogenes. During the early stage after the infection, CD103 + CD11b − DCs and Foxp3 + Tregs are signi cantly increased in short-term fasting mice. In the previous report 15 , Foxp3 + Tregs induced by CD103 + DCs are the safeguard of the host from excessive immune responses after pathogen invasion. We have suggested that the increase of Foxp3 + Tregs are occurred by increase of TGF-β2 expression in short-term fasting mice. It was reported that TGF-β directly promotes expansion of the Foxp3 + Treg in vivo 42 . Therefore, it is likely that the increase of Foxp3 + Treg during the early stage of infection could be a TGF-β-dependent expansion. The possibility for the expansion of Foxp3 + Tregs in short-term fasting mice by increase of TGF-β-producing CD103 + DCs should be veri ed further by transferring Foxp3 + Tregs into TGF-β de cient or DC-speci c IRF8 de cient mice for other possibilities. Furthermore, our result clearly showed the higher expression of aldh1a2 in CD103 + DCs from short-term fasting mice, which might have caused increase of Foxp3 + Tregs as well. A study suggested that RA is mainly produced by intestinal DCs and epithelial cells, and inhibition of RA receptor reduced the induction of Foxp3 + Tregs 43 . Thus, these ndings suggest that tolerogenic conditions made by short-term fasting might restrain overwhelming immune response and protect host from tissue damage at early time point of infection.
The results in the present study showed that CCR7 and CD205 are also signi cantly increased from shortterm fasting mice infected with L. monocytogenes, together with PD-L1. As aforementioned, PD-L1expressing DCs acted as tolerogenic DCs inducing Foxp3 + Tregs 30,31 . So we thought it will be intriguing to investigate the roles of CCR7 and CD205 in PD-L1-expressing DCs from mice with short-term fasting. It has been well reported CCR7 is one of the major chemokine receptors to regulate migration of DCs from tissues to lymph nodes 44 . And CD205 is directly associated to antiten uptake, then enhances the antigen-presentation via both MHC class I and MHC class II pathways 45,46 . Collectively, the results in the present study suggested that CD103 + DCs in short-term fasting mice may have the roles as, by highly expressing CCR7, CD205, and PD-L1, the most e ciently migrating DCs to lymph node to provoke systemic immune responses as well as maintain immune tolerance.
Based on the previous works, CD103 −

Animals and short-term fasting
Female BALB/c mice, 6 weeks old, were purchased from Orient Bio Inc., Korea. Mice were divided into two groups, one fed ad libitum and the other fasted for 24 h with water provided. In order for the mice to avoid eating their own feces during starvation, the mice were transferred into new bedding cages when the fasting started. All experimental procedures were approved by Institutional Animal Care and Use Committee at Seoul National University (Approval number: SNU-130510-4-1), Korea. We con rmed that all animal experiments were carried out by adhering ARRIVE guidelines. All animal experiments were performed and carried out in accordance with relevant guidelines and regulations.

Bacteria preparation and infection
Recombinant Listeria monocytogenes expressing ovalbumin (Lm-OVA) and its parental 10403s strain were kindly provided by Dr. Hao Shen (University of Pennsylvania, Philadelphia, PA, USA). Bacteria, cultured with brain heart infusion media for 8 h at 140 rpm on a shaking incubator at 30 °C, were harvested by centrifugation and thoroughly washed twice with PBS. Bacteria count was estimated by measuring optical density at 600 nm as previously described 47

Determination of bacterial colony
To determine bacterial burden, spleen, liver and mLNs were taken after the perfusion using PBS. Each organ was homogenized with PBS with 0.1% Triton x-100. To examine the number of Listeria in the blood, at least 200 µl of blood was collected by eye-bleeding and centrifuged at 6300 x g for 10 min to separate serum and cells. Serial diluents were plated on brain heart infusion agar plate for 12 to 16 h at 37 °C incubator and CFU was examined.

Statistical analysis
The mean value ± standard deviation was determined for each group. For comparison of means between two groups, the data were analyzed using two-tailed paired student's t-test and considered statistically signi cant when p-value was less than 0.05. Otherwise, it was mentioning in the gure legend.       Tolerogenic characteristics of CD103+ DCs in mice with short-term fasting followed by L. monocytogenes infection. Mice were fasted for 24 h and then infected with L. monocytogenes. (A) Expression of CCR7, PD-L1, CD205, CD86, MHC II, and CD62L was examined in CD103+ DCs. (B) The mRNA expression of TGF-β1, TGF-β2 and aldehyde dehydrogenase 2 (aldh1a2) was measured in CD103+ DCs. Statistical signi cance by unpaired Student's t-test. The representative results from 2-3 independent experiments, n=4-5 mice. Figure 7