Short-term fasting before living kidney donation has an immune-modulatory effect

Abstract

Background: Short-Term Fasting (STF) is an intervention reducing the intake of calories, without causing undernutrition or micronutrient-related malnutrition. It aims to systemically improve resilience against acute stress. Several (pre-)clinical studies have suggested protective effects of STF, marking the systemic effects STF can induce in respect to surgery and ischemia-reperfusion injury. In addition, STF also affects the number of circulating immune cells. We aim to determine the effect of STF on the abundance and phenotype of different immune cell populations.

Methods: Thirty participants were randomly selected from the FAST clinical trial, including living kidney donors, randomized to an STF-diet or control arm. In an observational cohort sub-study we prospectively included 30 patients who donated blood samples repeatedly during study runtime. Using flow cytometry analyses, immune cell phenotyping was performed on peripheral blood mononuclear cells. Three panels were designed to investigate the presence and activation status of peripheral T cells, B cells, dendritic cells (DCs) and myeloid cells.

Results: Eight participants were excluded due to sample constraints. Baseline characteristics showed no significant differences, except for fasting duration. Weight changes were minimal and non-significant across different time intervals, with slight trends toward long-term weight loss pre-surgery. Glucose, insulin, and β-hydroxybutyrate levels differed significantly between groups, reflecting adherence to the fasting diet. Flow cytometry and RNA sequencing analysis revealed no baseline differences between groups, with high variability within each group. STF changes the levels and phenotype of immune cells, reducing the abundance and activation of T cells, including regulatory T cells, increased presence of (naïve) B cells, and elevation of type 1 conventional DCs (cDC1s). In addition, a decrease in central memory T cells was observed.

Discussion: In this study, we observed significant changes due to fasting in B cells, T cells, and DCs, specifically toward less specialized lymphocytes, suggesting an arrest in B and T cell development. Further research should focus on the clinical implications of changes in immune cells and significance of these observed immunological changes.

Conclusion: STF results in reduced numbers and activation status of T cells and Tregs, increased presence of (naïve) B cells, and elevation of cDC1s.

Keywords: Short-Term Fasting; acute inflammatory response; caloric restriction; cell population analysis; immune response.

Figure 1

Patients from both treatment arms are highly comparable at start of treatment. (A) Schematic methodologic overview of the clinical procedures. The intervention group was instructed to follow a STF-diet, starting 60 hours before surgery. Participants drank ad libitum water, tea or coffee to maintain fluid balance, and to maintain electrolyte balance, they were allowed a max of 4 bouillon soups a day. After surgery, they resumed regular intake. (B) t-SNE plot that illustrates no sample segregation based on treatment among 88 FACS variables at Visit 1. (C) Volcano plot depicting the fold of change of 88 FACS variables between the two treatment groups at Visit 1. The x-axis represents the log2 fold change, whereas the y-axis represents -log10 P values. Variables on the right (positive) are higher in the intervention group, and those on the left (negative) are higher in the control group. (D, E) t-SNE plot that illustrates no sample segregation based on treatment among 88 FACS variables at Visit 2 (D) or Visit 3 (E).

Figure 2

Transcriptome analysis of blood cells from fasted versus control participants at Visit 2. (A) PCA plot showing no separation between the two treatment arms at Visit 2 but high variability among controls. (B) GeneSet Enrichment Analysis (GSEA) plots of Gene Ontology Biological Process (GO-BP) Regulation of Insulin Secretion Involved in Cellular Response to Glucose Stimulus. (C) Bubble plot summary of GSEA top 15 significant GO-BP Immune Pathways. Red dots have a positive normalized enrichment score (NES), meaning upregulated, and blue dots negative NES, thus downregulated. Size of dots indicates size of corresponding gene set.

Figure 3

Assessment of alterations in immune cell populations over time in the control group. (A) Volcano plot depicting the fold of change of 88 FACS variables between the Visit 1 and Visit 2 in the control arm. The x-axis represents the log2 fold change, whereas the y-axis represents -log10 P values. Variables on the right (positive) are increased at Visit 1, and those on the left (negative) are increased at Visit 2. (B-D) Line plots showing alterations in T cells (B), NK cells (C) and B cells (D). (E) Volcano plot depicting the fold of change of 88 FACS variables between the Visit 1 and Visit 3 in the control arm. The x-axis represents the log2 fold change, whereas the y-axis represents -log10 P values. Variables on the right (positive) are increased at Visit 1, and those on the left (negative) are increased at Visit 3. (F, G) Line plots showing alterations in B cells (F), and myeloid cells (G). Tcm, central memory T cells. * = p < 0.05, ** = p <0.01.

Figure 4

Alterations in immune cell populations over time and according to a STF-diet. (A) Volcano plot depicting the fold of change of 88 FACS variables between the Visit 1 and Visit 2 in the intervention group. The x-axis represents the log2 fold change, whereas the y-axis represents -log10 P values. Variables on the right (positive) are increased at Visit 1, and those on the left (negative) are increased at Visit 2. (B-D) Line plots showing alterations in T cells (B), B cells (C) and dendritic cells (D). (E) Volcano plot depicting the fold of change of 88 FACS variables between the Visit 1 and Visit 3 in the intervention group. The x-axis represents the log2 fold change, whereas the y-axis represents -log10 P values. Variables on the right (positive) are increased at Visit 1, and those on the left (negative) are increased at Visit 3. (F-I) Line plots showing alterations in B cells (F), dendritic cells (G), T cells (H) and regulatory T cells (I). (J-L) Bar plots showing differences in regulatory T cells (J), NK cells (K) and B cells (L) between the two treatment groups at visit 4 or visit 5, as indicated. Tregs, regulatory T cells; cDC, conventional dendritic cells; Tcm, central memory T cells. * = p < 0.05, ** = p <0.01.