DPP4 inhibition curbs systemic inflammation

Abstract

Background: Systemic inflammation is a critical clinical condition regularly observed in the context of surgery-induced trauma or infection. Systemic inflammation induces an ubiquitous activation of the vasculature and vascular dysfunction related to organ damage and adverse outcomes. The dipeptidyl peptidase-4 (DPP4) modulates the receptor preferences and activity of a multitude of humoral substrates mediating the systemic inflammatory response. We here determined whether DPP4 inhibition is a means to beneficially modulate systemic inflammatory responses affecting vascular and organ integrity.

Methods: In cardiac surgery patients medicated with DPP4 inhibitors, we used a systems biology approach for in-depth characterization of the perioperative immune response and assessment of macro- and microvascular dynamics compared to control patients. In parallel, we mechanistically evaluated the efficacy of DPP4 inhibition on modulating immune responses, capillary leakage, vasoplegia and endothelial transcriptomic profiles in mice with severe systemic inflammation.

Results: Preoperative oral intake of the DPP4 inhibitor sitagliptin modulated innate and adaptive immune phenotypes and was associated with augmented microvascluar integrity, reduced vasoplegia and improved clinical parameters of capillary leakage in patients undergoing cardiac surgery. In mice, DPP4 inhibition curbed the inflammatory response to a polymicrobial sepsis resulting in a massive reduction in endothelial gene activation assoicated with preserved vascular barrier function, augmented vasopressor responses and organ integrity.

Conclusions: We conclude that DPP4 inhibition may be a safe and potent means to curb immune responses to surgery or infection, resulting in a preservation of vascular integrity that translates into organ protection and improved clinical outcomes.

Trial registration: https://www.

Clinicaltrials: gov ; Unique identifier: NCT05725798, study start: 2022-02-01.

Keywords: Capillary leakage; Cardiac surgery; DPP4 inhibitor; Inflammation; Vasculature.

Fig. 1

Perioperative DPP4 inhibition modulates the inflammatory response to surgery. A: Recruitment of n = 14 patients undergoing elective cardiac surgery with n = 7 subjected to preoperative intake of the DPP4 inhibitor (DPP4i) sitagliptin. Analysis of the cellular and humoral immune response by flow cytometry and ELISA techniques. Designed with BioRender.com. B: Anti-inflammatory alterations induced by DPP4 inhibitor treatment on main PBMC populations, their subsets and surface markers measured by FACS compared to untreated control patients are marked in green, pro-inflammatory alterations in red, alterations with unknown effects in purple. BioRender.com. C: bh-SNE representations obtained from concatenated samples (n = 7 vs. 7) within the CD4 + panel 18 h after surgery. Quantity of CD4 + CD45RO + cells 18 h after surgery (n = 7 patients/group; mean ± standard error of mean (SEM); Mann-Whitney-test). Quantity of T helper 1-cells (TH1-cells) (CCR4- CCR6-CD183 + CD4 + memory T-cells) and T helper 17-cells (TH17-cells) (CCR4 + CCR6 + CD183- CD4 + memory T-cells) 18 h after surgery (n = 7; mean ± SEM; TH1 unpaired t-test; TH17 Mann-Whitney-test). CD146 expression within the CD4 + clusters. D: bh-SNE representations obtained from concatenated samples within the CD8 + panel 18 h after surgery. Quantity of CD8 + effector memory (EM) T-cells (n = 7; mean ± SEM; unpaired t-test). CD27 expression within the CD8 + clusters. E: Quantity of naive B-cells 18 h after surgery (n = 7; mean ± SEM; unpaired t-test). F: bh-SNE representations obtained from concatenated samples (n = 7 vs. 7) within the monocyte adhesion- and migration-panel 18 h after surgery. VCAM-1-expression within the clusters of the monocyte adhesion- and migration-panel. G: bh-SNE representations obtained from concatenated samples (n = 7 vs. 7) within the pro-inflammatory monocyte-panel 18 h after surgery. H, I: Expression of CD40, ICAM-1 and PECAM-1 on monocytes (n = 7; mean ± SEM; unpaired t-test). For the bh-SNE representations, unsupervised clustering by PhenoGraph identified cell clusters, as indicated by numbers. The color of the cluster signifies the log2-fold changes of cluster abundance in up- (red) or downregulation (blue) of the DPP4 inhibitor group compared to the control group. Asterisks indicate differences vs. control group (*p < 0.05)

Fig. 2

DPP4 inhibition modulates infection-associated inflammatory responses in mice. A: Schematic illustration of mice subjected to systemic inflammation by induction of a polymicrobial sepsis by cecal ligation and puncture (CLP). Created with BioRender.com. B: White blood cell, lymphocyte, monocyte and platelet count in murine blood 18 h after the induction of sepsis by CLP following the injection of the DPP4 inhibitor sitagliptin or vehicle serving as control intervention (n = 7–11 mice/group; mean ± SEM; one-way ANOVA/Bonferroni). C: Quantitative summary of significant changes in cytokine and chemokine levels 18 h after sepsis induction following administration of DPP4 inhibitor sitagliptin (n = 3–8 mice/group; mean ± SEM; one-way ANOVA/Bonferroni; Interleukin (IL), interferon (IFN), granulocyte-macrophage colony-stimulating factor (GM-CSF)). Asterisks indicate differences vs. sham- operated mice (*p < 0.05, **p < 0.01 and ***p < 0.001). Hashtags indicate statistically significant differences vs. septic mice (#p < 0.05, ##p < 0.01 and ###p < 0.001)

Fig. 3

DPP4 inhibition reduces vascular leakage in systemic inflammation. A, B: Functional characteristics of the sublingual microcirculation assessed in patients with preoperative DPP4 inhibitor intake or without serving as controls. Arrows mark vessels with compromised or intact microcirculation. BioRender.com. C: Comparison of extent of edema analyzed by fluid area measurements between the groups immediately and on the first postoperative day (POD 1) (n = 7 patients/group; mean ± SEM; one-way ANOVA/Bonferroni). D: Total postoperative crystalloid volume requirements based on passive-leg-raise test (n = 7 patients/group; mean ± SEM; unpaired t-test). E: Comparison of the proportion of perfused vessels (PPV) between the groups immediately and on POD1 (n = 6–7 patients/group; mean ± SEM; unpaired t-test). F: Comparison of the microvascular flow index (MFI) immediately and on POD1 (n = 6–7 patients/group; mean ± SEM; unpaired t-test). G: Differences in blood concentrations of p-selectin and ICAM-1 18 h postoperatively (n = 7 patients/group; mean ± SEM; un- paired t-test). H: Pulmonary edema visualized by hematoxylin and eosin-stained micrographs of murine lungs 18 h after sepsis induction and injection of DPP4 inhibitor or vehicle. Quantitative summary of ventilated pulmonary area 18 h after sepsis induction following administration of DPP4 inhibitor sitagliptin directly and with 6 h time delay (n = 6–9 mice/group; mean ± SEM; one-way ANOVA/Bonferroni). I: Lung tissue edema of septic mice treated with DPP4 inhibitor or respective control, indicated as tissue wet/dry ratio 18 h after sepsis induction (n = 3–5 mice/group; mean ± SEM; one-way ANO-VA/Bonferroni). J: Representative photographs showing Evans blue content of murine intestines presenting albumin that extravasates during tissue edema formation. K: Tissue edema of murine intestines after sepsis induction and DPP4i, measured as tissue wet/dry ratio 18 h after sepsis induction (n = 5–7 mice/group; mean ± SEM; one-way ANOVA/Bonferroni). Scale bars represent 10 μm. Asterisks indicate differences vs. patient control group/sham-operated mice (*p < 0.05, **p < 0.01, ***p < 0.001 or p-value = as indicated). Hashtags indicate statistically significant differences vs. septic mice (###p < 0.001)

Fig. 4

DPP4 inhibition reduces vasoplegia and protects organ integrity. A: Time course curve of norepinephrine requirements [µg/kg/min] within the first 24 h after surgery (n = 7 patients/group; mean ± SEM; Mann-Whitney-test). Graphic was created with BioRender.com. B, C: Response of murine third-order mesenteric resistance arteries to 10nM phenylephrine hydrochloride 18 h after sepsis induction in wild type mice subjected to DPP4 inhibition or control treatment (left panel) and in dpp4-/- mice (n = 5 mice/group; mean ± SEM; one-way ANOVA/Bonferroni). Graphic was created with BioRender.com. D: Representative pictures showing hepatic tissue edema and damage 18 h after CLP/sham surgery following treatment with DPP4 inhibitor or control. Liver tissue edema of septic mice treated with DPP4 inhibitor or respective control indicated as tissue wet/dry ratio (n = 6 mice/group; mean ± SEM; one-way ANOVA/Bonferroni). Quantitative summary of hepatic tissue damage (n = 3–8 mice/group; mean ± SEM; one-way ANOVA/Bonferroni). E: Representative pictures and quantitative summary of tubular injury 18 h after CLP/sham surgery following injection of DPP4 inhibitor sitagliptin (n = 3–5 mice/group; mean ± SEM; one-way ANOVA/Bonferroni). Scale bars of histological micrographs represent 100 μm. Asterisks indicate differences vs. patient control group/sham-operated mice (*p < 0.05, **p < 0.01 and ***p < 0.001). Hashtags indicate differences vs. septic mice (#p < 0.05 and ###p < 0.001)

Fig. 5

DPP4 inhibition modulates endothelial cell genomic profiles during sepsis. A-D: Effects of DPP4 inhibition on human pulmonary microvascular endothelial cell monolayer permeability upon exposure to DPP4 substrate procalcitonin (PCT) or TNF-alpha assessed in a two-chamber assay by fluorescence intensity of 70 kDa fluorescein isothiocyanate-labeled dextran macromolecules in lower chambers. Values are calculated relative (timesfold) to the mean of the control value defined as 1.0 (n = 5–9 independent experiments/group, one-way ANOVA/Bonferroni). Asterisks indicate differences vs. control (***p < 0.001). Hashtags indicate differences vs. respective PCT treatment (###p < 0.001). Principal component analysis (PCA) of RNA sequencing of endothelial cells exposed to procalcitonin with or without sitagliptin as DPP4 inhibitor. E: Endothelial cells were isolated from murine lungs (n = 5–6 mice/group) 18 h after cecal ligation and puncture (CLP) or sham surgery following treatment with DPP4 inhibitor or control. Endothelial RNA was subjected to bulk RNA sequencing. Summary of the number of differentially expressed genes (DEGs) in murine endothelial cells. F: Heatmap of expression levels of DEGs from contrasts. Values were scaled by row. red: up-regulated DEGs, blue: down-regulated DEGs. Each column in the heatmap represents one mouse. G, H: Top up- and downregulated genes related to gene ontology in septic vs. non-septic mice. I, J: VENN diagram deciphering the number of DEGs from the same contrasts and scatterplot showing sitagliptin results in less regulation of genes induced by sepsis. K, L: Up- and downregulated genes in septic mice subjected to treatment with sitagliptin in contrast to septic mice. Graphics were created with BioRender.com