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Pharmacology & Therapeutics 209 (2020) 107503 Contents lists available at ScienceDirect Pharmaco...

Pharmacology & Therapeutics 209 (2020) 107503 Contents lists available at ScienceDirect Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera Dipeptidyl peptidase 4 inhibitors and their potential immune modulatory functions Shiying Shao a, QinQin Xu a, Xuefeng Yu a, Ruping Pan b, Yong Chen a,⁎ a Division of Endocrinology, Department of Internal Medicine, Tongji hospital, Tongji medical college, Huazhong University of Science & Technology, Wuhan 430030, PR China b Department of Nuclear Medicine, Tongji hospital, Tongji medical college, Huazhong University of Science & Technology, Wuhan 430030, PR China a r t i c l e i n f o a b s t r a c t Available online 14 February 2020 Dipeptidyl peptidase 4 (DPP4) inhibitors (DPP4is) are oral anti-diabetic drugs (OADs) for the treatment of type 2 diabetes mellitus (T2DM) through inhibiting the degradation of incretin peptides. Numerous investigations have been focused on the effects of DPP4is on glucose homeostasis. However, there are limited evidences demonstrat- Keywords: ing their Potential modulatory functions in the immune system. DPP4, originally known as the lymphocyte cell Dipeptidyl peptidase 4 surface protein CD26, is widely expressed in many types of immune cells including CD4(+) and CD8(+) T Cluster of differentiation 26 cells, B cells, NK cells, dendritic cells, and macrophages; and regulate the functions of these cells. In addition, Type 2 diabetes mellitus DPP4 is capable of modulating plenty of cytokines, chemokines and peptide hormones. Accordingly, DPP4/ Immunotherapy CD26 is speculated to be involved in various immune/inflammatory diseases and DPP4is may become a new drug class applied in these diseases. This review focuses on the regulatory effects of DPP4is on immune functions and their possible underlying mechanisms. Further clinical studies will be necessitated to fully evaluate the ad- ministration of DPP4is in diabetic patients with or without immune diseases. © 2020 Elsevier Inc. All rights reserved. Contents 1. Introduction................................................ 1 2. Effect of DPP4/CD26 on the immune system................................. 2 3. Effects and underlying mechanisms of DPP4/CD26 inhibition on immunotherapy................ 4 4. Outlook................................................. 11 Acknowledgments.............................................. 11 References.................................................. 11 1. Introduction Morimoto, 2008). Human DPP4/CD26 consists of a short 6 amino acid intracellular domain, a transmembrane region, and an extracel- Dipeptidyl peptidase 4 (DPP4) is a transmembrane glycoprotein lular domain which possesses dipeptidyl peptidase activity and selec- with a molecular mass of 220–240 kDa, originally known as T cell tively cleaves off the N-terminal dipeptides from peptides with surface marker cluster of differentiation 26 (CD26) (Kameoka, proline, alanine or, to a lesser extent, serine at the penultimate Tanaka, Nojima, Schlossman, & Morimoto, 1993; Ohnuma, Dang, & position. Abbreviations: AT, Adipose tissue; BP, Bullous pemphigoid; CVD, Cardiovascular disease; CCL, C-C motif chemokine ligand; CXCL, Chemokine (C-X-C motif) ligand; CPRD, Clinical Practice Research Datalink; CD26, Cluster of differentiation 26; CSF, Colony-stimulating factor; CD, Crohn's disease; CXCR4, CXC chemokine receptor 4; DC, Dendritic cell; DPP4, Dipeptidyl peptidase 4; DPP4is, DPP4 inhibitors; EPC, Endothelial progenitor cell; EPO, Erythropoietin; FGF2, Fibroblast growth factor 2; G-CSF, Granulocyte-CSF; GM-CSF, Granulocyte- macrophage-CSF; GLP-1, Glucagon like peptide-1; GIP, Glucose dependent insulinotropic peptide; HCC, Hepatocellular carcinoma; HFD, High fat diet; IBD, Inflammatory bowel disease; IFN-γ, Interferon gamma; IL, Interleukin; LADA, Latent autoimmune diabetes in adults; MI, Myocardial infarction; NK, Natural killer; NOD, Non-obese diabetic; OADs, Oral antidiabetic drugs; PCa, Prostate cancer; Treg, Regulatory T; RA, Rheumatoid arthritis; RASF, RA synovial fibroblast; SGLT2, Sodium glucose cotransporter 2; SGLT2i, SGLT2 inhibitor; sDPP4, Soluble DPP4; STZ, Streptozotocin; SDF-1, Stromal cell-derived factor-1; Th, T helper; TLR2, Toll-like receptor 2; TGF-β, Transforming growth factor-β; TNF-α, Tumor necrosis factor-α; T2DM, Type 2 diabetes mellitus; UC, Ulcerative colitis. ⁎ Corresponding author at: Division of Endocrinology, Tongji Hospital, Huazhong University of Science & Technology, Jiefang Road 1095, Wuhan, Hubei Province 430030, PR China. E-mail address: [email protected] (Y. Chen). https://doi.org/10.1016/j.pharmthera.2020.107503 0163-7258/© 2020 Elsevier Inc. All rights reserved. 2 S. Shao et al. / Pharmacology & Therapeutics 209 (2020) 107503 DPP4/CD26 is widely expressed in different organs, including kid- 2.1. CD4(+) and CD8(+) T cells ney, gastrointestinal tract, liver and bone marrow, as well as on the sur- face of various cell types such as stromal, stem, epithelial, endothelial, It has been previously demonstrated that higher expression of CD26 and immune cells (Thul et al., 2017; Waumans, Baerts, Kehoe, on cell surface of CD4(+) T cells is correlated with their T helper type 1 Lambeir, & De Meester, 2015). Moreover, it also exists as a soluble (Th1)-like phenotype, whereas the lower expression of CD26 is associ- form (sDPP4/CD26) in body fluids (Varin et al., 2019), which is com- ated with their Th2-like phenotype (Willheim et al., 1997). Further- posed of the extracellular amino acids, and shows significant DPP4 ac- more, high CD26 surface expression is also correlated with the tivity (Casrouge et al., 2018; Durinx et al., 2000). production of Th1-type cytokines (Reinhold et al., 1997; Willheim It is known that the gut-derived incretins glucagon like peptide-1 et al., 1997). (GLP-1) and glucose dependent insulinotropic peptide (GIP) are es- DCs are the most potent antigen-presenting cells (APC) specialized sential for preservation of glucose homeostasis (Ahren & Hughes, in the initiation of immune responses by directing the activation and 2005; Baggio & Drucker, 2007), while DPP4 rapidly degrades circulat- differentiation of naïve T lymphocytes. Pacheco et al. reported that, in ing GLP-1 and GIP. Over a decade, DPP4 inhibitors (DPP4is), which a deaminase activity-independent way, ADA interacts with CD26 on are commonly called gliptins, work as valuable oral antidiabetic the T cell surface. The complex subsequently anchors to DC surface by drugs (OADs) in the treatment of type 2 diabetes mellitus (T2DM) an ADA-anchoring protein and then triggers costimulation, leading to through extending the half-life of native incretins (Marguet et al., an augmented T-cell activation with a Th1 pattern and a marked in- 2000; Mari et al., 2005). After the first DPP4i, sitagliptin, has been ap- crease in the production of Th1 proimmflamatory cytokines including proved by the Food and Drug Administration (FDA) for adults with interferon gamma (IFN-γ), tumor necrosis factor (TNF)-α, and interleu- T2DM in 2006, numerous gliptins, are generated, including kin (IL)-6 (Pacheco et al., 2005). On the contrast, CD26 knockout in mice saxagliptin, alogliptin, vildagliptin, linagliptin, teneligliptin, leads to a reduction in Th1 immune responses (Preller et al., 2007) but trelagliptin, and omarigliptin. an up-regulation of Th2-type cytokines, such as IL-4, IL-5, and IL-13 Investigations from over the past 2 decades have demonstrated (Yan, Gessner, Dietel, Schmiedek, & Fan, 2012). These findings further that DPP4 may have broad biological functions beyond glucose me- confirm the importance of CD26 in the proliferation and function of tabolism as evidenced by its various substrates and widespread ex- Th1 and Th2 cells. pression. Specifically, DPP4 cleaves a large number of cytokines, IL-17-producing CD4(+) T cells (Th17 cells), the proliferation of chemokines and peptide hormones involved in the regulation of im- which is regulated by IL-6, IL-23, and transforming growth factor mune system (Broxmeyer et al., 2012; Proost et al., 1999). It is also (TGF)-β, play an important role in the pathogenesis of autoimmune in- capable of modulating lymphocyte function in many aspects, espe- flammation; while regulatory T (Treg) lymphocytes exert their immu- cially in T cell activation and signal transduction. Accordingly, it is as- nosuppressive effect by means of direct cell-to-cell interaction or sumed that DPP4is have immune regulatory functions and potential through secretion of cytokines such as TGF-β and IL-10 (Shao, Yu, & therapeutic uses in the treatment of autoimmune and inflammatory Shen, 2018). diseases. It has been found that expression of enzymatically active CD26/DPP4 However, there are limited studies and clinical evidences demon- is higher in human Th17 cells than in Th1, Th2, or Tregs cells (Bengsch strating their immune modulatory functions and precise mechanism et al., 2012). Phenotypic analysis has revealed that CD26(++)CD4(+) underlying this process. This review focuses on these functions of T cells contain more than 90% of Th17 cells (Bengsch et al., 2012). DPP4/CD26 and its inhibitors, which may provide some new clues for CD26 knockout results in a significant reduction of the Th17 cytokines the clinical application of gliptins. such as IL-17 and IL-21 in mice with lung transplantation (Yamada et al., 2016). The author assumed that the modulation of Th17 cells by CD26 is via the costimulatory effects on signaling lymphocytic activa- 2. Effect of DPP4/CD26 on the immune system tion (Yamada et al., 2016). However, further investigation on down- stream signaling was not performed. IL-6 is important for T cell CD26 may exert its effect on the immune system through two mech- proliferation and Th17 cells show an IL6-dependent increase (Wang anisms. On the one hand, its enzymatic function results in a decomposi- et al., 2018; Winer et al., 2009). We have described that CD26 is corre- tion of targeted substrates into inactive and active fragments and exerts lated with augmented Th1 activation along with a marked increase of a direct effect on the immune response. On the other hand, CD26 acts as Th1 cytokines including IL-6. Pinheiro et al. found that sitagliptin treat- a potent costimulatory molecule in the process of T cell signal transduc- ment could completely abolish IL-6 expression by peripheral blood tion (Ohnuma et al., 2008). mononuclear cells (PBMCs) with the stimulation of phytohemaggluti- DPP4 exerts these non-catalytic functions via binding with caveolin- nin (PHA) from healthy volunteers and inhibit Th1/Th17 differentiation 1, fibronectin, adenosine deaminase (ADA), and CXC chemokine recep- in vitro (Pinheiro et al., 2017). Thus, we assume that IL-6 may also be in- tor 4 (CXCR4) (Cheng, Abdel-Ghany, & Pauli, 2003; Herrera et al., 2001; volved in CD26-mediated Th17 proliferation. Ohnuma et al., 2007; Ohnuma et al., 2008). The best-known signal On the contrary, CD26 expression is lower on the membranes of Treg transduction is the interaction between DPP-4 and ADA. The ADA is an cells (Mandapathil et al., 2010), which has been characterized as a neg- enzyme that catalyzes the hydrolytic deamination of adenosine to ino- ative selection marker for human Tregs (Garcia Santana, Tung, & Gulnik, sine (Martin, Huguet, Centelles, & Franco, 1995). It has long been 2014). Based on CD39/CD26 marker, analysis of human natural regula- known that high concentrations of adenosine inhibit the proliferation tory T cells (nTregs) has identified five different cell subsets, of T lymphocytes (Green & Chan, 1973). Only the ADA bound to CD26 representing a distinct stage of maturation respectively (Schiavon is functional and could counteract the inhihitory effect of extracellular et al., 2019). In addition, DPP4i significantly increases the Treg expan- adenosine. Accordingly, CD26/ADA/adenosine pathway is considered sion in non-obese diabetic (NOD) mice (Tian et al., 2010), indicating as an essential model in T-cell activation. Beyond that, the CD4(+) T that DPP4/CD26 inhibition may be responsible for the proliferation of cells, capable of transendothelial migration in vitro, appear to highly ex- Treg lymphocytes. Martinez-Navio identified that ADA promotes an press CD26, indicating a potential function of CD26 in T cell migration augmented generation of CD4(+)CD25(high)Foxp3(+) Tregs by a (Brezinschek, Lipsky, Galea, Vita, & Oppenheimer-Marks, 1995). mechanism that seems to be mainly dependent on its enzymatic activity Besides T lymphocytes, CD26 is identified to be expressed in B cells, (Martinez-Navio et al., 2011). Accordingly, CD26/ADA/adenosine is con- natural killer (NK) cells, dendritic cells (DCs) and macrophages as well sidered to exert an important regulatory role in the generation of Tregs. (Table 1). However, the immune regulatory function of DPP4/CD26 in It has been previously demonstrated that both CD8(+)CD26(high) these cells remains poorly characterized. and CD8(+)CD26(−) cells represent activated cell phenotypes and  S. Shao et al. / Pharmacology & Therapeutics 209 (2020) 107503 3 Table 1 Expression and function of DPP4/CD26 on immune cells. Immune Expression Function Reference cells Th1 cell High expression CD26 is correlated with the production of Th1 (Preller et al., 2007; Reinhold et al., 1997; Willheim et al., 1997) cytokines Th2 cell Low expression DPP4 inhibition leads to up-regulation of Th2 (Willheim et al., 1997; Yan et al., 2012) cytokines Th17 cell Highest CD26 expression in CD4 CD26 is correlated with the production of Th17 (Bengsch et al., 2012; Yamada et al., 2016) (+) T cell cytokines Treg cell Negative/low expression DPP4 inhibition leads to up-regulation of Tregs (Garcia Santana et al., 2014; Mandapathil et al., 2010; Schiavon et al., 2019; Tian et al., 2010) CD8(+) T High/negative expression CD26 mediates co-stimulation of CD8(+) T cells (Hatano et al., 2013; Ibegbu et al., 2009) cell B cell Low expression CD26 is correlated with B cell activation (Buhling et al., 1995; Klemann et al., 2009; Morimoto et al., 1989; Yan et al., 2003) NK cell Low expression CD26 is correlated with the proliferation and (Buhling et al., 1994; Shingu et al., 2003; Yan et al., 2003) cytotoxicity of NK cells DC Positive expression CD26 together with ADA stimulates T-cell (Gliddon & Howard, 2002; Zhong et al., 2013) proliferation Macrophage Positive expression DPP4 regulates M1/M2 macrophage polarization (Shah et al., 2011; Zhuge et al., 2016) Note: ADA, adenosine deaminase; DC, dendritic cell; DPP4, Dipeptidyl peptidase 4; NK cell, natural killer cell; Th1 cell, T helper type 1 cell; Th2 cell, T helper type 2 cell; Th17 cell, IL-17- producing cell; Treg cell, regulatory T cell. may offer a characteristic marker of successful memory development and cell cycle progression, thus concluding that CD26 is involved in (Hatano, Ohnuma, Yamamoto, Dang, & Morimoto, 2013; Ibegbu et al., the modulation of NK cell proliferation. DPP4/CD26 may also be in- 2009). CD28 is one of the molecules expressed in T cells, which provides volved in the natural cytotoxicity of NK cells, which is verified by the ob- co-stimulatory signals for T cell activation (Gamble et al., 2018). Inter- servation that NK cell cytotoxicity against tumour (MADB106) cells is estingly, CD26-mediated co-stimulation of CD8(+) T cells results in diminished in a DPP4/CD26-deficient F344 rat sub-strain, a model of greater cytotoxic effect than that induced by CD28 co-stimulation path- lung metastasis (Shingu et al., 2003). One of the underlying mechanisms way (Hatano et al., 2013). The modulation of CD26 on CD8(+) T lym- is that tumor target cells can directly adhere to NK cells via CD26. In ad- phocytes may use costimulatory transduction mediated by early dition, NK cells can exert their cytotoxicity via secretory lysosomes. In- growth response 2 (EGR2) and IL-10 (Hatano et al., 2015; Pinheiro terestingly, DPP4/CD26 has been found on the membrane of secretory et al., 2017). lysosomes in NK cells by mean of proteomic analysis (Casey, Meade, & Hewitt, 2007). Whether CD26 on secretory lysosomes could also medi- 2.2. B cells, and NK cells ate NK cell cytotoxicity is remained to be identified. In addition to its effects on CD4(+) and CD8(+) T cells, CD26 also 2.3. Dcs and macrophages contributes to the modulation of maturation, proliferation, and immu- noglobulin isotype switching of B cells (Buhling et al., 1995; Morimoto The first description of DPP4/CD26 expression on DCs was reported et al., 1989). Less than 5% of freshly isolated CD20(+) B cells express by Gliddon and colleagues from bovine afferent lymph and lymph nodes the CD26 antigen in the peripheral blood of healthy donors and com- (Gliddon & Howard, 2002). Both obese humans and rodents demon- mon variable immunodeficiency (CVID) patients. Upon activation by strated increased DPP4/CD26 expression on DCs from visceral adipose pokeweed mitogen, the fraction of CD26(+) human B cells can increase tissue (AT) (Zhong et al., 2013). It is considered that CD26 expression to around 50% (Buhling et al., 1995), suggesting an involvement of CD26 by DC confers the ability to modify macrophage-derived chemokine in B-cell activation. Specific suppression of DPP4 activity reduces the B (MDC) so that it may not stimulate Th2 polarized cells but attract and cell activation in a dose-dependent manner (Buhling et al., 1995). induce responses in Th1 polarized cells (Gliddon & Howard, 2002). Fur- DPP4 inhibitor could suppress the enzymatic activity and thereby trig- thermore, the CD26-ADA complex on DCs is capable of stimulating T cell ger certain signal transduction pathways leading to a decrease in DNA proliferation via adenosine degradation in mixed lymphocyte reaction synthesis of B cells (Buhling et al., 1995). Howbeit, the authors failed (MLR) assays using T-cell/DC cocultures (Zhong et al., 2013). These find- to identified the detailed molecular mechanisms. ings disclose a novel mechanism for the paracrine regulation of inflam- One of the most important functions of B lymphocytes is the secre- mation in AT by DPP4. tion of immunoglobulins (Igs). A significant change of Ig secretion by DPP4/CD26 expression on macrophages from visceral AT was also B cells in CD26-deficient mice was identified, which may be triggered identified in both high fat diet (HFD)-induced (C57BL/6) and genetically by a change of different cytokines including IFN-γ, IL-4, and IL-2, (Yan, obese (ob/ob) mice (Zhong et al., 2013). Long-term DPP4 inhibition by Marguet, Dobers, Reutter, & Fan, 2003); while no difference of Ig secre- alogliptin decreases visceral AT macrophage content (CD11b(+), tion was observed in another study (Vora et al., 2009). As published by CD11c(+), Ly6C(hi)) in a ApoE (−/−) mouse model (Shah et al., Morimoto, CD26 was found to be expressed on the CD4 memory/helper 2011). Similarly, Ikedo et al. identified that alogliptin inhibits the accu- (CD45RO + CD29+) population, which can respond to recall antigens mulation of macrophages in intracranial aneurysms in SD rats and and induce B-cell IgG synthesis (Morimoto et al., 1989). Buhling et al. they verified that such effect is independent of GLP-1 (Ikedo et al., measured the IgM concentrations from highly purified B cells with dif- 2017). Silencing expression of DPP4 by shRNA targeting hepatocytes ferent inhibitor concentrations and found a dose-dependent decrease in HFD induced obese mice also suppresses macrophage inflammation of IgM secretion (Buhling et al., 1995). Based on these findings, CD26 of visceral AT (Ghorpade et al., 2018). Furthermore, Zhong and col- mediated B-cell Ig secretion may either through T cell-dependent B- league reported that macrophage-expressing DPP4 binds ADA, resulting cell responses or direct effects on B cells. in T-cell proliferation via modulation of adenosine concentrations and Buhling et al. showed that freshly isolated human NK cells express then the development of adipose inflammation (Zhong et al., 2013). only low amounts of CD26 (Buhling et al., 1994). Furthermore, the au- All these findings disclose the significant role of DPP4/CD26 on macro- thors identified that specific DPP4 inhibitors suppress DNA synthesis phage proliferation and function. 4 S. Shao et al. / Pharmacology & Therapeutics 209 (2020) 107503 Additionally, it is found that DPP4/CD26 is predominantly expressed Table 2 in M1-polarized macrophages in white AT of HFD induced obese mice Known/potential factors truncated/regulated by DPP4/CD26. (Zhuge et al., 2016), indicating its effect on the regulation of M1/M2 Substrate Reference Macrophage Polarization. Macrophage inflammatory protein-1α (MIP- Chemokines 1α), also known as C-C motif chemokine ligand (CCL) 3, is a chemokine CCL2 (MCP-1) (Qin et al., 2018) and DPP4 substrate. It has been reported that cleavage by DPP4 converts CCL3 (MIP-1α) (Zhuge et al., 2016) MIP-1α into a most efficient chemoattractant (Proost et al., 2000). CCL5 (RANTES) (Oravecz et al., 1997) CCL11 (Eotaxin) (Hollande et al., 2019) DPP4i linagliptin fails to induce M2 macrophage polarization and CCL22 (MDC) (Gliddon & Howard, 2002; Proost et al., 1999) exert insulin-sensitizing effects in MIP-1α−/− mice (Zhuge et al., CXCL10 (IP-10) (Barreira da Silva et al., 2015; Proost et al., 2001) 2016), suggesting that DPP4 regulates M1/M2 polarization may be me- CXCL11 (I-TAC) (Proost et al., 2001) diated by MIP-1α. CXCL12 (SDF-1) (De La Luz Sierra et al., 2004; Janssens et al., 2017) Mig (Proost et al., 2001) 2.4. Cytokines, chemokines, and peptide hormones Cytokines IL-3 (Broxmeyer et al., 2012; O'Leary et al., 2017) GM-CSF (Broxmeyer et al., 2012) Although DPP4 is responsible for proteolytic cleavage of a wide G-CSF (Broxmeyer et al., 2012) range of substrates, most of DPP4-related researches have been focused Erythropoietin (Broxmeyer et al., 2012) on the incretin hormones GLP-1 or GIP for T2DM treatment (Ahren & FGF2 (Wesley et al., 2005) Hughes, 2005; Baggio & Drucker, 2007; Unniappan et al., 2006). In addi- IL-1⁎ (Ou et al., 2013) IL-2⁎ (Ou et al., 2013) tion to incretins, cytokines (Broxmeyer et al., 2012; O'Leary et al., 2017; IL-5⁎ (Ou et al., 2013) Wesley, McGroarty, & Homoyouni, 2005), chemokines (Barreira da Silva IL-6⁎ (Ou et al., 2013) et al., 2015; De La Luz Sierra et al., 2004; Hollande et al., 2019; Janssens IL-8⁎ (Ou et al., 2013) et al., 2017; Oravecz et al., 1997; Proost et al., 1999; Proost et al., 2001; IL-10⁎ (Ou et al., 2013) Qin et al., 2018), and some neuropeptides (Frerker et al., 2007; Guieu TGF-β# (Steinbrecher et al., 2001) IFNγ# (De Meester et al., 1994; Kameoka et al., 1993) et al., 2006) have been identified as its substrates (Table 2), thereby, TNF-α# (De Meester et al., 1994; Kameoka et al., 1993) allowing DPP4 to regulate immune responses. Plenty of chemokines and cytokines have been recognized as DPP4 Incretin hormones Gastric inhibitory peptide (Ahren & Hughes, 2005) targets. Among them, chemokine (C-X-C motif) ligand 12 (CXCL12, (GIP) also known as stromal cell-derived factor-1, SDF-1) is the most popular Gastrin-releasing peptide (Ahren & Hughes, 2005) one. It is a chemokine which can attract various progenitor cells, stem (GRP) cells, leukocytes, neurons, angioblast/endothelial cells, and tumor GLP-1 (Baggio & Drucker, 2007) GLP-2 (Baggio & Drucker, 2007) cells. Thus it is involved in plenty of processes such as angiogenesis, he- Peptide YY(1–36) (Unniappan et al., 2006) matopoiesis, and tissue repair (e.g., myocardial infarction (MI) and ischeamic stroke) (Bromage, Davidson, & Yellon, 2014; Kubota et al., Neuropeptides NPY (Frerker et al., 2007) 2016; Yang et al., 2018; Zhang et al., 2017). CXCL12/SDF-1 acts through Substance P (Guieu et al., 2006) the G protein-coupled CXCR4. Proteolytic cleavage of CXCL12 by DPP4 generates CXCL12(3–68) and results in a reduced CXCR4 affinity and a CCL, C-C motif chemokine ligand; CXCL, Chemokine (C-X-C motif) ligand; FGF2, fibroblast growth factor 2; G-CSF, Granulocyte-CSF; GIP, Gastric inhibitory peptide; GLP, hormones loss of its calcium-dependent signaling and chemotactic properties glucagon like peptide; GM-CSF, Granulocyte-macrophage-colony-stimulating factor; (De La Luz Sierra et al., 2004; Janssens et al., 2017). GRP, Gastrin-releasing peptid; IL, interleukin; IFNγ, interferon gamma; IP-10, IFN- In addition, DPP4 plays a more general role in regulating the ac- gamma-inducible protein-10; I-TAC, IFN-inducible T-cell alpha-chemoattractant; MCP-1, tivities of cytokines. DPP4 has been identified to truncate cytokines monocyte chemotactic protein 1; MDC, macrophage-derived chemokine; Mig, monokine induced by IFN-gamma; MIP-1α, Macrophage inflammatory protein-1α; NPY, such as fibroblast growth factor 2 (FGF2), IL-3, granulocyte- neuropeptideY; SDF-1, stromal cell-derived factor-1; RANTES, regulated on activation in macrophage (GM)-colony-stimulating factor (CSF), granulocyte (G)- normal T-cell expressed and secreted; TGF-β, transforming growth factor-β; TNF-α, CSF and erythropoietin (EPO), thus, resultantly decrease their activity tumor necrosis factor-α. and function (Broxmeyer et al., 2012; O'Leary et al., 2017; Wesley ⁎ Potential truncation by DPP4/CD26. # et al., 2005). Besides, there are a plenty of cytokines with the poten- Regulation rather than truncation by DPP4/CD26. tial truncation site of DPP4 including IL-1, −2, −5, −6, −8, and − 10 (Ou, O'Leary, & Broxmeyer, 2013). However, whether 3. Effects and underlying mechanisms of DPP4/CD26 inhibition on these cytokines have true DPP4 truncation sites needs to be specifi- immunotherapy cally determined via mass spectrometry and comparable analysis. Bi- ological assays in vitro and in vivo will be necessary to determine if DPP4is have been linked to the diseases as summarized in Table 3, the functional activity of truncated molecules differs from the full- including but not limited to autoimmune diabetes, inflammatory length form of the protein. bowel disease (IBD), rheumatoid arthritis (RA), allograft rejection, Furthermore, some cytokines regulated by DPP4 may be via compli- tumor, cardiovascular disease (CVD), asthma, infectious diseases and cated interactions between factors/immune cells rather than direct bullous pemphigoid (BP). However, the effect of DPP4is on these dis- truncation. For example, ADA-DPP4 interaction acts as co-stimulatory eases is controversial. signals during T cell receptor signaling, resulting in enhanced secretion of IFNγ and TNF-α (De Meester et al., 1994; Kameoka et al., 1993). Like- 3.1. DPP4/CD26 inhibition on autoimmune diseases wise, Steinbrecher et al. suggested that DPP4 suppression may stimulate TGF-β secretion from activated T cells, hence, have an anti- 3.1.1. DPP4/CD26 inhibition on autoimmune diabetes inflammatory effect in a mice model of experimental autoimmune en- Type 1 diabetes mellitus (T1DM) is an autoimmune disorder charac- cephalomyelitis (EAE) (Steinbrecher et al., 2001). terized by the destruction of pancreatic β cells and inadequate insulin Taken together, these cytokines, chemokines, and peptide hormones secretion (Roep & Tree, 2014). Under this circumstance, even slight mentioned in Table 2 are not meant to cover all identified factors but preservation of residual β cell mass may result in significant benefits rather to describe that there are a wide variety of biologically molecules in clinic (Palmer et al., 2004). Unfortunately, there are no immunomod- that may be truncated or regulated by DPP4/CD26. ulatory drugs that have been reported to induce/assist the disease  S. Shao et al. / Pharmacology & Therapeutics 209 (2020) 107503 5 Table 3 remission. Currently, DPP4is are widely applied as valuable mono- or Effect of DPP4 inhibition in autoimmune/inflammatory diseases. combination therapeutic OADs for T2DM. However, only a portion of Effect of DPP4 inhibition Reference clinical studies have indicated potential efficacy regarding glycemic Autoimmune HbA1c reduction in T1DM (Ellis et al., 2011; Farngren et al., control and β cell preservation in T1DM. diabetes patients 2012) Autoimmune diabetes is characterized by an increased Th1 immune No treatment efficacy in (Garg et al., 2013; Hari Kumar response and a decreased Th2 response. In addition, the imbalance of T1DM/LADA patients et al., 2013; Zhao et al., 2014) Th17/Treg is also involved in the pathogenic process (Shao et al., β cell preservation in LADA (Johansen et al., 2014; Zhao 2012). It has been described in Table 1 that DPP4 inhibition down- patients et al., 2014) No effect of β cell preservation in (Griffin et al., 2014; Hari Kumar regulates Th1-like phenotype, up-regulates Th2-type cytokines, stimu- T1DM/LADA patients et al., 2013) lates the proliferation of Tregs, and decreases IL-17 production. Thus, β cell preservation in animal (Conarello et al., 2003; Jelsing DPP4is are supposed to have the potential to treat autoimmune diabetes. model et al., 2012; Kim et al., 2012; In preclinical trials, inhibition of DPP4 activity has been shown to en- Kim et al., 2008; Pospisilik et al., 2003; Suarez-Pinzon et al., hance islet neogenesis and β cell survival, and reverse/delay the onset 2009; Takeda et al., 2012; Tian of diabetes in either streptozotocin (STZ)-induced animal model of et al., 2010) T1DM (Conarello et al., 2003; Pospisilik et al., 2003; Takeda et al., IBD Increased risk in T2DM patients (Abrahami et al., 2018; Kridin 2012) or NOD mouse model of autoimmune diabetes (Jelsing, Vrang, et al., 2018; Wang et al., 2019) van Witteloostuijn, Mark, & Klein, 2012; Tian et al., 2010). A combination Improved colitis in animal model (Ban et al., 2011; Salaga et al., 2017; Salaga et al., 2018) therapy of DPP4i and proton pump inhibitor (PPI) increases circulating RA Decreased risk in T2DM patients (Kim et al., 2015; Seong et al., levels of GLP-1 and gastrin in acutely diabetic NOD mice, resulting in 2019) the restoration of pancreatic insulin content, insulin secretion and No association in T2DM patients (Douros et al., 2018) normoglycaemia (Suarez-Pinzon, Cembrowski, & Rabinovitch, 2009). Increased disease severity in (Busso et al., 2005; Ospelt et al., Treatment of STZ-induced T1DM mice with DPP4i MK0431 before and animal model 2010) Allograft Rejection suppression in animal (Jung et al., 2006; Kim et al., after islet transplantation prolongs the survival of islet grafts (Kim rejection models of islet, lung, skin 2009; Zhai et al., 2007; Zhao et al., 2008). Differently, Kim et al. reported that β cell mass is enhanced transplantation et al., 2019) in NOD mice by the combination of Toll-like receptor 2 (TLR2) agonist Cancer No association in T2DM patients (Barnett et al., 2012; Green and DPP4i, but not by DPP4i or TLR2 agonist alone (Kim et al., 2012). et al., 2015; Leiter, et al., 2015; Mita, et al., 2016) Most of the previous animal studies considered that β cell preserva- Tumor suppression in vitro/in (Barreira da Silva et al., 2015; tion by DPP4is is attributed to an increase in GLP-1 (Conarello et al., animal models of HCC, breast Herrmann et al., 2014; Hollande 2003; Kim et al., 2008; Pospisilik et al., 2003; Suarez-Pinzon et al., cancer, melanoma, CML, and et al., 2019; Nishida et al., 2018; 2009; Takeda et al., 2012). Dysfunctional Tregs are thought to be a hall- multiple myeloma Qin et al., 2018) mark of T1DM (Geach, 2016). Preclinical data from Tian and colleagues Tumor development in vitro/in (Russo et al., 2018; Shingu et al., animal models of PCa, lung 2003; Wesley et al., 2005; Xie demonstrated that up-regulation of Tregs by DPP4 inhibition is associ- cancer, and CRC et al., 2017) ated with the remission of NOD mice (Tian et al., 2010). It is possible CVD Cardiovascular safety in T2DM (Aroor et al., 2018; Green et al., that DPP4 suppression results in the changes of chemokines in diabetic patients 2015;Scirica et al., 2013; White NOD mice and thus promotes the migration of Tregs to pancreas et al., 2013) Increased risk in HF in T2DM (Scirica et al., 2013; White et al., (Fig. 1A). patients 2013) Due to these significant results from animal experiments, numerous Cardioprotection in animal (Bostick et al., 2014; Hocher clinical studies are conducted to investigate the therapeutic effect of model et al., 2013; Kubota et al., 2016; DPP4is in patients with T1DM. Four placebo-controlled studies investi- Mulvihill et al., 2016; Shah et al., gated the efficacy of sitagliptin/vildagliptin treatment in T1DM (Ellis 2011; Connelly et al., 2013; Zhang et al., 2010) et al., 2011; Farngren, Persson, Schweizer, Foley, & Ahren, 2012; Garg No effect or impairment of (Mulvihill et al., 2016; Yin et al., et al., 2013; Hari Kumar, Shaikh, & Prusty, 2013). Two studies showed cardiac function in animal model 2011) significant HbA1c reduction of −0.27 and − 0.34% after DPP4is treat- Asthma Increased airway inflammation (Yan et al., 2012) ment for 8 or 4 weeks, respectively (Ellis et al., 2011; Farngren et al., in animal model Decreased airway inflammation (Schmiedl et al., 2010) 2012), while the other two studies didn't show any treatment-related in animal model differences (Garg et al., 2013; Hari Kumar et al., 2013). A meta- Infectious Increased risk of nasopharyngitis (Amori et al., 2007; Gamble analysis from Wang et al. revealed that the additional use of DPP4is re- diseases and urinary tract infection but et al., 2018) sults in a greater decrease in HbA1c levels compared to insulin mono- no association of upper therapy, although it is not significant (Wang, Long, et al., 2018). respiratory tract infections in T2DM patients Additionally, some trials investigated the effect of gliptins on β cell Potential adverse effect on MERS (Inn et al., 2018; Raj et al., 2013) preservation. In individuals with newly diagnosed T1DM, sitagliptin patients treatment for one year failed to result in any significant difference of Potential therapeutic effect on (Decalf et al., 2016; Riva et al., c-peptide secretion between groups (Hari Kumar et al., 2013). Likewise, HCV patients 2014) No effect on HIV patients (Dube et al., 2019; Goodwin Griffin and colleagues randomly assigned T1DM participants to receive et al., 2013) a combined therapy of oral sitagliptin and a PPI lansoprazole or matched BP Increased risk in T2DM patients (Aouidad et al., 2013; Arai et al., placebo for 12 months; however, the difference of c-peptide between 2018; Bene, et al., 2016; groups is not significant (Griffin, Thompson, Gottschalk, Kyllo, & Benzaquen et al., 2018; Douros Rabinovitch, 2014). These two trials estimated that gliptins are incapa- et al., 2019; Garcia et al., 2016; Kridin & Bergman, 2018; Lee ble of preserving β cell mass in autoimmune diabetes. Latent autoim- et al., 2019; Plaquevent, et al., mune diabetes in adults (LADA) is a special subtype of T1DM. 2019; Varpuluoma et al., 2018) Interestingly, a one-year prospective study conducted in LADA patients Note: BP, bullous pemphigoid; CML, chronic myeloid leukemia; CRC, colorectal cancer; demonstrated that a combined treatment of sitagliptin to these patients CVD, cardiovascular disease; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HIV, who receive insulin provides significant improvements in the parame- human immunodeficiency virus; IBD, inflammatory bowel disease; LADA, Latent autoim- ters of islet function (Zhao et al., 2014). Similarly, another DPP4i, mune diabetes in adults; MERS, Middle East respiratory syndrome; Pca, prostate cancer; linagliptin, contributes to a progressive increase in c-peptide levels in RA, rheumatoid arthritis; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus. patients with LADA during a 2-year study (Johansen et al., 2014). 6 S. Shao et al. / Pharmacology & Therapeutics 209 (2020) 107503 Fig. 1. The underlying mechanisms of DPP4 inhibition in autoimmune diabetes (A), inflammatory bowel disease (B), rheumatoid arthritis (C). Taken together, these existing studies fail to strongly support the Administration's Adverse Event Reporting System (FAERS) database clinical application of DPP4is in either glucose control or β cell preserva- (Wang et al., 2019). tion in T1DM patients. The contrary conclusions obtained from the Li et al. performed a meta-analysis of randomized controlled trials above clinic trials may be attributed to different baseline characteristics (RCTs) and no association between DPP4i and IBD was found. Howbeit, of the included patients (such as c-peptide levels, HbA1c, and disease sub-analysis identified that DPP4i OADs may reduce CD risk but in- duration), distinct follow-up lengths, different sample size, and other crease UC risk (Li et al., 2019). Of note, this analysis may have been un- variables. Another point worth noting is that, DPP4 suppression alone derpowered to detect such an association due to the limited number of may not be able to completely prevent autoimmune attack against β included trials/events and the statistical imprecision. Thus, we suggest cells under overt T1DM condition. Thus, additional immunological mea- that the association between DPP4is and IBD should be noted by physi- sures will probably be necessary for TIDM treatment in order to attenu- cians. It should be cautious when treating diabetic patients with IBD. ate the established autoimmunity. Besides, traditional animal models of T1DM are unlikely to provide 3.1.3. DPP4/CD26 inhibition on RA accurate predictions for clinical success in human studies because of RA is a chronic, systemic inflammatory disease with unknown etiol- the substantial genetic variation and the environment that is not pres- ogy and is characterized by progressive destruction of articular cartilage ent in laboratory rodents. Future researches should shift towards stud- and erosion of the underlying bone (Speiser, Ho, & Verdeil, 2016). ies in human beings with consistent baseline characteristics of Altered DPP4 activity was first noted in mice with RA in 1989 participants. In addition, larger sample size, longer follow-up duration, (Gotoh, Hagihara, Nagatsu, Iwata, & Miura, 1989). To gain insights into and the monitoring of immune parameters (such as T cells, B cells, the pathophysiological role of CD26 in arthritis, Busso et al. explored and cytokines) should be taken in consideration. DPP4 expression in experimental mice model of arthritis, murine antigen-induced arthritis (AIA), and found a reduced plasma DPP4 ac- 3.1.2. DPP4/CD26 inhibition on IBD tivity in those mice models (Busso et al., 2005). Secretion of CXCL12/ IBD, including Crohn's disease (CD) and ulcerative colitis (UC), is an SDF-1, the substrate of DPP4, by RA synovial fibroblasts (RASFs) is cru- autoimmune digestive system disease characterized by chronic, remit- cially involved in the inflammatory process by recruitment of CXCR4- tent or progressive inflammation in the gastrointestinal tract (Abbott, expressing T cells and monocytes from the periphery into the rheuma- Yazbeck, Geier, Demuth, & Howarth, 2006). Recent researches disclose toid synovium (Seki, Selby, Haupl, & Winchester, 1998). In CD26- a potential association of DPP4 inhibition with the risk of IBD in T2DM. deficient mice with AIA, the disease severity is increased due to a Duan and Zatorski reviewed the studies about the role of GLPs and lower DPP4 activity in synovial fluids, which results in an increase in DPP4is in IBD (Duan, Rao, Braunstein, Toomey, & Zhong, 2017; SDF-1 levels in those mice (Fig. 1C) (Busso et al., 2005). The major extra- Zatorski, Salaga, & Fichna, 2019). It is considered that T lymphocytes cellular proteolytic enzymes involved in cartilage resorption are matrix such as Tregs, macrophages, DCs, and proinflammatory cytokines may metalloproteinases (MMPs) and serine proteases (Proost et al., 2001). be involved in DPP4i-GLPs mediated gut immunity. Preclinical studies Ospelt et al. found that inhibition of serine protease activity of DPP4 pro- on animal models about the underlying mechanisms are limited with motes invasion of RASFs into cartilage in a mouse model of RA and leads only 3 studies included (Ban et al., 2011; Salaga et al., 2017; Salaga to an increase in SDF-1 levels in concert with its downstream effectors et al., 2018). Administration of DPP4 inhibitor EMDB-1 (Salaga et al., MMP-1 and MMP-3 in vitro (Ospelt et al., 2010). These results indicate 2017; Salaga et al., 2018) or ER-319711 (Ban et al., 2011) could mark- a central role of DPP4/SDF-1 signaling in protecting articular cartilage edly attenuate colitis in mouse models of experimental colitis, which against RASFs invasion. may be mediated by GLP-2 (Fig. 1B). The downstream signals of Busso et al. explored DPP4 expression in RA patients and also found a DPP4i-GLPs remain unclear, which need to be further explored. reduced plasma DPP4 activity (Busso et al., 2005). The concentration of Real-world evidences present an opposite conclusion to animal SDF-1 is greatly elevated in the synovial fluid from patients with RA in studies. A study of 283 T2DM patients treated with gliptins revealed contrast to its concentration in healthy individuals (Kanbe, Takagishi, that the prevalence of CD is significantly higher in patients with DPP4i & Chen, 2002). However, the functional role of DPP4/CD26 in human treatment than in controls (Kridin et al., 2018). A retrospective cohort observed in the present cohort findings contradicts that in animal stud- study using the United Kingdom Clinical Practice Research Datalink ies. In a large cohort of diabetic patients, those initiating DPP4i combina- (CPRD) found that new use of DPP4is over a median duration of tion therapy appeared to have a decreased risk of autoimmune diseases 1.6 years is associated with an increased risk of IBD (hazard ratio 1.75) including RA (hazard ratio 0.66) compared with those initiating non- compared to other antidiabetic medications (Abrahami et al., 2018). In DPP4i combination therapy (Kim et al., 2015). Similar result was addition, hazard ratios gradually increase along with longer durations found in a large population-based cohort study conducted by Seong of DPP4is usage, which reach a peak after three to four years (Seong, Yee, & Gwak, 2019). However, using the United Kingdom (Abrahami et al., 2018). Wang demonstrated a weak-to-moderate sig- CPRD, Douros conducted a cohort study among 144,603 patients with nal of IBD associated with DPP4i use through the U.S. Food and Drug T2DM initiating OADs between 2007 and 2016, and found that use of  S. Shao et al. / Pharmacology & Therapeutics 209 (2020) 107503 7 Fig. 2. The underlying mechanisms of DPP4 inhibition in allograft rejection (A), cancer (B), cardiovascular disease (C), and asthma (D). DPP4is is not associated with any increased/decreased risk of incident pulmonary transplantation in animal models (Jung et al., 2006; Zhai RA (Douros et al., 2018). Nevertheless, these results may suggest possi- et al., 2007). A recent study from Yamada group concluded that CD26 ble pharmacological pathways for prevention or treatment of RA by blockade promotes lung allograft acceptance due to reduced T cell infil- gliptins. tration, lower expression of IL-17 and IL-21, and increased expression of The contradictory findings between rodent DPP4 and human DPP4 IL-10, which is likely to be derived from alternatively activated macro- limit the usage of murine models for elucidating the function of phages (Yamada et al., 2016). These results expand the role of DPP4/ human DPP4 in RA. By now, there is no research investigating the mech- CD26 in alloantigen-mediated immune responses. anism of such distinction. We assumed that, except SDF-1, there should Moreover, CD26 knockout mice have been used to investigate the be other cytokines/immune cells regulated by DPP4/CD26 that partici- potential role of DPP4 in allogeneic skin graft rejection. It is shown pate in the inflammatory process of human RA. Future investigations that CD26 knockout mice display a reduced necrosis of grafts and a de- specifically focusing on the direct biological effects of DPP4 on human layed graft rejection with significantly reduced secretion levels of the RA will undoubtedly contribute to a better understanding of its role, cytokines such as IFN-γ, IL-2, IL-6, IL-4, IL-17, and IL-13 but increased as a possible therapeutic target, in RA. level of IL-10 after skin transplantation (Zhao et al., 2019). Additionally, a lower percentage of Th17 and CD8(+) T cells but a higher percentage 3.2. DPP4/CD26 inhibition on non-autoimmune diseases of Treg cells are detected in peripheral blood lymphocytes of CD26 knockout mice in the same study (Zhao et al., 2019), indicating that 3.2.1. DPP4/CD26 inhibition on allograft rejection CD26 deficiency leads to feebler rejection due to lower activation and The last and most effective therapeutic strategy for many end-stage less proliferation of host immune cells. diseases is organ transplantation. However, one of the biggest chal- So far, there is still few clinical studies focusing on the potential ef- lenges is to maintain the long-term survival of the grafts by preventing fect of DPP4i on allograft rejection. Future investigations in this field allograft rejection. are necessary and may expand the clinic application of DPP4is. Study by Kim et al. demonstrated that treatment with MK0431 in NOD mice reduces the effect of autoimmunity on islet graft survival, 3.2.2. DPP4/CD26 inhibition on cancer partially, by decreasing the homing of CD4(+) T cells into pancreatic Increasing evidences have demonstrated that the application of im- β cells through cAMP/PKA/Rac1 pathway (Fig. 2A) (Kim et al., 2009). munotherapy can improve the clinic outcome of cancer. Encouraging Moreover, there are several studies investigating whether the inhi- findings from experimental works regarding DPP4/CD26 suppression bition of DPP4 activity plays a role in acute pulmonary rejection. Treat- in melanoma, liver and breast tumors (Barreira da Silva et al., 2015; ment with AB192, a Pro-Pro-diphenyl phosphonate derivative, Herrmann et al., 2014; Hollande et al., 2019; Nishida, Hayashi, abrogates acute rejection and preserves early graft function after Morimoto, Sakamoto, & Yamada, 2018; Qin et al., 2018) pave the way 8 S. Shao et al. / Pharmacology & Therapeutics 209 (2020) 107503 for future clinical studies (Table 3). But in other locations, DPP4 inhibi- 5–7 days after MI (Hocher, Sharkovska, Mark, Klein, & Pfab, 2013; tion possibly leads to the tumor development (Russo et al., 2018; Kubota et al., 2016). Endothelial progenitor cells (EPCs) derived from Shingu et al., 2003; Wesley et al., 2005; Xie et al., 2017). the bone marrow are known to promote vascular repair and It has been found that both gene ablation and pharmacological inhi- neoangiogenesis. Importantly, it has been revealed that inhibition of bition of DPP4 notably prevents HFD-associated hepatocellular carci- DPP4 potentially enhances the delivery of EPCs towards injured vascu- noma (HCC) progression in rat model of carcinogen-triggered liver lar sites after MI in rats (Hocher et al., 2013), probably due to an in- cancer by down-regulating chemokine CCL2 production and angiogen- creased concentration of SDF-1 (Fig. 2C). A 4-week clinic trial in T2DM esis (Fig. 2B) (Qin et al., 2018). Hollande and colleagues have further in- patients treated with linagliptin identified a significant increase in vestigated the mechanism by which inhibition of DPP4 reduces tumor EPCs and SDF-1 (Fadini et al., 2010). In addition to EPCs, we have de- growth in HCC and breast cancer syngeneic mouse models and found scribed that SDF-1 could also attract other stem cells through the that administration of DPP4i sitagliptin results in higher concentrations CXCR4. Zhang et al. reported that the combination of overexpression of the eosinophil chemoattractant CCL11, a known target for DPP4- of CXCR4 in mesenchymal stem cells (MSC) with diprotin A, a DPP4 mediated truncation (Table 2), and increased migration of eosinophils inhibiter, in rat MI model enhances MSC recruitment and penetration into solid tumors (Hollande et al., 2019). Moreover, Barreira da Silva into ischemic myocardium via SDF-1 pathway, leading to the inhibition et al. have showed that melanoma growth is significantly delayed in of myocardial ischemia-induced apoptosis, tissue angiogenesis, and an DPP4 knockout mice of tumor-transplant model and the authors esti- improvement in heart function after MI (Zhang et al., 2010). Further- mated that CXCL10-mediated T cell trafficking may be involved more, the cardioprotective effect is abolished by the pretreatment (Barreira da Silva et al., 2015). with a CXCR4 antagonist or a signal transducer and activator of tran- On the contrary, using in vitro model system in metastatic prostate scription 3 (STAT3) inhibitor in mice with MI, indicating that DPP4 inhi- cancer cells, Wesley indicated that DPP4 may inhibit malignant pheno- bition may have direct protective effects on the post-MI heart through type of prostate cancer (PCa) by enhancing degradation of FGF2 SDF-1/CXCR4-mediated STAT3 signaling pathway (Kubota et al., (Wesley et al., 2005). In a rat model of lung metastasis, DPP4 can dras- 2016). Fadini also reported a profile of increased CD34(+)CD133(+) tically change the outcome of metastatic disease which may be partially progenitor cells, CD34(+)KDR(+) EPCs, and CX3CR1(bright) mono- mediated by NK cell cytotoxicity (Shingu et al., 2003), while DPP4 inhi- cytes along with significantly elevated SDF-1 in T2DM patients with 4- bition diminishes such effect. day treatment of linagliptin (Fadini et al., 2016), which further demon- Besides experimental studies, plenty of clinic trials have also been strates the potential favorable cardiovascular implications of gliptins. conducted for assessing possible cancer risk of DPP4is including In contrast, chronic administration of vildagliptin to normoglycemic sitagliptin (Green et al., 2015), alogliptin (Mita, et al., 2016), saxagliptin rats either prior to or after 12 weeks' induction of MI fails to avert the (Leiter, et al., 2015), and linagliptin (Barnett et al., 2012). All these trials reduction of ejection fraction or modify cardiac remodeling (Yin, Sillje, demonstrate that gliptins do not increase tumor risk in T2DM patients. Meissner, van Gilst, & de Boer, 2011). The inconsistent findings may Zhao performed a meta-analysis with a total of 72 trials enrolled and be attributed to the long duration of disease, and it is accordingly as- no significant associations are detected between the use of gliptins sumed that DPP4 inhibition has no substantial protective effects on car- and cancer development (Zhao et al., 2017), indicating the safety rather diac function in this well established long-term post-MI cardiac than benefits of gliptins in tumor. remodeling model. The different conclusions from animal models and clinic trials may Reduced interstitial fibrosis in hearts is observed in MK-0626 (a se- be attributed to relative weak effect of DPP4 on tumor immunity and lective DPP4i)-treated C57BL/6 J mice fed a high fat/high fructose diet the complexity of tumor microenvironment in human beings. Further for 16 weeks (Bostick et al., 2014). In consistence with these findings, studies that fully characterize the effects of DPP4i in various clinical set- Mulvihill et al. identified that young normoglycemic DPP4(−/−) mice tings will be required to comprehensively evaluate the administration also exhibit a significant reduction in cardiac fibrosis and a of DPP4i in cancer patients with T2DM. cardioprotective response after transverse aortic constriction (TAC) sur- gery (Mulvihill et al., 2016). Surprisingly, diabetic mice treated with 3.2.3. DPP4/CD26 inhibition on CVD MK-0626 exhibit modest cardiac hypertrophy, impairment of cardiac Patients with T2DM are at an increased risk of developing CVD, function, and dysregulated expression of genes and proteins involved which is the prevailing cause of death worldwide. Atherosclerosis, the in inflammation and cardiac fibrosis (Mulvihill et al., 2016). We as- main underlying factor of CVD, is considered to be a chronic inflamma- sumed that metabolic status may affect the cardiovascular benefits tory disease with various cell types involved in this pathogenic process from DPP4 inhibition, which is remained to be investigated. including differentially activated T and B lymphocytes, monocytes and Although clinical studies have emphasized cardiovascular safety, no macrophages, DCs, neutrophils, and endothelial cells (Rafieian-Kopaei, evidences of reduced major adverse cardiovascular events with differ- Setorki, Doudi, Baradaran, & Nasri, 2014). Abundant pre-clinical evi- ent DPP4is were observed (Aroor et al., 2018; Green et al., 2015; dences implicate the beneficial role of DPP4 inhibition in atherosclerosis Scirica et al., 2013; White et al., 2013). More than that, some cardiovas- and CVD, which has been summarized by several reviews (Aroor, cular outcome trials revealed an increase in hospitalization rates for Manrique-Acevedo, & DeMarco, 2018; Avogaro & Fadini, 2014, 2018; heart failure (HF) among subjects treated with saxagliptin or alogliptin Vedantham, Kluever, & Deindl, 2018; Zhong, Maiseyeu, Davis, & (Scirica et al., 2013; White et al., 2013). Rajagopalan, 2015). According to these results, it is speculated that the cardiovascular Macrophages play a central role in the development of atherosclero- benefits from DPP4 inhibition may be dependent on the disease dura- sis by the maintenance of the local inflammatory response, propagate tion (transient or long-term) and metabolic phenotype (diabetic or plaque development, and promote thrombosis (Barrett, 2020). Shah non-diabetic), which may explain, at least in part, the insignificant et al. identified that DPP4i decreases aortic plaque, exerts cardioprotective effect of DPP4is in T2DM patients. Further clinical stud- antiatherosclerotic effects and reduces inflammation via inhibition of ies to investigate these factors may be of importance. macrophages activation/recruitment in a LDL receptor-deficient mouse model, indicating important implications of the use of this class 3.2.4. DPP4/CD26 inhibition on asthma of drugs in atherosclerosis (Shah et al., 2011). Allergic asthma is a disease that causes the swelling and narrowing Sitagliptin has been found to attenuate the adverse remodeling fol- of airways, resulting in wheezing, shortness of breath and coughing lowing MI in Fischer F344 rats with STZ-diabetes (Connelly et al., (Umetsu, McIntire, Akbari, Macaubas, & DeKruyff, 2002). Whether 2013). Notably, DPP4 inhibition in normoglycemic rodents also signifi- CD26/DPP4 plays a role in the pathogenesis of asthma or allergic-like cantly improves cardiac function and decreases the infarct size within airway inflammation is still controversial.  S. Shao et al. / Pharmacology & Therapeutics 209 (2020) 107503 9 Determined by biopsies, the expression of CD26/DPP4 in the lamina immune-suppressive cytokine IL-10 and protective growth factor epi- propria of human bronchi is firstly described by the group of van der dermal growth factor (EGF) are significantly negatively and positively Velden (van der Velden et al., 1998); but there are no differences be- correlated with plasma sDPP4 concentration, respectively (Inn et al., tween asthmatics and healthy controls. Subsequently, a rat model of 2018). Thus, it is assumed that exogenous sDPP4 may have therapeutic asthma indicated a significant increase of CD26-expressing T cells in potential in MERS patients and that application of gliptins in MERS pa- the lungs, the amount of which arises along with the severity of airway tients may adversely affect the pathological and immune processes of inflammation (Skripuletz et al., 2007). Recently, Nieto-Fontarigo de- this disease. tected the higher number of CD26 molecules on CD4+ T cells from Conversely, increased sDPP4 activity was found in chronic infections both allergic and non-allergic asthma patients when compared to by hepatitis C virus (HCV), hepatitis A virus (HAV), and Epstein-Barr healthy subjects. However, circulating levels of sCD26 are reduced in virus (Andrieu et al., 2003). The pathogenesis of HCV infection is asthma patients, which may be explained by the expansion of CD26 strongly influenced by the nature of the host's antiviral immunity. It (−/low) T lymphocyte populations in peripheral blood (Nieto- has been described that CXCL10 can be truncated by DPP4 (Table 2). Fontarigo et al., 2019). Riva et al. reported that subjects developing chronic hepatitis C have A study from Yan et al. indicated an enhanced ovalbumin-induced higher concentrations of truncated CXCL10 and DPP4 activity; whereas airway inflammation in DPP4/CD26-deficient mice with increased Th2 DPP4 activity progressively decreases over time in patients who sponta- cytokines such as IL-4, IL-5, and IL-13 (Fig. 2D) (Yan et al., 2012). On neously clears the infection (Riva et al., 2014). A follow-up study was the contrary, another study reported that CD26 deficiency leads to a de- conducted to test the effects of DPP4 inhibition on CXCL10 processing crease in airway inflammation. They found a significantly increased in- in chronic HCV patients. Participants were treated daily with 100 mg flux of Tregs into the lungs in CD26-deficient rats and an increased IL-10 sitagliptin, which results in a significant decrease in truncated CXCL10 production in draining lymph node cells (Schmiedl et al., 2010). but a reciprocal increase in full length form (Decalf et al., 2016). These Currently, there is no population-based studies or analysis from data provide the direct evidence that therapeutic abrogation of DPP4 ac- CPRD on the association of DPP4is with asthma. Despite the inconsis- tivity by gliptins in humans can preserve the bioactive form of CXCL10, tence and limited evidences, the preclinical results indicate an impor- suggesting that DPP4i may be a novel strategy to target both the virus tant role of CD26 in regulating the allergic immune response, thus and the host (Fig. 3). raising the safety questions for clinical application of gliptins. It is highly In addition, DPP4is can block RANTES cleavage, thereby potentially suggested that the application of DPP4is to patients with asthma should facilitating human immunodeficiency virus (HIV) entry into T lympho- be carefully estimated. cytes (Lusso et al., 2011), and inhibit cleavage of SDF-1, potentially blocking HIV entry into T lymphocytes (Bleul et al., 1996). Accordingly, 3.2.5. DPP4/CD26 inhibition on infectious diseases it is significant to evaluate the immune and virological safety of DPP4 in- The risk of infectious diseases induced by DPP4i has been widely in- hibition in HIV. Dube et al. evaluated inflammation and immune vestigated. A meta-analysis including 29 clinic trials concluded that markers during treatment with the sitagliptin among virologically sup- DPP4is could increase the risk of infections for nasopharyngitis (risk pressed HIV patients without diabetes. It is found that sitagliptin has no ratio, 1.2) and urinary tract infection (risk ratio, 1.5) but not for upper effect on sCD14 levels, a biomarker of monocyte activation (Dube et al., respiratory tract infections (Amori, Lau, & Pittas, 2007). Using the UK- 2019). In addition, there are no differences in any of the immune based CPRD, Gamble also identified that initiation of a DPP4i is not asso- markers except a significant fall in CXCL10 (Dube et al., 2019). Similar ciated with an increased risk of respiratory tract infections compared to investigation was performed by Goodwin which indicated that, despite other glucose-lowering therapies (Gamble et al., 2018). However, there is no experimental studies that investigate the molecular/immune- related mechanisms in DPP4i-related bacterial infections. Newer anti-diabetic agent sodium glucose cotransporter 2 (SGLT2) inhibitor (SGLT2i) is very useful due to its potential benefits on HF and diabetic nephropathy (Heerspink et al., 2020; Marx, Grant, & Cosentino, 2020). Thus the combination of SGLT2i and DPP4i is used more and more widely in T2DM patients. It is reported that genital my- cotic infection is one of the most common adverse effects of SGLT2i (Adimadhyam et al., 2019). Of note, the risk of genital infections with combination therapy of Dapagliflozin and saxagliptin is lower than ob- served with dapagliflozin alone, suggestive of a protective effect of saxagliptin (Matthaei et al., 2015; Matthaei et al., 2016; Rosenstock et al., 2015), although the potential mechanism is unclear. However, a totally different conclusion is drawn by Min and colleagues. They ana- lyzed 7 RCTs and identified that the risk of genital infection increases (Min, Yoon, Moon, Hahn, & Cho, 2018). We speculate that different combinations of SGLT2is and DPP4is together with the add-on method (simultaneous combination/sequential combination) may contribute to the inconsistent the risk of infections. Further investigations to figure out the underlying mechanism should be of significance. Middle East respiratory syndrome coronavirus (MERS-CoV) is a β- coronavirus which is genetically associated to the severe acute respira- tory syndrome (SARS) coronavirus (Hilgenfeld & Peiris, 2013). Interest- ingly, sDPP4 is identified as a functional receptor for MERS-CoV through its interaction with the spike protein (Raj et al., 2013). It is known that the sDPP4 level in plasma varies depending on pathophysiological con- ditions (Lambeir, Durinx, Scharpe, & De Meester, 2003). Inn and col- leagues observed that sDPP4 in the plasma of MERS patients is significantly lower than those of normal volunteers. The levels of Fig. 3. The underlying mechanisms of DPP4 inhibition in infectious diseases. 10 S. Shao et al. / Pharmacology & Therapeutics 209 (2020) 107503 lowering SDF-1 levels, sitagliptin does not adversely affect immune or 3.3. DPP4/CD26 inhibition and BP virological status, or increase immune activation in nondiabetic HIV- positive adults (Goodwin et al., 2013). BP is a potentially severe autoimmune skin disease, the typical clin- ical features of which include the widespread blisters, often preceded by 3.2.6. DPP4/CD26 inhibition on other diseases or associated with urticarial or eczema-like lesions (Tasanen, T2DM patients present an increased risk of peripheral artery disease Varpuluoma, & Nishie, 2019). (PAD); however, the pharmacological options for PAD are limited (Yang Currently, DPP4i-associated BP is attracting increasingly attentions. et al., 2020). We have described in section 3.2.3 that SDF-1 is a chemo- Howbeit, the pathogenesis and underlying mechanisms remain unclear. kine that attracts EPCs and promotes angiogenesis. SDF-1 engineered to It has been shown that inhibition of DPP4 in F344 rats induces the infil- be resistant to DPP4/CD26 and delivered by nanofibers improves blood tration of eosinophils into the skin (Fig. 4) (Forssmann et al., 2008), flow in a mouse model of PAD (Segers et al., 2011). Both DPP4i MK-0626 which is a typical histopathologic feature of BP. The major BP (Shih et al., 2014) and sitagliptin (Huang et al., 2012) are found to in- autoantigen BP180, known as a transmembrane collagen XVII, is re- crease the number of circulating EPCs, elevate the level of SDF-1, and sponsible of anchoring epidermis into the underlying dermis. Under promote the neo-vasculogenesis in a hind limb ischemia mouse the condition of BP, anti-BP180 autoantibodies could impair BP180 model. All these results indicate that administration of DPP4is may function, leading to dermal-epidermal separation (Algaissi et al., have therapeutic potential as an inducer of vasculogenesis. 2019). It should be noted that DPP4 converts plasminogen to plasmin, Besides PAD, linagliptin significantly improves the outcome of func- which is known to cleave BP180 into 120 and 97 kDa fragments tional stroke in mouse model by a transient middle cerebral artery oc- (Hofmann et al., 2009; Nishimura et al., 2016). Accordingly, it is specu- clusion (MCAO) and the author concluded that the beneficial effect of lated that the suppression of DPP4 may be involved in the disruption of linagliptin may be accomplished in a SDF-1/CXCR4-dependent manner BP180 immunotolerance and the subsequent development of epitopes (Chiazza et al., 2018). for BP autoantibodies. Further studies are needed to elucidate the de- DPP4 also regulates hematopoietic stem cells (HSCs) and hemato- tailed mechanisms. poietic progenitor cells (HPCs) by truncating multiple CSFs (Table 2). Over the last few years, case reports and pharmacovigilance analyses It is reported by Broxmeyer et al. that inhibition of DDP4 augments have suggested a risk of BP after the application of DPP4is, and the the ability of G-CSF or GM-CSF to induce myeloid colony formation highest risk are observed with linagliptin and vildagliptin (Aouidad and EPO to induce erythroid colony formation in vitro (Broxmeyer et al., 2013; Bene, et al., 2016). Studies from European and French et al., 2012). Additionally, an accelerated hematopoietic recovery after pharmacovigilance databases were the first epidemiological investiga- myeloablative chemotherapy, radiotherapy, or stem cell transplantation tions which identify a high BP incidence in gliptins treated T2DM pa- is observed in DPP4-deficient mice and, to a lesser extent, pharmaco- tients (Bene, et al., 2016; Garcia, Aranburu, Palacios-Zabalza, logic inhibition of DPP4 with sitagliptin (Broxmeyer et al., 2012). Lertxundi, & Aguirre, 2016). More recently, Douros et al. conducted a Among the peripheral blood cells, the recovery of neutrophils, lympho- cohort study using the U.K. CPRD among 168,774 patients taking cytes, and monocytes is more significant than red blood cells (RBCs) OADs and found that current use of DPP4is doubles the risk of BP, albeit (Broxmeyer et al., 2012). Although there are potential concerns that the absolute incidence is low. Moreover, hazard risks gradually in- need to be addressed in clinical trials, treatment with DDP4is holds con- creased along with the extension of use, reaching a peak after siderable promise as a new strategy to stimulate hematopoietic recov- 20 months' application of gliptins (Douros et al., 2019). Likewise, a sim- ery after chemotherapy or stem cell transplantation. Furthermore, due ilar increase was also observed in a Japanese pharmacovigilance to the potential influence on EPO and RBC recovery, it will be interesting database, particularly among patients treated with vildagliptin, to investigate the hematopoiesis effect of gliptins in patients with dia- linagliptin, or teneligliptin (Arai, Shirakawa, Konishi, Sagawa, & betic nephropathy and anemia. Terauchi, 2018). Fig. 4. The underlying mechanisms of DPP4 inhibition in bullous pemphigoid.  S. Shao et al. / Pharmacology & Therapeutics 209 (2020) 107503 11 Several observational studies (all case-control designs) have also Declaration of Competing Interest been conducted and it was reported that a significantly increased or a trend towards an increased risk of BP is associated with the usage of The authors declare that there are no conflicts of interest. DPP4is with odds ratios ranging between 1.58 and 3.16 (Benzaquen et al., 2018; Kridin & Bergman, 2018; Lee, Lee, Yoon, & Kim, 2019; Acknowledgments Plaquevent, et al., 2019; Varpuluoma et al., 2018). Moreover, Benzaquen et al. has reported that application of DPP4is increases the risk of BP of This work was supported by a grant from Tongji Hospital in HuaZhong almost 3-fold, and the increase is associated with vildagliptin rather University of Science and Technology [grant number 2201103295 to Y than other gliptins (Benzaquen et al., 2018). It is affirmed by another Chen]; National Natural Science Foundation of China [grant number similar nationwide study from Korea that vildagliptin displays the 81100581]; a grant from the Bethune·Merck Diabetes Research Fund highest risk among the DPP4is (Lee et al., 2019). [grant number 2018 to S Shao]; and a grant from Cardiac rehabilitation Based on the above findings, vildagliptin appears strongly associated and metabolic therapy research fund [grant number 2018 to S Shao]. with BP. This medicine differs from other DPP4is since it has a relatively lower DPP4 selectivity (Baetta & Corsini, 2011). Accordingly, it is specu- References lated that off-target inhibition on other members of DPP family, such as Abbott, C. A., Yazbeck, R., Geier, M. S., Demuth, H. U., & Howarth, G. S. (2006). Dipeptidyl DPP8 and DPP9 may cause the pathophysiology of gliptin-related BP peptidases and inflammatory bowel disease. Advances in Experimental Medicine and (Lee et al., 2019). However, patients treated with linagliptin, which is Biology 575, 155–162. a high selective DPP4i, showed an equal risk of BP as well (Baetta & Abrahami, D., Douros, A., Yin, H., Yu, O. H. Y., Renoux, C., Bitton, A., & Azoulay, L. (2018). Dipeptidyl peptidase-4 inhibitors and incidence of inflammatory bowel disease Corsini, 2011), when compared with vildagliptin (Kridin & Bergman, among patients with type 2 diabetes: population based cohort study. BMJ 360, k872. 2018). Therefore, it is assumed that the DPP4i itself rather than the Adimadhyam, S., Schumock, G. T., Calip, G. S., Smith Marsh, D. E., Layden, B. T., & Lee, T. A. off-target effect contributes to the high risk of BP suffering. (2019). Increased risk of mycotic infections associated with sodium-glucose co- transporter 2 inhibitors: a prescription sequence symmetry analysis. 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Elevated different genetic characteristics, such as human leukocyte antigen human dipeptidyl peptidase 4 expression reduces the susceptibility of hDPP4 trans- (HLA) haplotypes. This question was recently worked on in a study con- genic mice to middle east respiratory syndrome coronavirus infection and disease. The Journal of Infectious Diseases 219, 829–835. ducted by Ujiie (Ujiie et al., 2018). The results suggested that HLA- Amori, R. E., Lau, J., & Pittas, A. G. (2007). Efficacy and safety of incretin therapy in type 2 DQB1*03:01 may be strongly associated with gliptin-related BP (Ujiie diabetes: systematic review and meta-analysis. JAMA 298, 194–206. et al., 2018). Andrieu, T., Thibault, V., Malet, I., Laporte, J., Bauvois, B., Agut, H., & Cahour, A. (2003). Sim- ilar increased serum dipeptidyl peptidase IV activity in chronic hepatitis C and other With the rapid growth of investigations on the potential association viral infections. 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However, 132, 2131–2157. regarding to the more recent findings, DPP4is might be considered to Ban, H., Bamba, S., Imaeda, H., Inatomi, O., Kobori, A., Sasaki, M.,... Fujiyama, Y. (2011). The be a double-edged sword. Apart from the metabolic benefit, the associ- DPP-IV inhibitor ER-319711 has a proliferative effect on the colonic epithelium and a minimal effect in the amelioration of colitis. Oncology Reports 25, 1699–1703. ated immunological effects induced by DPP4 inhibition, may bring out Barnett, A. H., Patel, S., Harper, R., Toorawa, R., Thiemann, S., von Eynatten, M., & Woerle, benefits or side effects under different conditions (Table 3). It is recom- H. J. (2012). Linagliptin monotherapy in type 2 diabetes patients for whom metfor- mended that physicians should be cautious about pre-existing inflam- min is inappropriate: an 18-week randomized, double-blind, placebo-controlled phase III trial with a 34-week active-controlled extension. Diabetes, Obesity & matory diseases in T2DM patients when DPP4is are initiated. Metabolism 14, 1145–1154. The controversial effects of DPP4is on autoimmune or inflammatory Barreira da Silva, R., Laird, M. E., Yatim, N., Fiette, L., Ingersoll, M. A., & Albert, M. L. (2015). diseases may be attributed to the wide tissue distributions and multi- Dipeptidylpeptidase 4 inhibition enhances lymphocyte trafficking, improving both natu- rally occurring tumor immunity and immunotherapy. Nature Immunology 16, 850–858. functions of DPP4/CD26. Tissue-specific regulation of DPP4/CD26 Barrett, T. J. (2020). Macrophages in atherosclerosis regression. Arteriosclerosis, should be considerable to figure out its immunomodulatory effects. In Thrombosis, and Vascular Biology 40, 20–33. addition, effects observed from animal models are inconsistent with Bene, J., Moulis, G., Bennani, I., Auffret, M., Coupe, P., Babai, S.,... French Association of Regional PharmacoVigilance, C (2016). Bullous pemphigoid and dipeptidyl peptidase clinical trials, which indicates that animal models may not be IV inhibitors: a case-noncase study in the french pharmacovigilance database. The completely appropriate to estimate clinical outcomes. Researches British Journal of Dermatology 175, 296–301. should focus on studies in human beings with more detailed subgroup Bengsch, B., Seigel, B., Flecken, T., Wolanski, J., Blum, H. E., & Thimme, R. (2012). Human designs, for example, according to the disease duration and metabolism Th17 cells express high levels of enzymatically active dipeptidylpeptidase IV (CD26). Journal of Immunology 188, 5438–5447. status. Future research would also be needed to determine the effect of Benzaquen, M., Borradori, L., Berbis, P., Cazzaniga, S., Valero, R., Richard, M. A., & DPP4i in the non-diabetic population. Feldmeyer, L. (2018). Dipeptidyl peptidase IV inhibitors, a risk factor for bullous pem- Nevertheless, gliptins do represent an exciting and novel drug class phigoid: retrospective multicenter case-control study from France and Switzerland. Journal of the American Academy of Dermatology 78, 1090–1096. for the treatment of autoimmune or inflammatory disease. Of note, it Bleul, C. C., Farzan, M., Choe, H., Parolin, C., C