The intramembrane protease presenilin homolog signal-peptide-peptidase-like 2a (SPPL2a) cleaves CD74. SPPL2a−/− mice accumulate N-terminal fragments of CD74 that impair membrane traffic within the endocytic system and alter B cell biology (56, 57). Since in SPPL2a−/− mice, CD74 signaling is inhibited, SPPL2a inhibitors may offer new pathways to inhibit CD74 signaling (58). Despite these recent advances, there is little or no information on the potential therapeutic or adverse effects of CD74 targeting strategies for kidney diseases and the available information is mainly derived from the study of CD74−/− mice, which may have developed adaptive mechanisms over development.

The role of soluble CD74 is unclear. Circulating soluble CD74 with MIF neutralizing activity was increased in primary biliary cirrhosis and was hypothesized to contribute to liver fibrosis (59). Indeed, soluble CD74 ectodomain prevents binding of MIF to cell surface receptors (10). However, MIF/sCD74 complexes were found to enhance MIF antioxidant activity (60). Autoantibodies against CD74 (anti-HLA class II-associated invariant chain peptide, CLIP) were found in 67% of ankylosing spondylitis patients, in 15% of systemic lupus erythematosus patients, and only in 0.8% of blood donors (61, 62).

# CD74 and Kidney Injury

There is a growing body of evidence linking CD74 to promotion or protection from kidney injury (**Figure 2**).

role of CD74 is unclear, but CD74 expression is increased in cystic epithelium.

# Expression of CD74 and Related Molecules in Kidney Injury

MIF has long been known to be upregulated in kidney injury (2). However, reports of increased CD74 expression are more recent and there is very little information on MIF-2.

Low CD74 expression may limit inflammatory and proliferative cell responses to MIF, and the increased renal cell CD74 expression observed during kidney injury (discussed below) may contribute, together with increased ligand availability, to elicit biological responses during kidney disease. Thus, overexpression of CD74 led to upregulation of NF-κB-dependent genes encoding cytokines in macrophages (63) and to NFκB activation and proliferation in human embryonic kidney cells (64).

CD74 is upregulated in tubular epithelial cells in at least some forms of chronic kidney disease (CKD), such as kidney graft rejection, diabetic and Fabry nephropathy and autosomal dominant polycystic kidney disease (ADPKD), and in podocytes in human diabetic nephropathy and Fabry nephropathy (11, 65, 66). A transcriptomics analysis also revealed increased kidney CD74 mRNA expression in human hypertensive nephropathy (11). CD74 is also increased in human clear cell renal cell carcinoma (67), in B cells, and kidney of lupus mice in parallel to progression of inflammation (68) and in the tubulointerstitium in anti-GBM nephritis, where it was expressed *de novo* in glomerular parietal epithelial cells and crescents (51). Interestingly, kidney cancer may be a complication of CKD-related acquired polycystic kidney disease (69). There is much less information on the expression of CD74 in acute kidney injury.

Migration inhibitory factor was recently identified as an important regulator of cyst growth and therapeutic target in ADPKD (3). MIF accumulated in cyst fluid of human ADPKD, promoted cystic epithelial cell proliferation and regulated apoptosis. In experimental murine polycystic kidney disease, MIF was required for renal inflammation and cyst expansion. CD74 expression was increased in *Pkd1* mutant renal epithelial cells and ADPKD kidneys, suggesting that MIF actions may imply CD74 activation. However, a CD74 antibody blocked the MIF-induced phosphorylation of ERK but did not modulate the MIF-induced inflammatory response of increased MCP-1 expression in culture (3).

The CD74 signaling machinery may also be upregulated in kidney disease. CD44 is rapidly upregulated after injury in acute and chronic experimental and human kidney disease including glomerulonephritis, diabetic nephropathy, cyclosporine nephrotoxicity, urate nephropathy, and ischemia–reperfusion kidney injury (70–76). Increased CD44 expression was localized to tubules, glomerular parietal epithelial cells, mesangial cells and infiltrating macrophages, T cells, and neutrophils (70, 72, 75–78).

Finally, the expression of at least some CD74 ligands is also increased in kidney injury. MIF expression is upregulated in acute and chronic, human, and experimental kidney diseases (2). However, there is no information on MIF-2 expression and kidney disease.

### CD74 Targeting and Kidney Injury

Neutralizing anti-MIF antibodies, small molecules or endogenous inhibitors or MIF−/− mice have shown that MIF aggravates anti-GBM glomerulonephritis (69–71), experimental IgAN (79), and lupus nephritis (80). More recently, the inhibitor of MIF/CD74 interactions RPS19 prevented the development of glomerular crescents, necrosis, inflammation, renal dysfunction, proteinuria, and the upregulation of MIF and CD74 in anti-GBM glomerulonephritis (51). However, MIF absence from mice did not protect from renal allograft rejection (81) or ureteral obstruction-induced kidney injury (82).

There is limited experimental and no clinical data on CD74 targeting in kidney injury. Given the multiple MIF and CD74 functions, studies are needed that explore whether targeting CD74 is therapeutic in models in which targeting MIF was beneficial. Information on the role of CD74 as a potential therapeutic target in tissue injury is mainly derived from CD74−/− mice. As discussed above, CD74 deficiency may be beneficial for some non-renal diseases, such as liver and heart disease, and deleterious in others, such as vascular injury. Specifically, liver fibrosis was increased in CD74−/− mice (34). An initial report found no protection from ureteral obstruction-induced kidney inflammation or fibrosis (82), but an abstract indicated that MIF targeting promoted interstitial fibrosis and inflammation following ureteral obstruction, whereas recombinant MIF reduced fibrosis (4). According to abstract reports, CD74 deficiency was also associated with increased interstitial fibrosis and inflammation following ureteral obstruction (day 5) and ischemia–reperfusion (day 21) (4). By contrast, CD74−/− mice are protected from glomerular injury induced by anti-GBM antiserum (Djudjaj JASN2015).

Since MIF or MIF-2 activation of CD74 is tissue protective in heart ischemia and liver injury induced by activation of the Fas receptor or metabolic disorders, a similar protective effect maybe hypothesized in ischemic acute kidney injury or metabolic kidney diseases characterized by increased CD74 expression, such as diabetic nephropathy or Fabry disease. Interventional studies should test these hypotheses and evaluate whether changes in fibrosis after kidney ischemia–reperfusion is secondary to an improved initial kidney injury or to a specific action of fibrosis mechanisms. These studies should differentiate between complete abrogation of CD74 expression (CD74−/−) and therapeutic downregulation of CD74. Incomplete blockade of the system may have different consequences than complete CD74 absence, given the potential proinflammatory effects of excess CD74 activation.

# Kidney Safety of Lethal Anti-CD74 Immunoconjugates

The anti-CD74 antibody hLL1 milatuzumab, alone or as an immunoconjugate, is undergoing clinical trials to treat malignancy (83, 84). Milatuzumab binds to CD74 and promotes internalization of the antibody-CD74 complex, thus delivering conjugated antitumoral agents inside tumor cells with high CD74 expression, but not to normal cells with low CD74 levels (85). Since CD74 expression is also increased in renal cells from injured kidneys, nephrotoxicity is a potential complication of antitumoral anti-CD74 therapy, especially in patients with prior kidney disease since the active chemotherapeutic agent may be released inside already injured, CD74-expressing renal cells. This may be of special concern for one of the indications under study, multiple myeloma, which frequently causes kidney disease.

# Summary and Conclusions

The role of CD74 in kidney injury has barely been explored and the scarce information available is derived from CD74-deficient mice that may not recapitulate the findings of targeting CD74 *de novo* in an adult. CD74 may contribute to or protect from tissue injury in a disease-specific manner. Thus, like MIF, CD74 may protect from experimental kidney interstitial fibrosis but promotes glomerular injury, while MIF (and potentially CD74) also promotes polycystic kidney disease. A few years ago, the situation was less complex. Given the longstanding preclinical evidence for a pathogenic role of MIF in glomerular kidney disease, anti-MIF strategies were tested in renal disease, although the sponsor decided to terminate a phase 1 trial of the anti-MIF monoclonal antibody imalumab in lupus nephritis (NCT01541670). However, MIF, MIF-2, and CD74 may be tissue protective or promote injury in an organ- and disease-specific manner and different forms of therapeutic manipulation of the system may be envisioned for different indications, from inhibiting to actually activating CD74 signaling. The potential impact of future intervention on CD74 for kidney disease is double. On one hand, therapeutic modulation of the system may be used to treat kidney disease. On the other, the kidney may suffer adverse effects from the therapeutic targeting on non-kidney diseases. Therapeutic approaches blocking MIF or CD74 signaling for non-renal indications may theoretically promote kidney fibrosis as an adverse effect, while therapeutic approaches activating

# References

the system may cause or aggravate glomerular injury. Thus, a better understanding of CD74 and the kidney is required for nephrologists and non-nephrologists. Future areas of research include the potential therapeutic interest of molecules aimed at increasing or decreasing CD74 activity for different forms of kidney disease, including acute and chronic kidney injury, the potential renal adverse effects of these approaches when used for non-renal indications, the impact on kidney disease of naturally occurring circulating soluble CD74 or CLIP and the kidney safety of antitumor therapeutic strategies delivering toxins into CD74-expressing cells or targeting CD74. Additional unknowns to be solved include the drivers and consequences of CD74 RIP in kidney cells, the significance for kidney disease of MIF-2 and of CD74 actions on mesenchymal stem cells, and whether AMPK is activated by CD74 in renal cells since, contrary to the heart, no difference in AMPK activation by acute ischemia was observed between MIF−/− and wild-type mice in the kidney, and this was attributed to lower CD74 expression in the kidney (86).

# Acknowledgments

Grant support: ISCIII and FEDER funds CP14/00133, PI13/00047, Sociedad Española de Nefrologia, ISCIII-RETIC REDinREN/RD012/0021, Comunidad de Madrid CIFRA S2010/ BMD-2378. Salary support: FIS to LV-R, Miguel Servet to MS-N. Programa Intensificación Actividad Investigadora (ISCIII/ Agencia Laín-Entralgo/CM) to AO.

established gamma-secretase inhibitors. *PLoS One* (2015) **10**:e0128619. doi:10.1371/journal.pone.0128619

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Valiño-Rivas, Baeza-Bermejillo, Gonzalez-Lafuente, Sanz, Ortiz and Sanchez-Niño. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# **The dipeptidyl peptidase family, prolyl oligopeptidase, and prolyl carboxypeptidase in the immune system and inflammatory disease, including atherosclerosis**

*Yannick Waumans, Lesley Baerts, Kaat Kehoe, Anne-Marie Lambeir and Ingrid De Meester\**

*Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium*

### *Edited by:*

*Heidi Noels, RWTH Aachen University, Germany Jürgen Bernhagen, RWTH Aachen University, Germany*

### *Reviewed by:*

*Rafael Franco, University of Barcelona, Spain Catherine Anne Abbott, Flinders University, Australia Mark Gorrell, University of Sydney, Australia*

### *\*Correspondence:*

*Ingrid De Meester, Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, Antwerp 2610, Belgium<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to Chemoattractants, a section of the journal Frontiers in Immunology*

> *Received: 21 April 2015 Accepted: 13 July 2015 Published: 07 August 2015*

### *Citation:*

*Waumans Y, Baerts L, Kehoe K, Lambeir A-M and De Meester I (2015) The dipeptidyl peptidase family, prolyl oligopeptidase, and prolyl carboxypeptidase in the immune system and inflammatory disease, including atherosclerosis. Front. Immunol. 6:387. doi: 10.3389/fimmu.2015.00387*

Research from over the past 20 years has implicated dipeptidyl peptidase (DPP) IV and its family members in many processes and different pathologies of the immune system. Most research has been focused on either DPPIV or just a few of its family members. It is, however, essential to consider the entire DPP family when discussing any one of its members. There is a substantial overlap between family members in their substrate specificity, inhibitors, and functions. In this review, we provide a comprehensive discussion on the role of prolyl-specific peptidases DPPIV, FAP, DPP8, DPP9, dipeptidyl peptidase II, prolyl carboxypeptidase, and prolyl oligopeptidase in the immune system and its diseases. We highlight possible therapeutic targets for the prevention and treatment of atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease.

**Keywords: dipeptidyl peptidase, prolyl oligopeptidase, fibroblast activation protein** α**, prolyl carboxypeptidase, inflammation, immunophysiology, atherosclerosis**

# **Introduction**

Research from over the past 20 years has implicated the dipeptidyl peptidase (DPP) family in various physiological processes and pathologies of the immune system. Usually only four prolyl-specific peptidases are considered: DPPIV (EC <IP_ADDRESS>), fibroblast activation protein α (FAP; EC 3.4.21.B28), and the more recently discovered DPP8 and DPP9 (EC 3.4.14). However, due to similarities in substrate specificity and structural homology, it is more relevant to consider a broader family that also includes prolyl oligopeptidase (PREP; EC <IP_ADDRESS>), dipeptidyl peptidase II (DPPII) (EC <IP_ADDRESS>), and prolyl carboxypeptidase (PRCP; EC <IP_ADDRESS>). First, DPPII and PRCP share the α/β hydrolase fold with the other DPPs and the catalytic triad is completely conserved in both enzymes (2). Moreover, DPPII can cleave several DPPIV substrates*in vitro* (3). Conversely, due to its substrate preference for tripeptides (4), DPPII could actually be considered as a prolyl carboxytripeptidase, emphasizing its similarities to PRCP. Another argument for considering a broader family stems from the fact that functional studies on the role of peptidases rely heavily on the use of enzyme inhibitors and many of the inhibitors used in earlier studies are now known to inhibit more than one family member. For example, early studies on DPPIV used inhibitors which we now know also inhibit DPPII, DPP8, DPP9, FAP, and/or PREP due to their sequential and/or structural similarity [e.g., Ref. (5–9)]. PRCP is known to be inhibited by KYP-2047 and Z-Pro-Prolinal at higher concentrations, which have often been used for the functional study of PREP [e.g., Ref. (10–12)]. **Table 1** summarizes the most commonly used DPP inhibitors and their selectivity compared to DPPIV. In view of the aforementioned reasons and for the sake of simplicity, we will use "DPP family" as a blanket term, which includes DPPII, PRCP, and PREP even though strictly speaking they are not DPPs. **Figure 1** provides a general overview of this broadly defined DPP family. The roles of various family

*Partly adapted from Deacon (28).*

*\*Z-Pro-Prolinal is currently used as an inhibitor for PRCP although it is in vitro 103 times more selective toward PREP.*

members in certain aspects of the immune system or immune dysfunction have been reviewed in the past [e.g., Ref. (13– 15)]. In this review, we provide a comprehensive discussion and update on the roles of DPPIV, DPPII, DPP8, DPP9, FAP, PREP, and PRCP in the immune system and inflammatory disease. We highlight the role of these enzymes in atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease, as the DPP family encompasses possible therapeutic targets for the prevention and treatment of this disease.

# **A Brief Guide to the DPP Family**

# **Dipeptidyl Peptidase IV**

The prototypical DPP, DPPIV (often DPP4 in medical jargon) cleaves off an N-terminal dipeptide from peptides with Pro or Ala on the penultimate position. Its localization as a soluble enzyme in body fluids, or anchored in the plasma membrane of cells provides it with the necessary access to cleave a wide range of bioactive peptides. As such, it can modify their biological activity. Glucagon-like peptide (GLP)-1 and -2, and glucosedependent insulinotropic peptide (GIP) (29, 30), substance P (31), neuropeptide Y (NPY) (32), stromal cell-derived factor-1α/β (SDF-1α/β or CXCL12) (33), granulocyte macrophage colony-stimulating factor (GM-CSF) (1), CXCL10 (34–36), and high-mobility group box 1 (HMGB1) (37) have been identified as physiological substrates, while others, such as RANTES, have been proposed based on *in vitro* experiments [e.g., Ref. (38)]. DPPIV also performs many of its physiological functions through interactions with other proteins, such as collagen, fibronectin, adenosine deaminase (ADA), caveolin-1, and the mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) (39–41). Some of those will be discussed in more detail below.

Dipeptidyl peptidase IV is well known for its role in glucose homeostasis. It has become a validated therapeutic target for the treatment of type 2 diabetes (T2D) (46). DPPIV inhibitors reduce the rate of GLP-1 inactivation (**Boxes 1** and **2**). It has also been shown to be involved in cancer biology. The role of the DPP family in cancer has been addressed in several other reviews (39, 47–51). Finally, DPPIV has recently come back into the center of attention as the receptor for the MERS coronavirus (52).

### **BOX 1 Incretins.**

The incretins are a group of glucose-lowering molecules produced by the intestines. The best known incretin is glucagon-like peptide-1 (GLP-1). This incretin is derived from proglucagon and secreted after a meal from L-cells in the distal ileum and colon. In the pancreas, it induces insulin secretion and biosynthesis while lowering glucagon secretion. In addition, GLP-1 increases the β-cell mass, thereby restoring insulin production. It is clear that GLP-1 also has functions outside glucose metabolism. Its receptor, GLP-1-R, is not only found in the pancreas but also expressed in brain, lung, kidney, stomach, and heart (42, 43). Recently, it was shown that stimulation after myocardial infarction reduces the infarct size (44, 45). Currently, GLP-1 agonists are approved for the treatment of type 2 diabetes. These incretin mimetics seem to have a slightly better efficacy as DPPIV inhibitors and lead more frequently to weight loss. Unfortunately, an important drawback for their therapeutic use is that they can only be administered by subcutaneous injection (46).

### **Fibroblast Activation Protein** α

Fibroblast activation protein α, also known as seprase can present itself as a type II transmembrane protein or as a shedded plasma protease (57). In the latter case, it is also known as antiplasmincleaving enzyme, which converts α2-antiplasmin into a more active form, suppressing fibrinolysis (58). Some of the known DPPIV substrates were later found to be cleaved *in vitro* by FAP as well (59), though any physiological relevance remains unclear.

Unlike DPPIV, FAP also possesses a gelatinase activity. This enables FAP to degrade proteins of the extracellular matrix (60). This is of particular interest with regard to its involvement in a number of pathological processes (47). FAP is highly induced during inflammation, activation of hepatic stellate cells in liver cirrhosis and strongly expressed by mesenchymal cells of remodeling tissue (47, 61). FAP is also a key regulator during tumor growth and metastasis (47). As all these processes require degradation of the extracellular matrix, FAP's involvement in these pathologies is most likely associated with its gelatinase activity (51). Its role in cancer biology has been reviewed before (47, 62). It is interesting to note that, so far, in clinical trials Talabostat has shown minimal or no clinical benefit for the treatment of metastatic colorectal cancer, advanced non-small cell lung cancer, or stage IV melanoma (63–65). It should be mentioned, however, that Talabostat is a broad-range inhibitor also targeting DPPIV, DPP8, and DPP9.

# **Dipeptidyl Peptidases 8 and 9**

Dipeptidyl peptidases 8 and DPP9 show DPPIV-like activity and share a very high-sequence similarity to each other (77% aa similarity, 57% aa identity) (24). These cytoplasmic enzymes have several isoforms. It has been a matter of debate whether all are expressed as protein in cells and, if so, whether they are active (66–69). Interestingly, the N-terminal extension of the longer DPP9 variant contains a nuclear localization signal and, indeed, this form localizes to the nucleus (69). DPP8 has been shown to cleave a number of DPPIV chemokine substrates *in vitro* (70). Another DPPIV substrate, NPY, has indirectly been shown to be

### **BOX 2 DPPIV inhibitors.**

Dipeptidyl peptidase IV inhibitors prolong the biological half-life of the incretins and are therefore used for the treatment of type 2 diabetes. Sitagliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin are DPPIV inhibitors currently available on the market for treatment of type 2 diabetes. Sitagliptin and alogliptin are highly selective toward DPPIV *in vitro*, whereas vildagliptin and saxagliptin are less selective with regard to DPP8 and 9, and linagliptin with regard to FAP (28). Their clinical efficacy and safety in the use of type 2 diabetes seem comparable as far as can be judged from the data available.

There is a growing interest toward a use outside type 2 diabetes as it has become clear that DPPIV inhibitors have pleiotropic effects. While negative effects have been found in heart failure (53), some studies suggest them as a possible therapeutic strategy in cardiovascular pathologies (28, 54). The SITAGRAMI trial and follow-up studies revealed that the combination of a DPPIV inhibitor with granulocyte-colony-stimulating factor or in monotherapy presents a therapeutic option after myocardial infarction (55, 56). As stated above, the mechanism is not yet clear but may be explained by a longer biological half-life of DPPIV substrates, glucagon-like peptide-1, B-type natriuretic peptide, and stromal cell-derived factor-1 α/β. All three peptides have a cardioprotective effect that is abolished by DPPIV-mediated cleavage. For an extensive review of the involved substrates, see Matheeussen et al. (43).

a DPP8 and DPP9 substrate as well (71). Efforts have been made to find intracellular DPP8 and 9 substrates using a peptidomic approach (72), but so far it has been hard to attribute physiological relevance to the possible substrates beyond the role of DPP8 and 9 in intracellular peptide turnover (73).

The physiological functions of DPP8 and DPP9 are still not properly understood. Mainly, a lack of available knockout animals, specific inhibitors, and substrates has hampered progress (24). A mouse model has been established with a targeted inactivation of DPP9 enzymatic activity (74), but homozygous DPP9 inactive neonates die within 8–24 h after birth. Despite these limitations, some indications toward their role are surfacing. Using immunohistochemistry, DPP8 and 9 were found associated with spermatozoids and spermatids and the short mRNA of DPP8 is predominantly expressed in testes (75, 76), suggesting a role in spermatogenesis and male fertility. Recent work has found SUMO1 to be an allosteric activator of DPP9 (77), whereas a small peptide corresponding to the interaction surface of SUMO1 is a non-competitive inhibitor of DPP8 and DPP9 (78). A genomewide association study has linked DPP9 to idiopathic pulmonary fibrosis (79).

Finally, a number of studies have shown a role for DPP8 and DPP9 in apoptosis (71, 80–83). Two studies showed that overexpression enhanced induced apoptosis and impaired cell adhesion and migration (80, 81). Conversely, DPP8/9 inhibition in tumor cells decreased the number of viable cells because of a decreased cleavage of pro-apoptotic NPY (71). In macrophages, inhibition caused a marginal, yet significant increase in apoptosis, independent of NPY cleavage (82). Interestingly, vildagliptin, a DPPIV inhibitor already on the market to treat type 2 diabetes, but with poorer selectivity toward DPP8 and 9, was shown to enhance parthenolide's anti-leukemic activity through its inhibition of DPP8 and 9, and not DPPIV (83).

# **Dipeptidyl Peptidase II and Prolyl Carboxypeptidase**

Prolyl carboxypeptidase, also called angiotensinase C or lysosomal Pro-X carboxypeptidase, is a lysosomal carboxypeptidase sharing strong sequence homology with the likewise lysosomal DPPII (4, 84). PRCP preferentially cleaves off the C-terminal amino acid when Ala or Pro is in the penultimate position, while DPPII targets N-terminal X-Pro or X-Ala dipeptides (85, 86). In addition to a structural similarity, PRCP and DPPII have partially overlapping substrate specificities due to DPPII's preference for tripeptide substrates (4). Perhaps surprisingly, Gly-Pro-pNA and Ala-Pro-pNA, two typical synthetic DPP substrates, have actually been used to perform PRCP activity measurements (87).

Prolyl carboxypeptidase is particularly known as one of the key enzymes of the renin–angiotensin system (RAS). It inactivates the vasoactive peptides angiotensin II and angiotensin III by cleaving off the C-terminal Phe (88). α-Melanocyt-stimulating hormone 1–13, an anorexigenic neuromodulator, is inactivated by PRCP, implying a role in body weight control (89). Based on the involvement of PRCP in the conversion of these peptide hormones, the enzyme has also been associated with diseases, such as hypertension, diabetes mellitus, obesity, inflammation, and cardiovascular dysfunction (90, 91).

Dipeptidyl peptidase II has no known natural substrates. The DPPIV substrate substance P has been shown to be cleaved by DPPII *in vitro* (3), but much less efficiently, casting doubt over any physiological relevance. It has been shown that inhibition or silencing of DPPII causes apoptosis of quiescent *G*0 lymphocytes (92–94). On the other hand, a highly specific DPPII inhibitor, UAMC00039, did not induce apoptosis, autophagy, or necrosis in human leukocytes (25, 95), but this study did not specifically look at quiescent cells or lymphocytes. Finally, changes in DPPII activity levels have been observed in a number of pathologies, such as neurodegenerative disorders, myopathies, cancer, and gastro-intestinal disorders (4).

# **Prolyl Oligopeptidase**

Prolyl oligopeptidase is an oligopeptidase with endopeptidase activity. It has been shown to be localized in the cytoplasm (96– 99), but given its ability to inactivate several neuropeptides*in vitro* by limited proteolysis (100–115), its involvement in the *in vivo* generation of immunoactive peptides *N*-acetyl-prolyl-glycylproline and *N*-acetyl-seryl-aspartyl-lysyl-proline (116, 117), and its presence in plasma (118, 119), it most likely also has an extracellular role.

Initial interest for PREP derived from the positive effects of PREP inhibitors on scopolamine-induced amnesia in rats (120– 123). PREP inhibition was also found to promote neuronal survival and neurite outgrowth of cerebellar granule cells (124). However, a recent study in mice shows that the lack of PREP *in vivo* causes a reduction of synaptic spine density in the hippocampal region along with reduced long-term potentiation and memory functions (125).

Many of PREP's functions are mediated through its interactions with other proteins. PREP is known to interact with GAP-43 (126, 127), α-tubulin (96), and GADPH (128). Its most studied interaction is with α-synuclein (126), reviewed in Ref. (129). PREP and α-synuclein have been shown to co-localize in cell models of stress and in the substantia nigra of post-mortem Parkinson's disease brain (11, 130). *In vitro,* the aggregation rate of α-synuclein increases in the presence of high concentrations of PREP, which is abolished through active site inhibitors of PREP and absent with a catalytically impaired PREP mutant (131). *In vivo*, PREP inhibition reduces α-synuclein aggregates in a cellular and animal model for Parkinson's disease (11).

# **The DPP Family in the Immune System**

# **The DPP Family in the Innate Immune System** DPP Family Members in Monocytes and Macrophages

The role of DPPIV in monocytes and macrophages has been somewhat contested. Whereas DPPIV's presence on monocytes and macrophages has been shown repeatedly in mice and rats (132–134), its expression in human monocytes and macrophages is less obvious. **Figure 2** shows an overview of the expression of DPPIV throughout the immune system. In visceral obesity, DPPIV expression is low on peripheral blood monocytes, macrophages, and dendritic cells, but it is upregulated *in vitro*

after differentiation and activation of isolated monocytes into macrophages or dendritic cells, and *in vivo* locally in adipose tissue (135). Interestingly, the authors showed that macrophage- or dendritic cell-associated DPPIV most likely binds ADA, promoting local degradation of adenosine, a T-cell proliferation suppressor, thereby inducing T-cell proliferation (135). Three other studies also found no to low DPPIV expression or activity associated with human monocytes and/or macrophages (82, 136–138). Others have investigated DPPIV in monocyte- or macrophage-like cell lines (136, 137, 139–144). In HL-60 cells, its expression has been found to be regulated by differentiation into macrophage-like cells (139). DPPIV inhibitor alogliptin can affect ERK activation, MMP1 and IL-6 secretion in U937 cells (140, 141). However, these studies employed alogliptin at concentrations lower than its IC50 for DPPIV. It is therefore questionable whether the observed effects were mediated by DPPIV at all. On the other hand, proliferation is reduced in the presence of a DPP inhibitor in U937 cells expressing high levels of DPPIV, but not in the same cell type expressing low levels of DPPIV (144). Moreover, the same inhibitor causes the former cells to secrete lower amounts of IL-1β, but higher amounts of TNFα (144). It could be that inhibition merely increases TNFα's half-life, as DPPIV has been implicated in its degradation in U937 cells (137). In THP-1 cells, DPPIV inhibitors alogliptin and sitagliptin both reduced these cells' chemotactic potential (142). DPPIV inhibitors sitagliptin and NVPDPP728 also reduced NLRP3, TLR4, and IL-1β expression and increased GLP-1R expression in THP-1 cells and this effect was blocked through PMA differentiation (143). Importantly, such cell lines have been derived from different types of myeloid leukemia, and as it is known that DPPIV expression is often dysregulated in cancer (47–51), the physiological relevance of these findings remains uncertain. FAP has been shown on tumor-associated macrophages in human breast cancer (145).

Dipeptidyl peptidase 8/9 activity has been found in human monocytes and U937 cells (136). DPP8 was found associated with activated microglia/macrophages in a rat model of cerebral ischemia (146). DPP8 and 9 are abundantly present in macrophage-rich regions of atherosclerotic plaques (82). Interestingly, DPP9 is upregulated after*in vitro* monocyte-to-macrophage differentiation. Moreover, inhibition or RNA silencing of DPP9 attenuates pro-inflammatory M1, but not M2, macrophage activation (82).

In rats, DPPII is expressed in tissue-resident macrophages (147, 148). Humans show DPPII activity in monocytes as well as U937 cells (25, 136). Human blood derived alveolar macrophages show high-PRCP activity (138, 149). Interestingly, in a mouse *in vivo* angiogenesis assay, macrophage infiltration into the wound was increased in mice with a PRCP deletion (150).

Prolyl oligopeptidase activity has been shown in mouse and rat peritoneal macrophages and in rat pulmonary macrophages (134, 151, 152). Its activity in mouse peritoneal macrophages is increased after thioglycollate ellicitation (134). In addition, PREP has been identified as a neurotoxic component in the supernatant of activated THP-1 cells, which are monocyte-like cells (153). Apparently, these cells secrete PREP upon activation with IFNγ and LPS and partly because of this, their supernatant is toxic to neuroblastoma SH-SY5Y cells, as shown through the use of PREPspecific inhibitors (153). PREP's mode of action in this remains unclear.

### DPP Family Members in Granulocytes

Recently, a study showed that DPPIV acts as a chemorepellent for human and murine neutrophils (154). Adding recombinant DPPIV to purified human neutrophils in an Insall chamber causes the neutrophils to migrate away from the higher concentration of DPPIV. This effect is blocked by DPPIV inhibitors, meaning that the effect is mediated through DPPIV's enzymatic activity, although a candidate substrate is not obvious. Moreover, in a mouse model of acute respiratory distress syndrome, oropharyngeal aspiration of DPPIV prevented accumulation of neutrophils in the lung (154). By contrast, PREP is involved in the generation of prolyl-glycyl-proline, a collagen fragment that is an efficient neutrophil chemoattractant (155). Human peripheral blood neutrophils contain PREP activity and are themselves capable of generating prolyl-glycyl-proline after LPS-activation, alluding to a self-sustaining pathway of neutrophil inflammation (116). PRCP is also abundantly expressed in human neutrophils (90).

The recruitment of eosinophils is affected by DPPIV activity. CCL11, also known as eotaxin, is a DPPIV substrate and cleavage by DPPIV prevents the activation of its receptor CCR3 (156). In rats, it was shown that administration of CCL11 results in eosinophil recruitment and this recruitment is significantly more effective in DPPIV-deficient F344 mutants (156).

Finally, DPPII activity has been reported in the granules of mast cells in several publications (147, 148, 157). It is released from peritoneal mast cells upon degranulation and is apparently inhibited by histamine and Zn2+ at concentrations present in the granules of mast cells (157).

## DPP Family Members in Natural Killer Cells

Dipeptidyl peptidase 4 is present in low amounts on freshly isolated human NK cells and its expression is only upregulated in a small subpopulation after IL-2 stimulation (158). In that study, it was also shown that DPPIV inhibition suppresses DNA synthesis and cell cycle progression of NK cells, but these effects may be DPP8/9 mediated as the inhibitors used in that study are now known to also inhibit DPP8/9 activity (159). Another study shows that DPPIV is actually only expressed by a small subpopulation of peripheral NK cells (160). The natural cytotoxicity of NK cells is not influenced by the presence or absence of DPPIV on their cell surface (158, 160). However, DPPIV-negative NK cells show significantly less CD16-dependent lysis than DPPIV-positive NK cells (160). Interestingly, NK cytolytic function against tumor cells was diminished in DPPIV-deficient rats in a model for lung metastasis (161).

**Figure 3** shows an overview of published data on the DPP family in the innate immune system.

# **The DPP Family in the Adaptive Immune Response**

### DPP Family Members in Humoral Immunity

Only about 5% of freshly isolated CD20-positive B cells express DPPIV, but this fraction grows significantly upon pokeweed mitogen (PWM) or *S. aureus* protein stimulation (162). Similar to NK cells, DPPIV inhibitors significantly suppress DNA synthesis in B-lymphocytes (162), but again these inhibitors are now known to also inhibit DPP8 and 9 (159). Mouse spleen-derived B-lymphocytes only express low amounts of DPPIV mRNA (163). DPP8 and 9 mRNA, on the other hand, are expressed at much greater levels in these cells, and they are upregulated in Raji cells, a B-lymphocyte-like cell line, after PWM, LPS stimulation or mitomycin c treatment, and downregulated after DTT treatment (163). DPP8 and 9 have also been shown immunohistochemically in human lymph follicular lymphocytes (164). DPPII activity has also been shown in human B-lymphocytes (25).

# DPP Family Members in Cell-Mediated Immunity

Dipeptidyl peptidase IV was originally described as a surface marker for T-lymphocytes, in which case it is better known as CD26, and later more specifically for a subset of CD4-positive memory cells, CD4+ CD45RO+ CD29+ cells, which respond maximally to recall antigen tetanus toxoid and induce B-cell IgG synthesis (165, 166). Indeed, CD26 surface expression is augmented along with the antigen sensitivity of a particular CD4+ T-cell clone (167). CD26high CD8+ T-cells belong to the early effector memory T-cell subset (168). CD26 is also a marker for T-cell activation (165, 169–171). CD26 expression on CD4+ Tcells correlates with TH1 responses. Stimuli that typically induce a TH1 phenotype tend to induce CD26 expression (172). Additionally, the CD4+ T cells capable of transendothelial migration *in vitro* are characterized by a bright expression of CD26 (173, 174), but CD26 does not seem to be actually involved in T-cell adhesion to endothelial cells or fibroblasts (175). Recently, it was shown that up to 98% of all TH17 cells show very high CD26 expression, with mean fluorescent intensity on these cells almost twice as high as on TH1 or TH2 cells. Therefore, the authors of this study suggest CD26 as a marker for TH17 cells (176). Conversely, CD26 has been proposed as a negative marker for the selection Treg cells due to its very low-surface expression on these cells (176–178).

CD26 is also a costimulatory molecule for T-cell activation. Crosslinking of CD26, along with CD3, stimulates T-cell activation and proliferation (168, 179, 180). CD26 can also directly activate T-cells in an alternative activation pathway, but this requires the presence of the TCR/CD3 complex (181–183). During costimulation, CD26 is mannose-6 phosphorylated and internalized, the latter of which is mediated in part by its interaction with M6P/IGFIIR (184). It then localizes to lipid rafts where it might interact with CD45, required for TCR signaling, facilitating colocalization of this molecule with TCR signaling molecules (185, 186). A number of candidate binding partners for costimulation have been proposed. ADA and CD26 are known binding partners (187). Even though ADA binding to CD26 does not seem to be essential for immune functions in humans (188), the nanomolar affinity of this interaction probably reflects its importance (189). Indeed, association with free ADA or ADA presented by ADA-anchoring proteins on dendritic cells seems to costimulate T-cells through CD26 binding (190, 191). On the other hand, it has been shown that soluble DPPIV enhances T-cell proliferation independent of its enzyme activity or ADA-binding capability (192). Interestingly, the ADA–CD26 interaction can be inhibited by HIV-1 external envelope protein gp120 and this requires interaction of gp120 with CXCR4 (189). In fact, evidence suggests a physical association between CXCR4 and CD26

on peripheral blood lymphocytes (193). Fibronectin is another known binding partner of CD26 involved in T-cell costimulation (194–196). Finally, CD26 interacts with caveolin-1 on monocytes. This interaction causes an upregulation of CD86 on these cells, which potentiates antigen-specific T-cell activation (197). Most studies seem to find no need for DPPIV's enzymatic activity for succesful costimulation, as evidenced through the use of inhibitors and catalytically impaired DPPIV mutants (198–201).

Dipeptidyl peptidase 8 and 9 are present in baboon spleen interfollicular T-lymphocytes and Jurkat T cells (164). They are upregulated in the latter after PWM and LPS, but not PHA, stimulation (163, 202, 203). Activation of PWM-stimulated T-cells is suppressed after DPPIV/8/9 inhibition. Moreover, DNA synthesis and T-cell proliferation are reduced, as well as production of IL-2, - 10, -12, and IFN-γ. This is due to an induction of TGF-β secretion (159, 204–207). Inhibition also upregulates CTLA-4 and downregulates DPPIV expression (206, 208). These observations might be physiologically relevant as endogenous inhibitors of DPPs are known which have similar effects in cell-based experiments as the synthetic inhibitors (209, 210).

Dipeptidyl peptidase II activity is higher in T-lymphocytes than in B-lymphocytes (25) and absence of DPPII steers T-lymphocytes toward a TH17 phenotype. T-lymphocytes of DPPII KO mice hyperproliferate and secrete IL-17 after CD3 crosslinking or after *in vivo* priming and *in vitro* antigen-specific restimulation (211). PREP activity has also been shown in mouse T-lymphocytes (212). Its activity is significantly higher in immature, double-positive thymocytes compared to mature, single-positive thymocytes, or peripheral T-cells. T-cells stimulated with Con A followed by IL-2 show a time-dependent increase in PREP activity and pretreatment of cells with a PREP inhibitor renders them resistant to activation-induced cell death (212).

**Figure 4** shows an overview of *in vitro* data on DPP involvement in primary human T cell activation.

# **The DPP Family in Inflammatory Disease**

The DPP family has been reported to be dysregulated or even involved in a number of inflammatory disorders. Expression levels of a number of family members are modulated in rheumatoid

**FIGURE 4 | Overview of** *in vitro* **data on DPP involvement in primary human T cell activation**. **(A)** M6P/IGFIIR associates with mannose-6-phosphorylated DPPIV causing it to associate with CD45 in lipid rafts. This facilitates co-localization with the TCR signaling molecules for T cell costimulation. **(B)** Interaction of ADA presented by ADA-anchoring proteins on dendritic cells with DPPIV on T cells

causes costimulation. **(C)** Interaction of DPPIV on T cells with caveolin-1 on monocytes induces the expression of CD86 on the latter. Interaction of CD86 with CD28 costimulates T cells. **(D)** Inhibition of DPP8/9 induces TGFβ in PWM-stimulated T cells. TGFβ attenuates T cell activation. **(E)** Inhibition or absence of DPPII steers T cells toward TH17 differentiation.

arthritis. Whereas the density of CD26 on peripheral T cells is increased in patients, it is low on synovial fluid T cells (213–215). DPPIV activity in plasma, serum, or synovial fluid of patients has also been found to be decreased, similar to results in several rat models of arthritis (216–222). Interestingly, rats resistant to induction of arthritis show higher plasma DPPIV levels (222). By contrast, DPPII and PREP activity are increased in serum or synovial fluid of arthritis patients (219–221). Likewise, FAP immunoreactivity is much higher in fibroblast-like synoviocytes of rheumatoid arthritis patients compared to osteoarthritis controls (223). DPPIV's involvement in rheumatoid arthritis has been studied, but remains unclear. On the one hand, inhibition can suppress development of arthritis in rats (224). Note, however, that effects mediated through other DPPs are hard to exclude as these inhibitors were developed before DPP8 and DPP9 were discovered. On the other hand, induced arthritis is more severe in DPPIV-deficient mice (216). This may be due to increased levels of circulating CXCL12 (216), a DPPIV substrate shown to be involved in rheumatoid arthritis. Several case reports in patients seem to suggest a link between the development of rheumatoid arthritis and the use of DPPIV inhibitors (225–227). PRCP has also been associated with rheumatoid arthritis as its activity was shown in synovial fluid isolated from arthritic joints (149).

Inflammatory bowel disease shows a distinct expression pattern of the DPP family. DPPIV serum or plasma activity seems to be lower in patients, whereas there is an increase of circulating CD26+CD25+ cells with a higher CD26 surface expression (228, 229). FAP is heavily expressed by myofibroblasts in the submucosa strictures in Crohn's disease, and is upregulated after stimulation with TNFα or TGFβ (230). In a mouse model, colonic DPPII and DPP8 mRNA and DPPII activity are increased, while colonic DPP8/9 activity only increases significantly in mice that are also DPPIV knockouts (231). In mouse models, inhibition or abrogation of DPPIV seems to at least partially ameliorate symptoms, possibly by increasing circulating GLP-2, impairing neutrophil recruitment, and maintaining Treg populations (231–236). Some of those beneficial effects may be mediated in part by the other DPPs, as additive effects were found for DPPIV KO and the DPP inhibitors (231, 234, 237). A recent study suggests that the ameliorative effects of DPP inhibitors are most likely not mediated through GLP-2 protection (238).

The DPP family has also been studied in neuroinflammation. Ischemia-induced neuroinflammation in rats prompts a distinct expression and activity pattern of the DPPs. In the days following ischemia, the brain of these rats undergoes a complex reorganization of DPP expression with changes in mRNA, protein, and activity levels of DPPII, 4, 8, and 9 in cortical neurons, microglia, and macrophages (146). Similarly, PREP seems to be associated with astrocytes and microglia in lesioned inflamed brains of rats (239). DPPIV and PREP also may be involved in multiple sclerosis. CD26+ T cells were found to correlate with disease scores (240). Soluble DPPIV levels are elevated in cerebrospinal fluid of patients (241). Plasma PREP activity, on the other hand, is lower in patients with relapsing–remitting or primary progressive multiple sclerosis and in clinically isolated syndrome (118, 119). Interestingly, PREP inhibition seems to aggravate symptoms in a mouse model of multiple sclerosis (118).

In systemic lupus erythematosus, DPPs also seem to be dysregulated. In mouse models, DPPII and PREP activities are increased in plasma, spleen, kidney, and liver, whereas DPPIV activity is decreased (221, 242). Human patients also show elevated DPPII and reduced DPPIV activities in serum, along with reduced numbers of CD26+ T cells (221, 243). Interestingly, serum DPPIV levels are inversely correlated with disease score (243). FAP immunoreactivity is decreased in the synovium of lupus patients (244).

Finally, DPPIV has been studied in psoriasis, an immunemediated chronic inflammatory disorder with primary involvement of skin and joints. Its mRNA, protein levels and activity are higher in psoriatic skin samples (245, 246). By contrast, serum DPPIV levels and activity seem to be lower in patients (247, 248), accompanied by a reduction of peripheral CD8+CD26+ T cells (249, 250). Two case reports suggest a link between the use of DPPIV inhibitor sitagliptin and psoriasis. While one woman developed a psoriaform eruption 6 days after starting sitagliptin treatment (251), another patient's psoriatic lesions gradually diminished and were effectively gone 3 months after the start of sitagliptin treatment (252).

# **The DPP Family in Atherosclerosis**

Dipeptidyl peptidase IV has recently received much attention for its potential as a therapeutic target for the treatment of atherosclerosis (**Box 3**) (253). This is not surprising considering the current use of DPPIV inhibitors in the treatment of T2D and the fact that T2D is associated with a higher risk for atherosclerosis (28, 254). In the ApoE*−*/*−* mouse model of atherosclerosis, DPPIV inhibition generally reduces plaque area and monocyte and macrophage plaque infiltration (255–257). A reduction in the number of plaque lesions or in smooth muscle cell content have also been observed (255, 256), as well as lower plaque MMP9 and higher plaque collagen levels, suggesting increased plaque stability (258). One study reported effects of DPPIV inhibition on atherosclerotic plaques of only diabetic ApoeE*−*/*−* mice (141), but more recently, Terasaki et al. found similar effects in non-diabetic and diabetic ApoE*−*/*−* mice (259). Likely, such differences can be explained by the fact that different DPPIV inhibitors were employed. Effects of DPPIV on atherogenesis similar to those observed in ApoE*−*/*−* mice have been reproduced in LDLR*−*/*−* mice (142, 260). In human atherosclerotic plaques,

### **BOX 3 Atherosclerosis.**

Atherosclerosis is the most common underlying cause of cardiovascular diseases and should be regarded as an inflammatory disease. It starts with dysfunction of the endothelium leading to the expression of leukocyte adhesion molecules, such as selectins and integrins. Locally produced proinflammatory cytokines attract the immune cells into the inner layer of the endothelium. However, not only leukocytes are found in the plaque but also low-density lipoprotein particles (LDL) and their oxidized counterparts (oxLDL). In the plaque, monocytes differentiate into macrophages, phagocytose the oxLDL and turn into so-called pro-atherogenic foam cells. This process leads to a self-sustaining, local inflammation leading to plaque growth, and migration of smooth muscle cells into the core. A plaque is defined as stable as long as it is contained by a thick fibrous cap. However, the latter is slowly degraded by the proteolytic enzymes from the leukocytes. This eventually leads to rupture and the formation of arterial thrombi (264, 265).

DPPIV immunoreactivity could only be found on endothelium of neovessels (82). It was recently found that DPPIV activity may be a predictor for the onset of atherosclerosis in otherwise healthy Chinese individuals (261). Another prospective study investigated the influence of vildagliptin or sitagliptin treatment on intima-media

thickness, a surrogate marker for atherosclerosis. This study found that treatment with vildagliptin or sitagliptin reduced intima-media thickness, suggesting that DPPIV inhibition might be beneficial in atherosclerosis in humans as well (262). Moreover, treatment naïve T2D patients treated with alogliptin

for 3 months saw a significant decrease in their circulating atherogenic lipids (263).

It has been suggested that DPPIV inhibitors' anti-atherogenic effects are mainly mediated through decreased monocyte infiltration, as DPPIV inhibitors suppress monocyte activation and chemotaxis *in vitro* (142, 258). DPPIV inhibition also reduces *in vitro* foam cell formation in exudate peritoneal macrophages from ApoE*−*/*−* mice (255). Moreover, soluble DPPIV stimulates *in vitro* proliferation of smooth muscle cells and this can be reduced through the addition of a DPPIV inhibitor (256, 260). Finally, active circulating GLP-1 levels are augmented and this improves endothelial dysfunction (259, 266). Probably, DPPIV inhibition improves atherosclerosis through a combination of all these mechanisms. Indeed, incretin antagonists only partially attenuate the anti-atherogenic effects of DPPIV inhibition, suggesting that other mechanisms beyond incretin preservation are in play (259). Interestingly, monocyte–endothelial cell adhesion is abrogated by an anti-SDF-1α antibody *in vitro* (267). LDL seems to induce SDF-1α expression and leads to smooth muscle cell proliferation and inhibition of cell apoptosis (267, 268). SDF-1α is a DPPIV substrate, which loses its biological activity after cleavage (216). As DPPIV inhibition seems to improve atherosclerosis, whereas intact SDF-1α appears to be deletorious, it could be argued that SDF-1α cleavage by DPPIV does not play a major role in atherosclerosis.

Dipeptidyl peptidase 8 and 9 have been found to be abundantly present in the macrophage-rich regions of human atherosclerotic plaques and considering DPP9's role in macrophage activation, it might potentially be involved in atherogenesis (82). FAP expression is enhanced in some, but not all types of human atheromata. It is found on smooth muscle cells, and its expression correlates with macrophage burden, probably due to the fact that TNFα upregulates FAP in smooth muscle cells *in vitro*. As it is mainly associated with collagen-poor regions and can digest type I collagen and gelatin *in vitro*, FAP probably contributes to plaque instability (269).

# **References**

Interestingly, many of the studies reviewed above show the potential of targeting DPP family members for the treatment of atherosclerosis (see **Figure 5**). FAP inhibition might reduce plaque instability by decreasing collagen breakdown; DPP9 inhibition is likely to attenuate M1 macrophage activation, reducing the local inflammatory cascade; DPPIV inhibition may decrease monocyte infiltration, foam cell formation, improve endothelial dysfunction, and reduce smooth muscle cell proliferation; and finally, PREP inhibition might reduce neutrophil infiltration, preventing endothelial dysfunction, and monocyte infiltration. All of this shows the possibilities of repositioning DPPIV inhibitors, currently being used to treat type 2 diabetes, as well as the potential of targeting other members of the DPP family.

# **Conclusion**

Caution should be taken when interpreting results from literature data based on DPP inhibitors, especially from older studies. It is now known that, under the experimental conditions used, many of these inhibitors are not specific for one particular family member. The reported findings, however, remain interesting. This review has shown extensive involvement of members of the DPP family in the immune system. It is clear that these enzymes hold great potential as targets for the treatment of certain inflammatory disorders. Particularly, the possibility of targeting DPP family members for the prevention and treatment of atherosclerosis warrants further investigation.

# **Acknowledgments**

This work was supported by the University of Antwerp (GOA2009–2012) and the Flanders Research Foundation (FWO; Grant G0141.12). YW and KK are research fellows of the Flanders Research Foundation. Images were created based on Servier Medical Art licensed under Creative Commons Attribution 3.0 Unported License.

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signal targeting the active peptidase to the nucleus. *Cell Mol Life Sci* (2014) **71**(18):3611–26. doi:10.1007/s00018-014-1591-6

each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro. *Eur J Biochem* (1983) **137**:75–87. doi:10.1111/j. 1432-1033.1983.tb07798.x

region of the hippocampus, impaired LTP, and spatial learning and memory. *Cereb Cortex* (2013) **23**:2007–14. doi:10.1093/cercor/bhs199

injured liver. *World J Gastroenterol* (2013) **19**:2883–93. doi:10.3748/wjg.v19. i19.2883

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Waumans, Baerts, Kehoe, Lambeir and De Meester. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Dipeptidyl peptidase-4 regulation of SDF-1/CXCR4 axis: implications for cardiovascular disease

*Jixin Zhong\* and Sanjay Rajagopalan\**

*Division of Cardiovascular Medicine, University of Maryland, Baltimore, Baltimore, MD, USA*

Dipeptidyl peptidase-4 (DPP4) is a ubiquitously expressed protease that regulates diverse number of physiological functions. As a dipeptidase, it exerts its catalytic effects on proteins/peptides with proline, alanine, or serine in the penultimate (P1) amino acid residue from the amino terminus. The evidence to date supports an important effect of DPP4 in catalytic cleavage of incretin peptides and this perhaps represents the main mechanism by which DPP4 inhibition improves glycemic control. DPP4 also plays an important role in the degradation of multiple chemokines of which stromal cell-derived factor-1 (SDF-1, also known as CXCL12) is perhaps an increasingly recognized target, given its importance in processes, such as hematopoiesis, angiogenesis, and stem cell homing. In the current review, we will summarize the importance of DPP4-mediated enzymatic processing of cytokines/chemokines with an emphasis on SDF-1 and resultant implications for cardiovascular physiology and disease.

Keywords: SDF-1, CXCR4, dipeptidyl peptidase-4, cardiovascular, chemokine

# Introduction

Dipeptidyl peptidase-4 (DPP4) is a type-II integral transmembrane glycoprotein that has recently gained attention owing to its role in the catalytic degradation of incretins and as a receptor for entry for the Middle Eastern respiratory syndrome (MERS) virus. DPP4 is best known for its catalytic function, whereby it proteolytically cleaves a number of peptide and protein substrates. It exhibits a strong preference for peptides with proline, serine, or alanine as the penultimate amino acid from the amino terminus. The N-terminus of glucagon insulotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) consist of Tyr–Ala and His–Ala, respectively, rendering them excellent substrates for DPP4. In addition to its role in modulation of incretin peptides, such as GLP-1 and GIP, DPP4 regulates immune responses via cleavage of many cytokines and chemokines, including stromal cellderived factor-1 (SDF-1, also known as CXCL12), involved in immune function and physiological functions, such as angiogenesis. In this review, we will focus on the regulatory aspects of DPP4 on chemokines, such as SDF-1, and its potential implications in the pathogenesis and management of cardiovascular disease.

# An Overview of DPP4 Biology

Dipeptidyl peptidase-4 also called cluster of differentiation-26 (CD26) or adenosine deaminasebinding protein (ADA-binding protein), is a member of S9b peptidases. The S9b family consists of structurally homologous proteolytic enzymes, including DPP2 (also called quiescent cell proline dipeptidase, QPP), DPP4, DPP8, DPP9, and fibroblast activation protein (FAP). DPP4 was first

### *Edited by:*

*Heidi Noels, Institute for Molecular Cardiovascular Research, Germany*

### *Reviewed by:*

*Hideki Nakano, National Institute of Environmental Health Sciences, USA Ji Ming Wang, National Cancer Institute at Frederick, USA*

### *\*Correspondence:*

 *Jixin Zhong and Sanjay Rajagopalan, Division of Cardiovascular Medicine, University of Maryland, Baltimore, 20 Penn Street, Health Science Facility II, Room S022, Baltimore, MD 21201, USA<EMAIL_ADDRESS>srajagopalan@medicine. umaryland.edu*

### *Specialty section:*

*This article was submitted to Chemoattractants, a section of the journal Frontiers in Immunology*

*Received: 06 July 2015 Accepted: 03 September 2015 Published: 25 September 2015*

### *Citation:*

*Zhong J and Rajagopalan S (2015) Dipeptidyl peptidase-4 regulation of SDF-1/CXCR4 axis: implications for cardiovascular disease. Front. Immunol. 6:477. doi: 10.3389/fimmu.2015.00477*

identified as a new dipeptide naphthylamidase in 1966 (1) and subsequently found to be identical to T cell activation antigen CD26, ADA-binding protein, mouse thymocyte-activating molecule, and rat liver membrane glycoprotein gp110 (2). DPP4 consists of a short N-terminal intracellular domain (6 residues), a 22-residue-long transmembrane α-helix domain (23 amino acids), and a large C-terminal extracellular domain. The C-terminal extracellular domain is responsible for its catalytic activity and binding to a number of ligands, such as ADA and matrix proteins (3). The catalytic activity of DPP4 depends on its dimerization and glycosylation of specific residues (2). DPP4 can also assemble into tetramers on the cell surface, which may involve the linkage of dimers located on the surface of two different cells, enabling it to function as a cell–cell communication molecule. In addition to its membrane-bound form, DPP4 also circulates as a soluble form in the plasma, which lacks the cytoplasmic and transmembrane domain with preserved catalytic activity. Soluble DPP4 (sDPP4) is a homodimer with a molecular weight range of 210–290 kDa (4), but can form higher molecular weight assemblies migrating as 900-kDa complexes (5). Whether sDPP4 is cleaved from the membrane or is secreted is unclear. For instance, studies investigating viral liver infection suggested that sDPP4 is shed from membrane-bound DPP4 (6). sDPP4 has, however, also been detected in the lumen of secretory granules in pancreatic α cells and in the exocytic secretory lysosomes of natural killer cells (7, 8). sDPP4 is commonly elevated in many disorders, such as solid tumors, reactive airways disease, hepatitis C, type 2 diabetes, and obesity (6, 9, 10).

### Tissue Distribution and Cell Specific Expression and Phenotype of *Dpp4*−/− Mice

Dipeptidyl peptidase-4 is widely distributed throughout the body (**Table 1**), with particularly high expression on the apical surface of endothelial and differentiated epithelial cells. Bone marrow cells, brush border of the small intestine, proximal tubular cells, and glomerular cells in the kidney express high levels of DPP4 as well (11). DPP4 is present on endothelial cells and fibroblasts throughout the body. Among hematopoietic cells, DPP4 is expressed at the highest level on T cells with lower levels in monocytes and dendritic cells (12). DPP4 expression increases

as monocytes differentiate into antigen-presenting cells as well as during T cell activation (12, 13). DPP4 is expressed at high levels in kidney, spleen, lung, pancreas, and prostate (14). Mice lacking the gene encoding DPP4 are refractory to the development of obesity and hyperinsulinemia and demonstrate improved post-prandial glucose control (15, 16). Mice deleted for *Dpp4* are fertile and appear healthy. Only slight decrease of body weight in *Dpp4*−/− mice was observed compared to wild types. They have normal fasting blood glucose level, but shows reduced glycemic excursion after an oral glucose challenge (16). Increased intact insulinotropic form of GLP-1 and circulating insulin were seen in *Dpp4*−/− mice after oral glucose stimulation (16). Pair feeding and indirect calorimetry studies indicate that reduced food intake and increased energy expenditure accounted for the resistance to high fat diet-induced obesity in the *Dpp4*−/− mice. Ablation/ deletion of DPP4 is associated with improved metabolic control with improved insulin sensitivity, reduced pancreatic islet hypertrophy, and protection against streptozotocin-induced loss of β cell mass and hyperglycemia (15). Pharmacological inhibition of DPP4 enzymatic activity improves glucose tolerance in wildtype but not in *Dpp4*−/− mice. Interestingly, DPP4 inhibitor's also improve glucose tolerance in *Glp1r*−/− mice, indicating that DPP4 contributes to blood glucose regulation by controlling the activity of GLP-1 as well as additional substrates (16).

# DPP4 in Cytokine Processing

Dipeptidyl peptidase-4 has been shown to be able to cleave a number of chemokines and cytokines, including SDF-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), erythropoietin (Epo), regulated on activation normal T-cell expressed and presumably secreted (RANTES, also known as CCL5), macrophage-derived chemokine (MDC, also known as CCL22), eotaxin (also known as CCL11), monokine induced by IFN-γ (MIG, also known as CXCL9), IFN-γ-induced protein-10 (IP-10, also known as CXCL10), and interferon-inducible T-cell α chemoattractant (ITAC, also known as CXCL11) (**Table 2**). The regulation of cytokine levels through catalytic cleavage may influence their levels in tissue domains. The differential contribution of DPP4 in the regulation of each of these cytokines is obviously dependent on the levels of expression of DPP4, which may vary depending on the tissues, the cells predominantly expressing the cytokine of interest and the disease context. Additionally, circulating or cell free DPP4 may also contribute to catalytic activity. DPP4-mediated truncation of RANTES abolishes the chemotactic activity to monocytes but not to T cells (27). Eotaxin (CCL11) is an eosinophil chemotactic protein and has been shown to be involved in allergic responses. DPP4 truncation of eotaxin inactivates its chemotactic activity for eosinophils. DPP4-truncated eotaxin (3–74) shows impaired binding and signaling through CCR3. In addition, truncated eotaxin suppresses calcium signaling and chemotaxis of intact eotaxin (28). DPP4 inhibition by either genetic deletion or pharmacological inhibition, enhances eotaxin-induced mobilization of eosinophils into the blood and recruitment into the injury/injection site (29). MDC (CCL22) is a chemoattractant for monocytes, dendritic cells, NK cells, and chronically activated

### TABLE 2 | Cytokine substrates of DPP4.

*a Bold letters indicate dipeptides to be cleaved by DPP4.*

T lymphocytes (30, 31). DPP4-truncated MDC displays preserved chemotactic activity toward monocytes, but less potency toward lymphocytes and dendritic cells (32). CXCR3 interacts with MIG (CXCL9), IP10 (CXCL10), and ITAC (CXCL11), all of which are targets of DPP4. Cleavage of those chemokines by DPP4 attenuates their chemotactic activity, with the cleaved products serving as endogenous antagonists for CXCR3 binding (33).

Dipeptidyl peptidase-4 is also involved in the inactivation of multiple colony-stimulating factors (CSFs) and thus regulates hematopoietic stem cells (HSCs) and hematopoietic progenitor cell (HPC) function. The proliferative action of GM-CSF, G-CSF, IL-3, and Erythropoietin (Epo) on HPCs is enhanced in DPP4 knockout mice or by pretreatment with a DPP4 inhibitor (35). Catalytic inhibition of DPP4 or DPP4 deficiency promotes the engraftment of HSCs and HPCs after bone marrow transplantation in mice (35). DPP4-truncated CSFs may suppress the activity of their respective full-length CSF via antagonism (35).

# SDF-1 and CXCR4

Stromal cell-derived factor-1 is an 8-kDa peptide that is encoded by *Cxcl12* (36). It is a chemoattractant for T lymphocytes, bone marrow stem cells [such as HSC, endothelial progenitor cell (EPC), and mesenchymal stem cells (MSCs)], endogenous cardiac stem cells (CSCs), and adipose-derived regenerative cells (37–39). There are several isoforms of SDF-1 (SDF-1α–ζ), resulting from alternative splicing of its mRNA (40). Among these isoforms, SDF-1α is the best described. SDF-1α is expressed in many tissues, including bone marrow, heart, liver, kidney, thymus, spleen, skeletal muscle, and brain (36, 40–43). In the cardiovascular system, SDF-1α is expressed in stromal cells, endothelial cells, and cardiomyocytes (44, 45). SDF-1 is typically inactivated by exopeptidases, such as DPP4, matrix metalloproteinase (MMP)- 2, and -9 (34). Unlike cleavage of SDF-1 by DPP4 at position 2–3, MMPs cleave SDF-1 at position 4–5, leading to the loss of its binding activity to CXCR4 (46). The relative contribution of each of these peptidases in regulation of SDF-1 levels is unclear. CXCR4 is an alpha-chemokine receptor specific for SDF-1 and belongs to a family of G-protein-coupled receptors. CXCR4 is expressed on a range of progenitor cells (including hematopoietic, endothelial, and CSCs) and thus is important for cell migration and organ development during embryogenesis (39, 40, 47). Mice deficient for either CXCR4 or SDF-1 display abnormal B-lymphocyte, hepatic, and cardiac (ventricular septal defects) development, and die *in utero* (48–50). Loss-of-function CXCR4 mutations in humans also causes impaired neutrophil mobilization and B-cell lymphopenia (51). In addition to CXCR4, CXCR7 has also been suggested to be an important receptor for SDF-1 (52, 53). However, the relative contribution and interactions of CXCR4 and CXCR7 is not fully elucidated. The involvement of CXCR7 in cardiovascular disease, if any, is also not yet known (39). DPP4 may also play a more general role in regulating CSF activity and stem cell homing (35). It was previously believed that disruption of the interaction between CXCR4 receptor expressed by hematopoietic progenitors and SDF-1 expressed by bone marrow stromal cells is sufficient to detach anchored progenitors from their bone marrow niches, leading to their rapid mobilization to the peripheral blood. AMD3100 (also termed plerixafor) inhibits SDF-1-mediated migration *in vitro* by blocking the chemokine binding to its major receptor CXCR4 (54). AMD 3100 mobilizes immature progenitor cells from the bone marrow into the blood and has been approved for clinical mobilization in lymphoma and multiple myeloma patients undergoing autologous transplantation. When combined with G-CSF, AMD3100 synergistically augments mobilization of progenitor cells, with increased *in vitro* migration to SDF-1 gradients and facilitates repopulation of transplanted non-obese diabetic/severe combined immunodeficient mice (55). AMD 3100 has recently been shown to directly induce SDF-1 release from CXCR4+ human bone marrow osteoblasts and endothelial cells, with SDF-1 release from these cells into the circulation, representing a pivotal mechanism essential for steady-state egress and rapid mobilization of HPCs (56).

# DPP4 and SDF-1/CXCR4 Axis in Cardiovascular Disease

# SDF-1/CXCR4 and DPP4 Inhibition in Stem Cell Homing and Engraftment

The SDF-1/CXCR4 axis has been shown to be critical in tissue repair in multiple organ systems, including the eye, heart, kidney, liver, brain, and skin. Specific to the heart, the SDF-1/CXCR4 axis has been shown to be essential for cardiogenesis (57, 58). SDF-1 is now well known as a key regulator of stem cell migration to sites of tissue injury (44, 59). SDF-1 was first identified by Askari et al. as a key regulator of stem cell migration to ischemic cardiac tissue (44). CD34+ stem cells express the SDF-1 receptor CXCR4 at high levels (37, 60). During myocardial infarction, SDF-1 levels are elevated 1 h after infarction and return to baseline at day 7 and further reduced to a low level thereafter (44). Overexpression Zhong and Rajagopalan DPP4 in chemotaxis and cardiovascular disease

of SDF-1 in ischemic cardiomyopathy by either engineered cellbased or plasmid-based approach improved cardiac function in rats via enhancing stem cell homing and promoting revascularization of the infarct area (61, 62). Therefore, the ability to express SDF-1 locally is believed to enhance the vasculogenic potential of adult cardiac progenitor cells (63). However, the enhancement of endogenous stem cell-based repair appears to be blunted due to the short half-life of SDF-1 at the time of acute myocardial infarction owing to its degradation by proteases (44). As a major enzyme mediating the degradation of SDF-1, DPP4 may represent a potential target for improving stem cell homing with stem cell-based therapy. Preservation of SDF-1 by DPP4 inhibition has been shown to promote stem cell repopulation and homing to ischemic tissues. DPP4 inhibitors diprotin A or Val–Pyr, enhance chemotaxis of HSCs and HPCs and greatly increase homing and engrafting capacity of HSCs (64, 65). Pretreatment of HSC with DPP4 inhibitor diprotin A, enhanced their repopulation ability in lethally irradiated mice (66). Enhancement of engraftment of human CD34+ cord blood cells with DPP4 inhibition has also been observed in xenogeneic mouse recipients (NOD/SCID or NOD/SCID/beta 2null) (67, 68). Pretreating either donor cells *in vitro* or recipients *in vivo* is able to enhance the engraftment of stem cells (66, 69). In a lung transplantation model, systemic DPP4 inhibition by vildagliptin increases SDF-1 levels in plasma, spleen, and lung, accompanied by a significant increase of stem cells in the lung grafts. DPP4 inhibitor-treated mice also shows less alveolar edema compared with untreated recipients (70). Liebler showed that DPP4 inhibition enhances SDF-1/CXCR4 axis and increased the retention of human bone marrow-derived cells in the injured lungs of immune deficient mice by 30% (71). In addition to SDF-1, DPP4 inhibition also enhances bone marrow engraftment by preserving G-CSF and GM-CSF. Both G-CSF and GM-CSF are substrates for DPP4, with inhibition of DPP4 promotes bone marrow engraftment not only through SDF-1 but also CSF-dependent mechanisms (35). G-CSF and GM-CSF in turn may also increase the expression of DPP4 on CD34+ cells, which results in their decreased responsiveness to SDF-1 (72).

# SDF-1/CXCR4 and Regulation by DPP4 in Angiogenesis

Angiogenesis and vasculogenesis are an immensely complex process that requires the coordinated action of a multitude of cells, transcription factors, and cytokines working in concert in a precisely choreographed manner. It is widely believed that these processes can be recapitulated in the adult through the participation of a progenitor cell population of which EPCs are perhaps the best described and widely believed to be important building blocks for the assembly of functional vasculature in adults. While the origins of EPCs are still controversial, what is clear is that these cells have the capacity to differentiate into mature endothelial cells (73). Implantation of *ex vivo*-expanded EPC has been shown to improve neovascularization of injured tissues in animal models (74–76). SDF-1 plays a pivotal role in the trafficking and homing of EPCs to ischemic tissues (77–79). SDF-1 levels increase in plasma and ischemic tissue shortly after ischemic injury, in response to hypoxia which upregulates HIF-1α (79). HIF-1α upregulates SDF-1, by binding to the promoter of SDF-1 and initiating its transcription (80). *Ex vivo* priming with SDF-1, enhances the proangiogenic potential of EPC as evidenced by improved blood flow recovery when transplanted into a nude mouse model of hind-limb ischemia (81). Disease states, such as diabetes associated with upregulation of DPP4, may represent prototypical conditions associated with defective homing and integration of EPC's owing to rapid degradation of SDF-1 (82). Kanki et al. reported that SDF-1 could be cleaved by DPP-4 in both plasma and ischemic heart tissue (34). Shih demonstrated an improvement in EPC number and endothelial nitric oxide synthetase (eNOS) expression after DPP4 inhibition by MK-0626 (83).

# Therapeutic Applications of SDF-1 and DPP4 Inhibition-Mediated Prolongation of SDF-1 Effects in Cardiovascular Disease

Transient engineered cell-based or plasmid-based overexpression of SDF-1 in ischemic cardiomyopathy has been shown to improve cardiac function in animal models (62). In a study that compared the effects of SDF-1 overexpressed on MSCs alone or mesenchymal stem cells engineered to overexpress SDF-1 (MSC-SDF) on cardiac function in Lewis rats after acute myocardial infarction, tail vein infusion of MSC and MSC-SDF-1, 1 day after acute myocardial infarction, led to improved cardiac function by echocardiography by 70.7 and 238.8%, respectively, compared with saline controls. The beneficial effects of MSC-SDF transplantation were suggested to be mediated through preservation rather than regeneration of cardiac myocytes within the infarct area (84). Cardiac progenitor cell and CXCR4 expression on cardiac myocytes are required for further local trophic effects of MSC (85). The mechanism of action of SDF-1 overexpression in myocardial infarction and heart failure are likely multifactorial, including both systemic and direct trophic effects. An important effect of SDF-1 is its effect on the recruitment of CSCs to the infarct and infarct border zone (59). Delivery of MSCs engineered to overexpress SDF-1 at the time of acute myocardial infarction has been shown to lead to improvement in cardiac function (61). The myocardial repair initiated by endogenous stem cell appears blunted because of the natural short-term expression of SDF-1 at the time of acute myocardial infarction. In light of these effects in regulation of SDF-1, DPP4 inhibition has been suggested to be of potential benefit in cardiovascular diseases, such as myocardial infarction and peripheral artrerial disease. In combination with G-CSF, DPP4 inhibition augments myocardial regeneration and improves cardiac function after myocardial infarction in mice (86, 87). In combination with CXCR4 overexpression, diprotin A treatment has shown to improve myocardial function and repair of infarcted myocardium (88). A bioengineered protease-resistant form of SDF-1 has shown greater potency in promoting blood flow recovery after hind-limb ischemia (89) and improving cardiac function as well as capillary density in the infarcted heart (34). Dual injection of G-CSF and sitagliptin resulted in the mobilization of progenitor cells and relieved the symptom of end-stage heart failure in a 19-month-old boy (90). Protease-resistant forms of SDF-1 display an enhanced potency in improving blood flow in experimental peripheral artery disease and myocardial infarction (34, 89). It has been shown that parathyroid hormone treatment after myocardial infarction improves survival and myocardial function with potential involvement of enhanced homing of bone marrow-derived stem cells. Huber et al. demonstrated that parathyroid hormone serves as a DPP4 inhibitor and increases cardiac SDF-1 level, which in turn enhances CXCR4+ bone marrow-derived stem cell homing to ischemic heart and attenuates ischemic cardiomyopathy after infarction (91). Haverslag showed SDF-1 preservation by DPP4 inhibitor increases monocyte extravasation and thus accelerating perfusion recovery without detrimental side effects on plaque stability in atherosclerosis-prone *ApoE*−/− mice (92). **Figure 1** depicts modulation of SDF-1 levels in the myocardium by DPP4 inhibition and enhancement of myocardial angiogenesis by DPP4 levels. In a porcine model of HF, delivery of a plasmid SDF-1 with an endomyocardial injection catheter demonstrated safety at doses up to 100 mg while improving cardiac function and vasculogenesis up to 90 days post-injection at doses of 7.5 and 30 mg (59). In a Phase I dose escalation study with 12 months follow-up in ischemic cardiomyopathy, 17 subjects in New York Heart Association class III heart failure, with an ejection fraction ≤40% on stable medical therapy, were enrolled to receive 5, 15, or 30 mg of plasmid SDF-1 via endomyocardial injection. The primary end points for safety and efficacy were at 1 and 4 months, respectively. The primary safety end point was a major adverse cardiac event while efficacy end points were changes in quality of life, New York Heart Association (NYHA) class, 6-min walk distance, single photon emission computed tomography, N-terminal pro-brain natruretic peptide, and echocardiography at 4 and 12 months. The primary safety end point was met. At 4 months, all of the cohorts demonstrated improvements in 6-min walk distance, quality of life, and NYHA class (93). Stromal cell-derived factor-1 plasmid treatment for patients with heart failure (STOP-HF) was a Phase II, double-blind, randomized, placebo-controlled trial to evaluate safety and efficacy of a single treatment of plasmid SDF-1 delivered via endomyocardial injection to patients with ischemic heart failure. The primary endpoint was a composite of change in 6 min walking distance and Minnesota Living from Heart Failure Questionnaire from baseline to 4 months follow-up. The primary endpoint was not met (*P* = 0.89). For the patients treated with pSDF-1, there was a trend toward an improvement in left ventricular ejection fraction at 12 months (placebo vs. 15 vs. 30 mg ΔLVEF: −2 vs. −0.5 vs. 1.5%, *P* = 0.20). Patients in the first tertile of EF (<26%) that received 30 mg of pSDF-1 demonstrated a 7% increase in EF compared with a 4% decrease in placebo (ΔLVEF = 11%, *P* = 0.01) at 12 months (94). Although the reasons for the overall failure are currently unclear, the differential benefit in those with advanced left ventricle dysfunction raises the possibility of differential mechanisms that would be operational in more advanced patients. These include the possible overexpression of CXCR4 in cardiac myocytes in the infarct border leading to a negative inotropic state (95). The transient overexpression of SDF-1 in ischemic cardiomyopathy has been shown to lead to long-term down-regulation of cardiac myocyte CXCR4 expression, re-recruiting the contractile function of the border zone (61). Patients with greater left ventricle dysfunction are likely to have a greater volume of myocardial tissue under stress; therefore, a greater demonstrable response to SDF-1 overexpression. Another important reason could be that the upregulation of DPP4 in the border zone of the infarct or around ischemic areas may have resulted in rapid degradation of SDF-1 limiting the efficacy of such an approach. Since both the Phase I and Phase II studies were performed in the absence of DPP4 inhibition, it could be speculated that the results may have been different if the trials had been performed either in the presence of a DPP4 inhibitor.

cells from bone marrow to infarcted tissues. CXCR4, chemokine (C–X–C motif) receptor 4; DPP4i, DPP4 inhibitor; MI, myocardial infarction; SDF-1, stromal-derived factor-1.

# Conclusion and Future Directions

Due to the importance of SDF-1/CXCR4 axis in the stem cell and progenitor cell survival and function, understanding this axis and molecules that modulate their production and action will be of utility for the treatment of cardiovascular disease. There are a number of clinically approved drugs, including DPP4 inhibitors and parathyroid hormone, which have the ability to enhance SDF-1/CXCR4 responsiveness and may improve the outcome of cardiovascular diseases. Several recent large scale clinical trials have indicated that unlike most other oral anti-diabetic drugs

# References

that promote cardiovascular disease, DPP4 inhibitors are safe from cardiovascular standpoint despite lack of evidence showing beneficial effect (96–98). To what extent SDF-1/CXCR4 axis contributes to this effect requires further investigation.

# Funding

This work was supported by grants from AHA (15SDG25700381 and 13POST17210033) and Mid-Atlantic Nutrition Obesity Research Center (NORC Pilot & Feasibility Program) to Dr. Zhong.

Th2 lymphocytes and is produced by monocytes on stimulation with Th2 cytokines IL-4 and IL-13. *J Immunol* (1998) **161**:5027–38.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Zhong and Rajagopalan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# **DPP4 in diabetes**

### *Diana Röhrborn, Nina Wronkowitz and Juergen Eckel\**

*Paul-Langerhans-Group for Integrative Physiology, German Diabetes Center, Düsseldorf, Germany*

Dipeptidyl-peptidase 4 (DPP4) is a glycoprotein of 110 kDa, which is ubiquitously expressed on the surface of a variety of cells. This exopeptidase selectively cleaves Nterminal dipeptides from a variety of substrates, including cytokines, growth factors, neuropeptides, and the incretin hormones. Expression of DPP4 is substantially dysregulated in a variety of disease states including inflammation, cancer, obesity, and diabetes. Since the incretin hormones, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide (GIP), are major regulators of post-prandial insulin secretion, inhibition of DPP4 by the gliptin family of drugs has gained considerable interest for the therapy of type 2 diabetic patients. In this review, we summarize the current knowledge on the DPP4–incretin axis and evaluate most recent findings on DPP4 inhibitors. Furthermore, DPP4 as a type II transmembrane protein is also known to be cleaved from the cell membrane involving different metalloproteases in a cell-type-specific manner. Circulating, soluble DPP4 has been identified as a new adipokine, which exerts both para- and endocrine effects. Recently, a novel receptor for soluble DPP4 has been identified, and data are accumulating that the adipokine-related effects of DPP4 may play an important role in the pathogenesis of cardiovascular disease. Importantly, circulating DPP4 is augmented in obese and type 2 diabetic subjects, and it may represent a molecular link between obesity and vascular dysfunction. A critical evaluation of the impact of circulating DPP4 is presented, and the potential role of DPP4 inhibition at this level is also discussed.

### *Edited by:*

*Heidi Noels, Institute for Molecular Cardiovascular Research, Germany*

### *Reviewed by:*

*Yasuo Terauchi, Yokohama City University, Japan Noriyasu Hirasawa, Tohoku University, Japan*

### *\*Correspondence:*

*Juergen Eckel, Paul-Langerhans-Group for Integrative Physiology, German Diabetes Center, Auf'm Hennekamp 65, Düsseldorf D-40225, Germany<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to Chemoattractants, a section of the journal Frontiers in Immunology*

> *Received: 06 May 2015 Accepted: 13 July 2015 Published: 27 July 2015*

### *Citation:*

*Röhrborn D, Wronkowitz N and Eckel J (2015) DPP4 in diabetes. Front. Immunol. 6:386. doi: 10.3389/fimmu.2015.00386* **Keywords: CD26/DPP4, soluble DPP4, type 2 diabetes mellitus, incretins, DPP4 inhibitors/gliptins, multifunctional enzyme**

# **Introduction**

Dipeptidyl-peptidase (DPP) 4, which is also known as CD26, is a ubiquitously expressed glycoprotein of 110 kDa, which was first characterized by Hopsu-Havu and Glenner (1). DPP4 is a type II transmembrane protein, which is also cleaved off the membrane and released into the circulation by a process called shedding (2, 3). The importance of DPP4 for the scientific and medical community raised substantially since the approval of DPP4 inhibitors for the treatment of type 2 diabetes mellitus (T2DM). These so-called gliptins increase the incretin levels and therefore prolong the post-prandial insulin action. Since soluble DPP4 is characterized as an adipokine (4) and also correlates with parameters of the metabolic syndrome (5), it might also be an important molecular biomarker. DPP4 is a multifunctional enzyme, which serves as a binding partner for numerous peptides, among which are adenosine deaminase (ADA) and extracellular matrix proteins (2, 6, 7). Moreover, as a serine protease, DPP4 cleaves numerous substrates, which further amplifies its complexity of action. Thus, DPP4 is involved in signaling processes, immune cell activation, and its dysregulated expression and release is associated with numerous diseases.

In the present review, we wanted to emphasize the complex function of DPP4 with special focus on its association to T2DM. Furthermore, we wanted to offer a different perspective of the current view of DPP4 beyond the inhibition of its protease activity (8–10). The first part of the present review is dealing with general information about DPP4 and its numerous biological functions in regard to T2DM and its treatment. The last section collects the current knowledge about how DPP4 with its pleiotropic functions, as described before, affects several organs, thereby playing a pivotal role in the development of T2DM and its comorbidities.

# **General Information on DPP4**

### **Biology of DPP4**

The following part will deal with the domain architecture and respective relevance of these domains for the functionality of DPP4.

Dipeptidyl-peptidase 4 (EC<IP_ADDRESS>) is a type II transmembrane protein, which groups together with fibroblast-activation protein α (FAP), the resident cytoplasmic proteins, DPP8 and DPP9, and the non-enzymatic members, DPP6 and DPP10, to the serine peptidase subfamily S9B. All of these proteins share a typical α/β hydrolase fold (2, 6). The DPP4 protein consists mainly of 4 domains: a short cytoplasmic domain (1–6), a transmembrane domain (TMD) (7–28), a flexible stalk segment (29–39), and the extracellular domain (40–766), which can be further separated by a highly glycosylated region, the cysteine-rich region, and the catalytic region (**Figure 1**).

As a member of the type II transmembrane proteins, DPP4 contains a typical signal peptide, which is necessary for the targeting to the endoplasmatic reticulum and the initiation of the translocation across the cell membrane. In contrast to the classically secreted proteins, the signal peptide is not cleaved off, but serves as a membrane anchor. We were able to show that the circulating form of DPP4 (sDPP4), which lacks the cytoplasmic domain and the transmembrane region, is cleaved off the membrane of human adipocytes and smooth muscle cells in a process called shedding by the involvement of matrix metalloproteases (MMPs) (3).

Within the TMD, it could be shown that proline residues play an important role for the translocation of membrane-anchored proteins, such as DPP4. Chung and colleagues (11) studied single proline substitution throughout the TMD of DPP4. They were able to show that translocation and integration into the membrane are determined by the hydrophobicity, conformation, and also the location of proline within the TMD. Furthermore, the position of proline relative to other prolines and the location of highly hydrophobic residues within the TMD are important for correct translocation and membrane integration of DPP4.

In addition to the TMD, the glycosylation of DPP4 is also important for the correct trafficking of DPP4. Carbohydrates account for approximately 20% of the total molecular mass of DPP4 and cause heterogeneity of this protein depending on the location on different cell types. Two highly conserved glutamate residues (205 and 206) within the glycosylated region are essential for the activity of DPP4 (12). Interestingly, six of the nine N-glycosylation sites are located within the glycosylated region. These glycosylation sites are mostly conserved among species. They are necessary for folding, stability, and intracellular trafficking (13). Other modifications like sialylation and/or O-glycosylation have an impact on targeting DPP4 to the cell membrane. Sialyation of DPP4 increases significantly with age, and hypersialyation occurs in patients with HIV infection (14).

Not only glycosylation and residues within the TMD are important for the cellular function of DPP4 but also dimerization. DPP4 can be found as monomer, homodimer, or even as homotetramer on the cell surface of cells. DPP4 needs dimerization for enzymatic activity, and this is the predominant form of DPP4 (15). Dimerization occurs upon interaction with DPP4 itself or with other binding partners, e.g., FAP (16, 17), and occurs via interaction with the cysteine-rich region. Through its interaction with several proteins, DPP4 can act also in an enzymatic activityindependent way. Through this interaction, DPP4 is linked to various mechanisms like immune response and tumor invasion. The heterodimerization and interaction with different binding partners will be discussed in a later section.

The serine in the active site of DPP4 is located in the sequence Gly–Trp–Ser–Tyr–Gly and is part of the catalytic triad (Ser 630, Asp 708, His 740) within the catalytic region of DPP4. DPP4 is an exopeptidase, which cleaves dipeptides from the penultimate Nterminal position of its substrates and thereby either inactivates these peptides and/or generates new bioactive compounds (7). There are numerous different DPP4 substrates known to date and they will be addressed in a separate section within this review.

### **DPP4 Expression and Its Regulation**

Dipeptidyl-peptidase 4 is ubiquitously expressed on numerous different cell types among which are epithelial cells, fibroblasts, and leukocyte subsets. Mechanisms that regulate DPP4 gene transcription and enzymatic activity are not fully understood so far and may be dependent on the studied cell type.

The human DPP4 gene is located on chromosome 2, spans 70 kb, and consists of 26 exons (2). The DPP4 promoter region contains consensus sites for different transcription factors like NFκB, SP-1, EGFR, and AP-1 factor NF-1 (18). At least in chronic b lymphocytic leukemia cells, it could be shown that there is a consensus interferon γ-activated sequence (GAS), which is a binding motif for STAT1. The interferons, α, β, and γ, stimulate STAT1α binding to this region and thus lead to an increased DPP4 expression and activity (19). Interleukin (IL) 12, which is a key factor in differentiation of naïve T-cells into the Th1 subtype, is also able to upregulate DPP4 expression. Therefore, DPP4 is important in immune cell activation (20, 21). Our group was able to show that release of soluble DPP4 is increased upon TNFα stimulation and insulin *in vitro* (4). However, IL-12 and TNFα also seem to play a regulatory role in translation and translocation of DPP4. In activated lymphocytes, IL-12 upregulates DPP4 translation whereas TNFα decreases cell surface expression, which might be due to elevated sDPP4 release (22). Also transcription factors, such as HIF-1α and HNFs, target DPP4 expression (23), which fits to the observation of our group that hypoxia induces DPP4 release in human smooth muscle cells, which might be mediated by MMPs (3).

# **Non-Enzymatic Interactions of DPP4**

Through its cysteine-rich region, which is separated from the catalytic region, DPP4 is able to interact with different proteins, and further broadens its spectrum of activity and highlights its multifunctional role in different processes.

### Binding Partners of Membrane-Bound DPP4

The best-studied interaction in this regard is certainly the binding of DPP4 and ADA. It was already identified in 1993 by Morrison and colleagues (24). Importantly, the interaction of DPP4 and ADA preserves the enzymatic function of both binding partners. It has been shown that residues 340–343 of DPP4 are essential for the interaction with ADA. Regulation of the DPP4/ADA interaction occurs, e.g., via tetramerization of DPP4 or glycosylation at Asn281, which interferes with ADA binding (25). Also, the HIV envelope glycoprotein, gp120, which interacts with DPP4 on lymphocytes via its C3 region, is able to inhibit the association with ADA (2). Upon ADA binding, activation of plasminogen-2 occurs, which raises plasmin levels. This leads to a degradation of matrix proteins and an activation of MMP, thereby indicating that the interaction of DPP4 and ADA might be involved in tissue remodeling (26).

Furthermore, ADA catalyzes the irreversible deamination of adenosine and 2*′* -deoxyadenosine and is therefore a crucial player in the cellular and humoral immunity. Via interaction with CD45, the complex of ADA and DPP4 enhances T-cell activation. Interestingly, DPP4 is also able to promote T-cell proliferation independent from ADA binding or even its enzymatic activity (27). Zhong et al. were able to show that the interaction of DPP4 and ADA on dendritic cells might potentiate inflammation in obesity upon activation and proliferation of T-cells, which could be competitively inhibited by exogenous sDPP4, but not by inhibiting DPP4 enzymatic function (28). Furthermore, ADA activity is elevated in T2DM patients and may serve as a marker of inflammation and obesity (29).

Beside its role in inflammation, adenosine is also an important player in glucose homeostasis. Already in 1988 it was shown that, by lowering endogenous adenosine levels, ADA contributes to a reduced insulin sensitivity of glucose transport stimulation (30). Additionally, adenosine seems to facilitate insulin action in adipocytes (31). Another study could show a correlation of increased ADA activity in T2DM with fasting plasma glucose, HbA1c, aspartate, and alanine aminotransferase (ALT). DPP4 inhibitors exert no additional effects on ADA activity despite glycemic control or HbA1c-dependent effects (32). All these studies emphasize that the effects of ADA/DPP4-interaction are independent of DPP4 enzymatic activity.

Another known interaction partner of DPP4 is Caveolin-1, which is present on antigen-presenting cells (APCs) and binds to residues 630 and 201–211 of DPP4 expressed on T-cells. Thereby, an upregulation of CD68 occurs and initiates a signaling cascade, which might be implicated in the pathogenesis of arthritis, and may be relevant for other inflammatory diseases as well (33). Intracellular signaling is also initiated by DPP4 via interaction with Caspase recruitment domain containing protein 11 (CARMA-1) (6).

Another well-known interaction of DPP4 is with extracellular matrix proteins like collagen and fibronectin (34, 35). The interaction of DPP4 with fibronectin was revealed via nitrocellulose binding assays in rat hepatocytes and seems to play a role in the interaction of these cells with the ECM and with matrix assembly (36). Interaction of DPP4 with FAPα leads to a local degradation of ECM and thus migration and invasion of endothelial cells (37).

# Potential Receptors for sDPP4

Since DPP4 is shedded from the membrane of cells with intact enzymatic and cysteine-rich region, it can also exert biological functions in a paracrine or endocrine manner. These functions might also involve intracellular signaling events in the targeted cells. Therefore, it would be of great importance to know receptors of sDPP4 to better understand the multiple role of sDPP4 on different cells and in different disease conditions where serum levels are elevated. However, there is not much known about DPP4 receptors so far.

Ikushima et al. were able to show that DPP4 needs to associate with mannose-6 phosphate/IGF-IIR to exhibit its function as Tcell activator. This is due to the fact that for this activation, internalization of DPP4 is necessary, but DPP4 lacks a signal for exocytosis. The binding with M6P/IGF-IIR occurs via M6P residues in the carbohydrate moiety of DPP4 and the complex is then internalized and able to exert its biological function (38).

Our group showed that at least in human vascular smooth muscle cells, protease-activated receptor 2 (PAR2) might be activated by sDPP4. We were able to show that sDPP4-mediated ERK activation and proliferation, as well as upregulation of inflammatory cytokines could be prevented by silencing of PAR2. The same was shown by use of a specific PAR2 antagonist. We propose that sDPP4 acts as an activator of PAR2, since a sequence within the cysteine-rich region of DPP4 is highly homologous to the auto-activating tethered ligand of PAR2 (39).

# **Genetic Alterations of DPP4 and Predisposition to T2DM-Associated Diseases**

There are only few studies aiming to identify modifications in the DPP4 gene and their association with T2DM. Some of these are reviewed in the following section.

In 2009, Bouchard et al. analyzed single nucleotide polymorphisms (SNPs) in the DPP4 gene and searched for association with blood pressure, lipids, and diabetes-related phenotypes in obese individuals, to verify whether DPP4 gene polymorphisms could explain the individual risks of obese patients to develop metabolic complications. Three of the analyzed SNPs showed significant association with plasma total-cholesterol levels or plasma triglyceride level or total cholesterol level. But none of the polymorphisms or cardiovascular disease risk factors showed a significant correlation with DPP4 mRNA levels in omental adipose tissue. Therefore, the authors concluded that, at least in their studied group, DPP4 gene polymorphisms seem to be unrelated to the inter-individual risk of developing obesity-related metabolic complications (40).

In another study, visceral adipose tissue DNA of 92 severely obese, non-diabetic female patients was analyzed for methylation rate in the DPP4-promoter CpG island and compared between different DPP4 polymorphisms. These cytosine- and guanine-rich regions are prone to epigenetic modification like methylation, and thus inactivate or activate transcription of certain genes. Different methylation levels of the DPP4 gene were identified in three DPP4 SNPs. Interestingly, the methylation level was negatively associated with DPP4 mRNA abundance and positively with plasma total/HDL-cholesterol ratio. These observations suggest that plasma lipid profile is improved by a higher methylation status of the investigated CpGs (41). Two years later, the same group analyzed DPP4 gene methylation levels between obese subjects with and without the metabolic syndrome in visceral adipose tissue. They observed no significant difference in the percentage of methylation levels of the CpGs within or near the second exon of the DPP4 gene between non-diabetic severely obese subjects with or without metabolic syndrome. However, they were able to show a correlation between plasma cholesterol levels and the percentage of methylation when the subjects were classified into quartiles (42). This further underpins a link between epigenetic modification of the DPP4 gene and plasma lipid metabolism.

Aghili et al. analyzed 875 patients with angiographically documented coronary artery disease (CAD), and divided them in two subgroups dependent of their myocardial infarction (MI) status. By a genome-wide association study, loci, which predispose to MI, were assessed and associated with SNPs in the DPP4 gene. They found that polymorphisms in the DPP4 gene increase the risk of MI and progression of atherosclerosis in terms of plaque stability in patients with already existing CAD. Especially, one SNP was identified in both dominant and additive inheritance modes, which associates with low plasma DPP4 levels and which may increase the risk of MI in CAD patients (43).

Dyslipidemia, which is characterized by excessive lipids in the blood, is a common feature of T2DM. The status of this risk factor is quantifiable by the measurement of apolipoprotein B (ApoB) in the blood. In a very recent study by Baileys and colleagues, they aimed to identify novel SNPs associated with ApoB level. Especially in South Asians, who tend to develop risk factors for T2DM and MI at younger ages and lower BMI, they found an association of a DPP4 SNP with ApoB level (44).

# **The DPP4 Deficiency in Animal Models**

To date, there are several studies dealing with the question, which role DPP4 plays *in vivo.* Animal models are useful tools to study the involvement of DPP4 in different organs. Upon triggering different diseases like insulin resistance (IR) or MI, it is possible to understand the role of DPP4 in these comorbidities of T2DM.

### **DPP4 Deficiency in Rats**

A major part of the literature is dealing rather with DPP4-KO in rats than in mice. Most research groups work with the F344/DuCrj (DPP4-deficient) strain. Rats developing IR due to high-fat diet (HFD) feeding showed improved HOMA-IR values and blood glucose levels in oral glucose tolerance test (oGTT) and more active glucagon-like peptide-1 (GLP-1) and insulin in plasma (45). The same improved glucose tolerance with increased GLP-1 and leptin levels was found in DPP4-depleted Dark Agouti rats with diet-induced obesity (46). Another research group also found improvement in serum lipid profile despite increased visceral fat. They also performed insulin tolerance tests (ITT) in addition to GTT and saw an increased phosphorylation of Akt and reduced expression of gluconeogenic genes, concluding that DPP4-KO improved insulin sensitivity. Furthermore, the KO rats showed increased adipocyte maturation by increased expression of genes involved in triglyceride uptake and in PPARγ expression and increased adiponectin and leptin levels. In addition, adipose tissue is less inflamed illustrated by lower TNFα, IL-6, PAI1, and CCL7 levels (47). The observed effects were attributed to elevated glucose-dependent insulinotropic polypeptide (GIP) levels in the KO rats. Furthermore, the same group could also show attenuated liver damage under HFD challenge in the KO rats due to improved bile secretory function. They postulate that the enhanced export of bile acids out of hepatocytes and a reduction of bile acid synthesis via inhibition of CYP7A1, which converts cholesterol to bile acids, were mediated by increased GLP-1 in DPP4-KO rats (48). Interestingly, at least Yasuda and colleagues also saw a significant reduced food intake in the KO rats irrespective of the diet (45), which might be due to changed receptor specificity of neuropeptide Y (NPY), which was shown to be more potent in KO rats to influence food intake and feeding motivation (49). Although several independent working groups saw increased NPY levels in KO rats (49–51), the effect on food intake is controversial (45, 50). When diabetes is induced via streptozotocin (STZ) treatment in F344/DuCrj-DPP4-deficient rats, onset of hyperglycemia was delayed, but KO rats showed impaired creatinine clearance and more severe dyslipidemia, which might be caused by a dysregulated expression of factors involved in steroid and lipid metabolism (52, 53). The authors concluded that DPP4 might be responsible for preservation of renal function. Another effect of the whole-body KO of DPP4 in rats is induction of behavioral changes like a blunted stress phenotype (46, 51) and also effects on the immune-regulatory system like blunted NK cell and T-cell function and differential leukocyte subset composition or altered cytokine levels (46, 47).

# **DPP4 Deficiency in Mice**

Most of the observations already described in deficient rats are also true in whole-body DPP4-KO mice. Marguet et al. showed enhanced glucose tolerance, lower plasma glucose, and higher plasma insulin and GLP-1 after a 15 min oral glucose load without further characterizing the diet, age, or sex of the used C57BL/6 DPP4-KO mice (54). Conarello and colleagues found less weightgain independent of the diet, and marked hypertrophy in the HFD-fed KO mice in epididymal white (eWAT) and brown adipose tissue (BAT). Importantly, they admitted that the reduction in caloric intake accounted for ~70% of the observed changes in bodyweight. Although they still observed differences in the bodyweight between KO and WT when they used pair-feeding, they carried out their further analysis in *ad libitum* fed mice and it is therefore difficult to judge the influence of DPP4 irrespective of bodyweight. However, they found improved insulin sensitivity and islet morphology, and improved liver biology in respect to lipid content and marker gene expression (55). The observation that DPP4 might be involved in the immune-regulatory system was also investigated in DPP4-KO mice, which were treated with pokeweed mitogen that stimulates growth and proliferation of Bcells. DPP4 seems to be involved in maturation and migration of immune cells, cytokine secretion, and percentages of spleen lymphocytes (56).

All these studies have in common that they use whole-body KO animals. The disadvantage here is that one cannot distinguish between direct effects of the KO and side effects caused, for example, by different immune cell status or decreased caloric intake. To really decipher the role of DPP4 in different tissues and their crosstalk with other target tissues, it is of great importance to study tissue-specific KO models.

Because of this and because we were the first to describe DPP4 as a novel adipokine linked to parameters of the metabolic syndrome (4), we decided to develop an adipose tissue-specific KO mouse model. The AT-specific DPP4-KO mouse was generated using a Cre-lox strategy under control of the aP2 promoter on the C57BL/6J background. Interestingly, we found out that KO mice gained significantly more weight, fat, and lean mass under HFD with no effect on energy expenditure or food intake. However, KO mice showed improved HOMA-IR and lower fasting insulin. The observations that within AT, KO mice display a shift toward significantly more smaller adipocytes, and an increased expression of M2 macrophage marker genes points toward a beneficial role of DPP4 deletion in adipose tissue remodeling during HFD (57, 58).

# **Enzymatic Function of DPP4**

Dipeptidyl-peptidase 4 exerts its enzymatic action by clipping dipeptides from the penultimate position of its substrates. The active center, which is housed in an internal cavity, is surrounded by the β-propeller domain and the catalytic domain. Inhibitors and substrates enter/leave the active center by a so-called "side opening" (59, 60). The following section deals with known substrates of DPP4 in respect of T2DM, and with DPP4 as a drug target for T2DM treatment, which will include current knowledge on DPP4 inhibitors and the impact of DPP4 on organs involved in complications of T2DM.

# **DPP4 Substrates**

In theory, numerous peptides are potential DPP4 substrates since they contain the cleavable amino acid sequence at their penultimate position, but not for all of them it could be shown that DPP4 is able to cleave them *in vivo*. There seems to be a size limitation at least for cytokines, where DPP4 is more prone to cleave substrates of around 24 amino acids (aa) length. Furthermore, the substrate recognition is also dependent on the aa sequence around the penultimate position (61, 62). It turned out to be difficult to find physiological targets of DPP4 in the literature, reasons of that are excellently summarized in a recent review by Mulvihill and Drucker (6). We decided to focus here on the (potential) substrates of DPP4, which might play a role in T2DM or its complications. The list of DPP4 substrates mentioned here is not fully complete and aims to highlight the importance of DPP4 in T2DM also beyond its well-known incretin effect.

### Incretin Hormones

The incretin hormones account for approximately 50% of the insulin secretion after meal intake and are secreted from the gut within minutes after the meal intake. Through binding to distinct receptors on beta cells in the pancreas, they stimulate insulin secretion and suppress glucagon release depending on the blood glucose level. Most potent in their glucose-lowering action are glucagon-like peptide 1 (GLP-1) and GIP. Both peptides belong to the same glucagon peptide superfamily and share significant aa character.

Glucagon-like peptide-1 is secreted from L-cells of the gut into the bloodstream. Upon binding to G-protein-coupled receptors on the beta cells, intracellular cAMP level is elevated and the protein kinases, Epac1 and 2, are activated, which leads to an increase of insulin secretion. Furthermore, GLP-1 enhances beta-cell mass by mediating proliferation and differentiation and inhibiting apoptosis (8). By inhibiting gastric emptying, GLP-1 also improves blood sugar excursion, delays food absorption, and is therefore a regulator of satiety and appetite also through the hypothalamus (63).

Glucose-dependent insulinotropic polypeptide is a 42 aa peptide, which mainly originates from enteroendocrine K cells (64). Subjects with diabetes or impaired glucose tolerance show significantly reduced levels of meal-stimulated circulating GIP and the levels are negatively correlated with the severity of IR in the patients (65, 66). GIP has, in contrast to GLP-1, no effect on glucagon secretion, but also regulates fat metabolism in adipocytes (67).

Since inhibition of DPP4 due to genetic deletion or use of DPP4 inhibitors was shown to elevate GLP-1/GIP levels in numerous studies, this effect is the main focus of developing therapeutic targets for treatment of T2DM. There are numerous reviews, which focus on DPP4- and GLP-1-mediated effects, and this topic will not be further discussed here.

### Stromal Cell-Derived Factor-1α/CXCL12

Stromal cell-derived factor-1 (SDF-1) is a chemokine that promotes angiogenesis and attracts endothelial progenitor cells (EPC) by binding to its receptor C–X–C motif chemokine receptor type 4 (CXCR4). SDF-1 is thus discussed in the literature as important mediator of cardioprotective effects addressed to the use of DPP4 inhibitors (further discussed in "DPP4 Substrates: SDF-1- and BNP-Dependent Effects of DPP4 Inhibitors"). It is a well-known physiological target of DPP4 (68, 69). SDF-1α also plays a role in diabetes itself by protecting stem-cell-derived insulin-producing cells from glucotoxicity under high glucose conditions (70) or promoting pancreatic beta-cell survival in mice via Akt activation (71). Furthermore, it was shown that some genetic variants of SDF-1α are associated with late stage complications in T2DM patients (72, 73).

# NPY and PYY

Neuropeptide Y and peptide YY are members of the polypeptide family. They are highly expressed in the hypothalamus but are also present in peripheral tissues like islets. NPY regulates energy balance, memory, and learning, while PYY reduces appetite, inhibits gastric motility, and increases water and electrolyte absorption in the colon (74). Both NPY and PYY play a role in betacell survival and in glucose homeostasis (74). NPY is able to suppress insulin secretion acutely (75). Both polypeptides have in common that DPP4 truncation shifts their receptor specificity and thus alters their biological role in different cellular processes. *In vitro* experiments in adipocytes could show that DPP4 inhibition has an impact on lipid metabolism mediated by NPY (76, 77).

### Substance P

Substance P is a physiological target of DPP4, which is sequentially converted to SP (3–11) and SP (5–11) *in vivo* in F344–DPP4 positive rats (78). SP is a neurotransmitter and modulator, which is involved in neurogenic inflammation. Serum levels in diabetes are controversially discussed with one study showing a decrease in diabetic patients (79), and another one showing an increase in fasting blood samples with correlation to diabetic risk factors like BMI and blood pressure (80). This discrepancy in serum levels could be addressed to the fact that it is not always stated which form of SP (full length versus truncated) is measured. However, SP was shown to promote IR *in vitro* in human preadipocytes by interacting with proteins that are involved in the inhibitory phosphorylation of IRS-1. Furthermore, SP can directly inhibit insulin-dependent glucose metabolism in rat adipocytes (81). SP also promotes diabetic corneal wound healing, as shown by Yang and colleagues (82).

### Brain Natriuretic Peptide

Brain natriuretic peptide is responsible for vasodilation, natriuresis, and suppresses renin secretion. It is so far only a predicted DPP4 substrate, which was cleaved *in vitro* by DPP4 to BNP (3–32). This truncation was inhibited by a DPP4 inhibitor in a dose-dependent manner (83). Truncated forms of BNP with lower enzymatic activity are discussed as an indicator of heart failure severity. In 2013, dos Santos et al. could show an improved cardiac performance in sitagliptin-treated rats, which they attributed to increased levels of active BNP (84).

### Pituitary Adenylate Cyclase-Activating Polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) is very rapidly degraded by DPP4 to the fragments (3–27), (5–27), and (6–27). These fragments lack PACAPs insulinotropic ability, but are no feasible treatment options for T2DM because of their actions on glucose homeostasis and glucagon secretion (85). Several studies have shown that PACAP is a powerful stimulator of insulin secretion, which enhances glucose uptake in adipocytes and augments antilipolytic action of insulin (86, 87). After DPP4 inhibitor treatment in mice, PACAP-induced insulin secretion was enhanced (88). However, a proof that PACAP also plays a role in humans is lacking so far.

### Regulated on Activation, Normal T-Cell Expressed and Secreted/CCL5

Regulated on activation, normal T-cell expressed and secreted (RANTES) recruits leukocytes into inflammatory sites and is cleaved by DPP4 to RANTES (3–68). Due to this truncation, RANTES (3–68) is not able anymore to increase cytosolic calcium concentrations and to induce chemotaxis of human monocytes *in vitro*. This is explained by a shift in receptor subtype-specificity toward enhanced activation of CC-motif-chemokin-receptor 5 (CCR5) (89). Elevated serum levels of RANTES in T2DM are associated with post-prandial hyperglycemia (90). Interestingly, RANTES and its receptor CCR5 are important mediators of obesity-induced inflammation, which was shown in CCR5-KO mice (91). Levels of RANTES and CCR5 were reduced in adipose tissue of obese patients upon exercise (92). RANTES reduces glucose-stimulated GLP-1 secretion *in vitro* and *in vivo* in mice, by acting most probably through the intestinal glucose transporter SGLT1 (93).

### Eotaxin/CCL11

Eotaxin mediates mobilization of eosinophils into the bloodstream, which was shown to be increased in DPP4-deficient F344 rats (94). DPP4 cleaves eotaxin to eotaxin (3–74). However, there was no significant correlation of eotaxin seen in patients with T2DM or impaired glucose tolerance in the KORA cohort (95).

# **DPP4 as a Drug Target for the Treatment of T2DM** Deactivation of DPP4 Enzymatic Activity *DPP4 inhibitors*

Major DPP4 substrates are the so-called incretin hormones, which are key regulators of post-prandial insulin release. DPP4 inhibition leads to greater bioavailability of these proteins and therefore prolongs the half-life of insulin action. The majority of effects seen upon DPP4-inhibitor treatment are ascribed to an increase in GLP-1 levels. Because of this, DPP4 became a major target for the treatment of T2DM. This section deals with the most recent knowledge around DPP4 inhibitors, their mode of action – if known – and the newest developments in the inhibition of DPP4 enzymatic activity. There are numerous modifications and potential optimizations of the five so far approved gliptins reported. However, most of them are not in clinical trials yet and not much is known about their advantage in a head to head comparison to established gliptins. Therefore, we decided to focus on the most recent data on approved gliptins in this review. The data are also summarized in **Table 1**.

Dipeptidyl-peptidase 4 inhibitors lower DPP4 activity by 70–90%. They do not pass the blood–brain barrier and have no direct effect on satiety or on altering gastric emptying (8). The benefit for diabetes therapy clearly is their indifference on bodyweight gain and the low risk of hypoglycemia. There are five gliptins approved so far for clinical use, namely sitagliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin. Three more gliptins, teneligliptin, anagliptin, and trelagliptin are only approved in the Japanese and Korean market. Despite the same mode of action, the different gliptins diverge in their pharmacodynamic and pharmacokinetic properties, which might be clinically relevant for some patients (9, 96). The peptide mimetic compounds, vilda-, saxa, and teneligliptin, were identified by replacement experiments of peptide-based substrates, whereas the non-peptide mimetic compounds, sita, alo-, and linagliptin, were derived from initially found inhibitors of random screenings. The diverse chemical structures also explain the unique binding modes of the inhibitors to DPP4 (10).

The six inhibitors have been classified into three classes depending on their different binding modes in the DPP4 active center (10). Class 1 contains vilda- and saxagliptin, which only bind to the S1 and S2 subsites and form a covalent bond with the nitrile group of their cyanopyrrolidine moiety and Ser630 of DPP4. Saxagliptin has a fivefold higher activity in blocking DPP4 than vildagliptin. Group 2 contains alo- and linagliptin, which also interact with the S1*′* subsite or even in case of linagliptin with the S2*′* subsite. The uracil rings of both gliptins induce a conformational change in the Tyr547 of the S1*′* subsite. Because of the additional interaction of linagliptin with S2*′* subsite, it has an eightfold higher activity than alogliptin. The third class has the highest inhibitory function toward DPP4, because both sita- and teneligliptin interact with the S2-extensive subsite of the DPP4 active center, and an increasing number of interactions seems to increase the potency of the gliptin (10). Teneligliptin, which is only approved for T2DM treatment in the Japanese and Korean market so far, also has a unique structure characterized by a J-shape and an anchor-lock domain, which explains the strong inhibitory function and the low IC50 value of this drug [for review, see Ref. (97)]. The binding of the DPP4 S2-extensive subsite of some inhibitors also guarantees a high specificity toward DPP4 since other close-related peptidases like DPP8, DPP9, and FAP lack this subsite. All DPP4 inhibitors have in common that they build salt bridges with Glu-residues in the S2 subsite (10). At least for sitagliptin, it is also known that it lowers the level of free fatty acids (FFA) and thereby also comprises insulinsensitizing properties (98). Furthermore, sitagliptin was shown to have potent anti-inflammatory properties by suppressing expression of pro-inflammatory genes in mouse and humans (98, 99). In patients with renal impairment, which is a very common complication of T2DM, sitagliptin is more suitable than sulfonylureas (100). Anagliptin, which is only approved since 2012 in the Japanese market, seems to have serum lipid-lowering and antiatherogenic actions as well, which makes it unique among the gliptins approved so far (101, 102). Anagliptin has an IC50 of 3.3 nM and its main excretion route is renal elimination (101, 103). However, since this gliptin is only on the market since 2012,

### **TABLE 1 | Summarized properties of gliptins**.

comparative head-to-head trials and data on the long-term use are missing at the moment. Shinjo and colleagues demonstrated that anagliptin exerts anti-inflammatory effects on macrophages and adipocytes *in vitro* and on inflamed mouse livers *in vivo*. In this very recent study, anagliptin was more potent in its antiinflammatory actions than sitagliptin (104). To improve the quality of life of T2DM patients, development of novel agents now is more and more focused on long-acting agents. There are two more gliptins, which only have to be applied once weekly, namely SYR-472 (trelagliptin) and MK-3102 (omarigliptin) (105, 106). Trelagliptin is approved in Japan since 2015.

Although some authors claim that DPP4 inhibitors are only beneficial in early stages of diabetes, this could be rebutted by the work of Kumar and Gupta (107). They could show beneficial effects of three gliptins (sita-, saxa-, and vildagliptin) in lowering HbA1c also in patients with longstanding T2DM for more than 10 years. Thus, DPP4 inhibition also plays an important role irrespective of the duration of diabetes.

What has to be mentioned in respect of the beneficial roles of DPP4 inhibitors is that more and more studies about their beneficial pleiotropic effects are upcoming, which are also discussed in the following section of this review dealing with different organs. There are reports that gliptins themselves have effects on lipid profile and blood pressure as well as on inflammatory processes (108). In addition to the incretins, there are some DPP4 substrates, like SDF-1α, which might explain potential cardioprotective effects, which are discussed for gliptins. However, cardiovascular outcomes are still widely debated and controversially evidenced. Ongoing long-term studies will further shed light on the respective role of DPP4i beyond glucose homeostasis. Furthermore, one has to keep in mind that also DPP4 has direct effects independent of its enzymatic activity, like activation of downstream signaling events upon receptor binding, which are not well understood so far. Which role DPP4 inhibition plays on T2D relevant organs/comorbidities will be the topic of the following sections.

### *Alternative modes of DPP4 inhibition*

Very recently, Pang and colleagues published a different strategy to inhibit DPP4 activity. They used DPP4-targeted immune therapy by vaccines in a C57BL/6J mouse model and were able to show comparable effects like in treatments with gliptins regarding GLP-1 plasma levels and post-prandial glucose excursion and insulin sensitivity in HFD-fed mice. Furthermore, they observed no side effects on immune cell activation by the DPP4 vaccine. An advantage of this method is the long-lasting effect of the vaccine in the mouse model, which could, if transferable to human patients, be a convenient alternative to the daily intake of gliptins (109).

Further research in developing alternatives toward Gliptins especially for long-acting medications would be an interesting new approach to improve lifestyle of patients.

### Incretin-Based Therapies: Comparing DPP4i and GLP-1 Analogs