When extracellular MIF binds to its primary receptor cluster of differentiation 74 (CD74) (Leng et al., 2003) on the cell membrane, co-receptors including CD44 or C-X-C chemokine receptors (CXCRs; CXCR2, CXCR4, and CXCR7) (Bernhagen et al., 2007; Alampour-Rajabi et al., 2015) are also required to activate downstream signaling pathways (Shi et al., 2006). Once the CD74/CD44 complex is activated by MIF through the proto-oncogene tyrosine-protein kinase (SRC) (Leng et al., 2003), mitogen-activated protein kinase (MAPK) family members such as the extracellular signal-related kinase 1/2 (ERK1/2), phosphoinositide 3-kinase (PI3K), and protein kinase B (PKB, also known as AKT) are phosphorylated and subsequently activated (Leng et al., 2003; Shi et al., 2006; Lue et al., 2007; Gore et al., 2008). Sustained ERK1/2 activation promotes cancer cell invasion and inhibits cell death (Mitchell et al., 1999). AKT activation leads to phosphorylation of the proapoptotic proteins including Bcl-2 agonist of cell death (BAD) and transcription factor forkhead box O-3a (FoxO3a), promoting cancer cell survival (Lue et al., 2007). MIF-induced cyclo-oxygenase-2 (COX-2)/prostaglandin E2 (PGE-2) activation enhances tumor growth, cancer cell viability, and metastasis. MIF downregulates tumor suppressor protein p53, leading to inhibition of p53-dependent apoptosis, accumulation of mutation, and proliferation of cancer cells (Hudson et al., 1999; Mitchell et al., 2002). ERK1/2 and PI3K/AKT also activate transcription factors including nuclear factor-kappa B (NF-κB) and activator protein-1 (AP1), which result in the release of pro-inflammatory cytokines such as IL-6, IL-8, IL-10, and TNF-α. IL-6 and IL-8 also exhibit pro-tumourigenic functions including promotion of tumor formation by enhancing proliferation, reducing apoptosis, and promoting invasiveness (Taniguchi and Karin, 2014; Johnson et al., 2018). Besides, MIF inhibits p53-mediated apoptosis in macrophage with the induction of increased cytoplasmic phospholipase A2 (PLA2), arachidonic acid, COX2 and PGE2, which sustains the macrophage pro-inflammatory function (Mitchell. et al., 1999; Mitchell et al., 2002). MIF production is upregulated in hypoxic conditions associated with tumor development and progression (Winner et al., 2007). During hypoxia, MIF binding to CD74 contributes to hypoxia inducible factor 1 alpha (HIF1α) activation and stabilization, which then upregulates the expression of angiogenic growth factors including vascular endothelial growth factor (VEGF), lysyl oxidase (LOX), and connective tissue growth factor (CTGF) (Coleman et al., 2008; Oda et al., 2008; Xu et al., 2008; O’reilly et al., 2016). Therefore, MIF supports tumor growth through significantly enhancing angiogenesis. These pathways provide mechanistic bases to explain the role of MIF in the action of pro-inflammatory effects and cancer progression.

MIF can individually bind to CD74 or CXCR2/4 independent of whether the other receptor type is co-expressed (Bernhagen et al., 2007; Schwartz et al., 2009). Extracellular MIF triggers mononuclear (monocytes, neutrophils, and T cells) cell chemotaxis via MIF/CXCR2 and MIF/CXCR4. Monocyte arrest elicited by MIF depends on CD74 and the complex formed by MIF/CXCR2 or MIF/CXCR4 in the context of inflammation and atherosclerosis (Bernhagen et al., 2007). This effect can be suppressed by either a MIF genetic deficiency or antibodies to MIF, CXCR2 or CD74 (Bernhagen et al., 2007). Moreover, MIF can directly interact with CXCR7 (Alampour-Rajabi et al., 2015), resulting in MIF/CXCR7-mediated functional responses that include promotion of human CXCR7 internalization, activation of MIF-mediated ERK1/2 zeta-chain associated protein kinase-70 signaling pathway as well as B cell chemotaxis. It is not completely elucidated whether CD44 is also involved in the receptor complexes of CD74 with the CXCRs. Therefore, tissue responsiveness to MIF depends on its specific expression of MIF receptors and co-receptors, (i.e. CD44 and CXCRs).

In addition to its extracellular activities, it is reported that endocytic MIF can transcriptionally and post-transcriptionally override the immunosuppressive effects of glucocorticoids (Calandra and Roger, 2003). Other studies (Roger et al., 2001; Galvão et al., 2016; Lang et al., 2018; Shin. et al., 2019) published in the past few years collectively support a regulatory effect of endogenous MIF on Toll-like receptor 4 (TLR4) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling pathways. These are thought to play a critical role in AP for developing further pancreatic injury and systemic inflammation (Sharif et al., 2009; Sendler et al., 2020). In response to the stimulation by LPS and Gram-negative bacteria (canonical TLR4 activators), the MIF-deficient macrophages were found to be hyposensitive (reduced production of pro-inflammatory cytokines, e.g., TNF-α and IL-6) (Roger. et al., 2001), highlighting a role for MIF in modulation of TLR4 downstream signaling pathways.

MIF as a regulator of the NLRP3-mediated inflammatory response has been described in recent studies (Lang et al., 2018; Shin. et al., 2019). One study showed that MIF colocalized with ASC (apoptosis-associated speck-like protein containing a CARD), vimentin and NLRP3, potentially modulating the interaction between NLRP3 and vimentin to facilitate the NLRP3 inflammasome assembly, leading to acceleration of downstream cytokine release and pyroptosis (Lang et al., 2018). Accordingly, depletion or inhibition of MIF in macrophages and dendritic cells resulted in the inhibition of IL-1α, IL-1β, and IL-18 release in response to NLRP3-activating stimuli. Another study illustrated the link between MIF and NLRP3 activation in human peripheral blood monocytes using U1 small nuclear ribonucleoprotein immune complex, a NLRP3 inflammasome activator (Shin et al., 2012; Shin. et al., 2019), which can stimulate MIF and IL-1β production in human monocytes. MIF levels were increased in synovial fluid and positively associated with IL-1β in a murine acute gout model and human patients (Galvão et al., 2016). Taken together, these studies highlight the role of MIF in modulating activation of downstream events through TLR4/NLRP3 signaling pathways.

Moreover, intracellular MIF binds to JAB1 and results in tumor cell cycle progression and proliferation. MIF-JAB1 interaction also stabilize HIF1α by preventing its hydroxylation (Winner et al., 2007), resulting in increased expression of pro-angiogenic factors such as VEGF and IL-8 (Oda et al., 2008). It subsequently promotes tumor angiogenesis.

Results from studies investigating MIF and experimental AP are summarized in Table 1. Investigation of MIF in AP began in 2003, when Sakai and colleagues demonstrated a functional role for MIF in experimental models of AP induced by taurocholic acid (TCA-AP), caerulein (CER-AP), and choline-deficient, ethionine-supplemented diet (CDE-AP) (Sakai et al., 2003). In isolated peritoneal macrophages from ascitic fluid of TCA-AP rats, co-incubation with anti-MIF antibodies significantly inhibited IL-8 production (Sakai et al., 2003). MIF levels of serum, ascitic fluid, and lung tissue, but not pancreas or liver, were significantly increased in the TCA-AP in rats. Similarly, MIF levels in the lung were also significantly increased in the CDE-AP in mice. As for CER-AP in rats, the MIF levels were only markedly elevated in pancreatic ascites and thus peritoneal macrophages were considered to be the cellular sources of ascitic MIF. In 2006, Matsuda et al. (Matsuda et al., 2006) reported that plasma and lung MIF levels were increased by 7-fold and 4.7-fold, respectively, in a severe AP model induced by three injections of caerulein and a low dose of LPS in mice (CER/LPS-AP). The MIF levels in pancreatic tissue and serum were increased in l-arginine-induced AP (ARG-AP) in mice (Ohkawara et al., 2017) and their expression was up-regulated in the intrahepatic bile duct cells in a sodium taurocholate-induced AP (STC-AP) in rats (Wang et al., 2019).

Compared with wild type littermates, Mif ^−/− mice reduced pancreatic and serum pro-inflammatory indices as well as severity in ARG-AP (Zhu et al., 2020) and acute lung injury in CER/LPS-AP (Matsuda et al., 2006). Pre-treatment with anti-MIF antibody decreased lung TNF-α levels in the TCA-AP (Sakai et al., 2003) and suppressed the AP-induced elevation of lung TLR4 expression in CER/LPS-AP (Matsuda et al., 2006). Furthermore, anti-MIF antibody significantly reduced lethality in TCA-AP (Sakai et al., 2003), CDE-AP (Sakai et al., 2003), and CER/LPS-AP (Matsuda et al., 2006). MIF specific inhibitor ISO-1 ((S, R)3-(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester) significantly mitigated pancreatic (Zhou et al., 2018), lung (Zhou et al., 2018), liver (Guo et al., 2018), and kidney (Li et al., 2019) injury with simultaneous reduction of MIF and a spectrum of pro-inflammatory mediators in STC-AP in pregnant rats. In addition, administration of anti-protease-activated receptor-2 antibody (CER/LPS-AP) (Matsuda et al., 2006), glucocorticoid agonist (ARG-AP) (Paszt et al., 2008), chlorogenic acid (ARG-AP) (Ohkawara et al., 2017), and ginkgo biloba extract (STC-AP) (Xu et al., 2014) also reduced the severity of AP and circulating, pancreatic, or alveolar macrophage MIF levels in experimental models.

Clinical studies of MIF and AP are displayed in Table 2. Clinical studies (Sakai et al., 2003; Makhija et al., 2007; Rahman et al., 2007; Dambrauskas et al., 2010; Deng et al., 2017) that measured circulating MIF levels collectively and consistently demonstrated that the admission circulating MIF levels were proportionally associated with severity of AP patients. Sakai et al. (Sakai et al., 2003) determined serum MIF levels of 28 patients with mild AP and 18 with severe AP within 72 h disease onset in parallel with 12 healthy individuals, revealing that severe AP were associated with markedly higher serum MIF levels compared to mild AP and healthy controls (both p < 0.01). But there was no statistical difference between mild AP and controls. Similar results were observed in the study conducted by Rahman et al. (Rahman et al., 2007), although the serum samples were collected earlier at 24 h after AP onset. In addition, serum MIF levels were significantly raised in patients who developed pancreatic necrosis or multiple organ failure, indicating MIF could act as a sensitive biomarker to predict local and systemic complications for AP. Another study (Dambrauskas et al., 2010) comprising 108 AP patients (60 mild, 48 severe) also found that serum MIF levels within 72 h disease onset could be used as discriminator of severe and necrotizing AP. A study performed by our group (Deng et al., 2017) identified MIF as an early predictor for discriminating AP patients who had persistent organ failure (n = 20) from those without (n = 50) and healthy controls (n = 10) with an area under the receiver operating characteristic curve (AUC) of 0.90 (95% confidence interval [CI], 0.81–0.96). A most recent study (Shen et al., 2021) demonstrated that MIF at a cut-off value of 2.3 ng/ml has the best discriminative power (AUC, 0.950; 95% CI, 0.914–0.987) for predicting severe AP which was higher than Acute Physiology and Chronic Health Evaluation II (AUC, 0.899), Bedside Index for Severity in Acute Pancreatitis (AUC, 0.886), and serum IL-6 (AUC, 0.826). Therefore, MIF could be used as a potential early severity predictor in patients with AP and this needs further validation.

Previous studies described the ‘G’ to ‘C’ single nucleotide polymorphism (at −173 position) of MIF in patients with systemic-onset juvenile idiopathic arthritis (Donn et al., 2001) and sarcoidosis (Amoli et al., 2002), and a CATT repeat microsatellite (at -794 position) to be associated with lower disease severity in rheumatoid arthritis (Baugh et al., 2002). Accordingly, Makhija et al. (Makhija et al., 2007) compared the MIF gene polymorphism of a United Kingdom cohort of 164 AP patients with 197 healthy controls. It is shown that the distribution of MIF-173 genotype was significantly different between the two groups (p = 0.046), whilst there was no difference regarding the distribution of MIF-794 microsatellite genotypes and alleles. However, these findings warrant confirmation from a larger population.

The in vitro and in vivo studies of MIF and pancreatic cancer are outlined in Tables 3, 4. The levels of MIF expression vary in pancreatic cancer cell lines with high over-expression in PT-45, CFPAC-1, PaCa2, and Capan-1 cells (Ct < 6.5) (Denz et al., 2010). MIF over-expression is associated with reduced E-cadherin expression and increased vimentin expression, indicative of epithelial-to-mesenchymal transition characteristics thus enhanced invasiveness in pancreatic cancer cell lines (Funamizu et al., 2013). Besides, MIF over-expression is also associated with increased proliferation and reduced sensitivity to gemcitabine (Funamizu et al., 2013; Yang et al., 2016). Activation of PI3K/Akt (Denz et al., 2010; Guo et al., 2016; Yang et al., 2016; Wang et al., 2018) and ERK (Guo et al., 2016; Wang et al., 2018) mediated signaling pathways were demonstrated to be involved in the process. MIF knockdown by siRNA attenuated proliferation and invasion along with increased apoptosis in pancreatic cancer cell lines via upregulation of p53 and downregulation of ERK1/2 and AKT phosphorylation (Denz et al., 2010; Guo et al., 2016; Wang et al., 2018). MIF inhibitor (4-iodo-6-phenylpyrimidine; 4-IPP) reduced proliferation and colony formation in PANC-1 cells (Guo et al., 2016).

A recent study (Suresh et al., 2019) found that recombinant MIF treatment significantly enhanced tumor growth via promoting angiogenesis in a hamster pancreatic cancer model induced by subcutaneous inoculation with HapT1 cells. Conversely, MIF knockdown or inhibition (ISO-1) reduced overall cell growth in HapT1 cells.

Intriguingly, MIF has been linked to exosomes in PDAC. Costa-Silva et al. (Costa-Silva et al., 2015) demonstrated that MIF levels in plasma exosomes isolated from a mouse pancreatic cancer model were markedly increased compared to non-cancerous controls, even at an early stage of pancreatic intraepithelial neoplasia. The process of MIF-expressing PAN02 exosome “education”, (whereby naive, wild-type mice were injected retro-orbitally every other day for three weeks with 5 μg of PAN02-derived exosomes), induced liver pre-metastatic niche formation in treated mice which was also inhibited by MIF knockdown.

Yang et al. (Yang et al., 2016) demonstrated the role of MIF–miR-301b–NR3C2 axis in the pathogenesis of PDAC. Over-expression of MIF induced a marked increase of miR-301b and reduction of NR3C2 levels, resulting in profound proliferation, migration, and invasion of pancreatic cancer cell lines (PANC-1 and Capan-2). While inhibition of miR-301b abolished MIF-induced suppression of NR3C2 in vitro, Mif ^−/− mice were associated with reduced metastasis and improved survival in a pancreatic cancer mouse model (LSL-Kras ^G12D , LSL-Trp53 ^R172H/+ , Pdx-1-Cre) in vivo.

Clinical studies of MIF and pancreatic cancer are presented in Table 5 . Collectively, these clinical studies have determined the circulating MIF levels (Winner et al., 2007; Fredriksson et al., 2008; Chen et al., 2010; Kondo et al., 2013; Tan et al., 2014; Schinagl et al., 2016) and MIF expression in dissected pancreatic tissue (Cui et al., 2009; Denz et al., 2010; Funamizu et al., 2013; Tan et al., 2014; Guo et al., 2016; Schinagl et al., 2016; Yang et al., 2016; Wang et al., 2018) of PDAC patients and healthy controls. All the studies reported that MIF levels in serum/plasma or pancreatic tissue were significantly higher in PDAC patients than healthy controls or paired non-cancerous tissue with preferable diagnostic utility, except for one study (Fredriksson et al., 2008) in which there was 30% increase in PDAC without significant difference compared with healthy controls (p = 0.15). Higher MIF expression in tumor tissue was associated with worse survival of PDAC patients (Funamizu et al., 2013; Wang et al., 2018).

In addition, Denz et al. (Denz et al., 2010) reported that MIF mRNA expression in pancreatic tissue was higher in PDAC than chronic pancreatitis, both were higher than normal controls. Chen et al. (Chen et al., 2010) compared the serum MIF in PDAC patients and controls including AP, chronic pancreatitis and healthy donors, showing that MIF had an area under the curve of receiver operating characteristic of 0.78 in discriminating pancreatic cancer from controls. Tan et al. (Tan et al., 2014) focused on diabetes mellitus-associated pancreatic cancer (DM-PC) and found that MIF expression in pancreatic tissues of DM-PC was markedly higher when comparing to chronic pancreatitis or pancreatic cancer without DM. Similarly, serum MIF levels were also higher in new-onset than long term DM-PC, pancreatic cancer without DM, or new-onset T2DM patients (all p < 0.001).

Pharmacological inhibition of MIF with small molecule inhibitors have shown promise in the suppression of inflammation in various animals models such as severe sepsis (Calandra et al., 2000), rheumatoid arthritis (Mikulowska et al., 1997), allergic airway inflammation (Amano et al., 2007), colitis (Ohkawara et al., 2002), glomerulonephritis (Brown et al., 2002), and chronic obstructive pulmonary disease (Russell et al., 2016). Development of MIF inhibitors has been comprehensively and elegantly reviewed elsewhere (Xu et al., 2013; Kok et al., 2018; Trivedi-Parmar and Jorgensen, 2018). Small inhibitory agents of MIF have also been widely demonstrated in experimental cancer models, including lung cancer (Mawhinney et al., 2015), bladder cancer (Choudhary et al., 2013), adenoid cystic carcinoma (Liu et al., 2013), melanoma, and colon cancer (Ioanou et al., 2014). Briefly, small-molecule inhibitors of MIF primarily focus on rational structure-based design that target the MIF tautomerase activity and MIF-CD74 binding (Brown et al., 2009; Xu et al., 2013; Dickerhof et al., 2014; Spencer et al., 2015; Trivedi-Parmar and Jorgensen, 2018). The drought of a reliable, consensus-based in vitro assays for MIF biological activity has been a significant challenge to the development of small molecule MIF inhibitors. Further work should focus on developing a robust high-throughput clinically relevant MIF bioassay that can be applied for second-pass screening, glucocorticoid override, cellular proliferation, and cytokine release detection to expedite the discovery of efficient small molecular MIF inhibitors (Bloom et al., 2016b). In AP and PDAC, only ISO-1 and 4-IPP have been investigated in experimental animal models so far with limited data available, more endeavors are required to test small molecular MIF inhibitors in AP and PDAC in vitro and in vivo thus to consolidate the evidence for clinical translation.

In AP, two studies (Sakai et al., 2003; Matsuda et al., 2006) have demonstrated the efficacy of anti-MIF antibody in CER/LPS and CDE diet AP models, respectively, revealing a promising therapeutic target. A newly established series of nanobodies (NbE5 and NbE10) have shown to attenuate lethality in in vivo septic shock model (Sparkes et al., 2018), is of great interest to be applied in other conditions of inflammatory end-organ damage, such as severe AP. A number of MIF monoclonal antibodies developed by researchers at Baxter have revealed significant anti-MIF activity in human PC3 prostate cancer cell lines and in vivo xenograft model (Hussain et al., 2013). In vitro, BaxG03, BaxB01, and BaxM159 reduced cell growth and viability by inhibiting ERK1/2 and AKT pathways. The antibodies also inhibited MIF-promoted migration and invasion. In vivo, treatment with anti-MIF antibodies reduced tumor growth in a dose-dependent manner. Recently, a phase 1 clinical trial using imalumab (Bax69) in solid tumors and metastatic colorectal adenocarcinoma demonstrated that imalumab has a maximum tolerated dose of 37.5 mg/kg every 2 weeks and a biologically active dose of 10 mg/kg weekly (Mahalingam et al., 2020). Further investigation is warranted to define the role of anti-MIF antibody as a treatment strategy for pancreatic cancer. Of note, the development of antibodies may be mitigated by their short half-life, high costs associated with production, and potential immunogenicity.

As MIF relies largely on CD74 to regulate the downstream cellular events, treatment targeting CD74 holds great potential to inhibit MIF signaling. A humanized anti-CD74 monoclonal antibody, milatuzumab has shown to significantly prolong the survival duration of multiple myeloma xenograft mice models (Stein et al., 2004; Stein et al., 2009). It has reached phase 1 clinical trial in multiple myeloma (Kaufman et al., 2013) and systemic lupus erythematosus (Wallace et al., 2016), indicating no severe adverse effects. Apart from CD74, inhibitors against CXCL2 (Reparixin) and CXCL4 (Plerixafor) are also of interest for further investigation (Steinberg and Silva, 2010; Goldstein et al., 2020). However, as MIF/CD74 pathway also plays an important role in wound repair by activating pro-survival and proliferative pathways that protects the host during injury (Farr et al., 2020), complete inhibition of CD74 could cause some unpredictable side effects which need precaution.

First of all, MIF knockout animals are long lived with no characteristic health issues, giving the opportunity for the initiation of MIF-related gene therapy (Harper et al., 2010). MIF related gene therapy such as Mif gene knockout/knockdown, DDT gene knockdown, or CD74 gene knockdown has exhibited great potential in pre-clinical studies of AP (Matsuda et al., 2006; Zhu et al., 2020) and PDAC ( Winner et al., 2007; Denz et al., 2010; Funamizu et al., 2013; Tan et al., 2014; Costa-Silva et al., 2015; Guo et al., 2016; Yang et al., 2016; Wang et al., 2018; Suresh et al., 2019). Furthermore, it was demonstrated that a xenograft model of head and neck squamous cell carcinoma with MIF knockdown was more sensitive to cisplatin and 5-fluorouracil treatment than control (Kindt et al., 2013). On the other hand, overexpressing MIF in pancreatic cancer cells reduced the sensitivity to gemcitabine (Funamizu et al., 2013). Taken together, it supports that MIF downregulation may potentiate the effect of chemotherapy agents in cancer. There remain ongoing opportunities to develop additional MIF suppression therapies for clinical evaluation.