The multifaced role of the macrophage-migration-inhibitory-factor (MIF)-family in organ fibrosis

Abstract

The macrophage-migration-inhibitory-factor (MIF)-family consists of the structurally homologous proteins MIF, D-dopachrome tautomerase (D-DT), and D-DT like (D-DTL). While MIF is the most well-described member, much less is known about D-DT, and very little about D-DTL. Here, we provide an overview of the structure, similarities, and biological functions of these proteins. MIF and D-DT can have both protective and aggravating effects on various diseases depending on the disease type, involved organ, cell type, and disease stage. Given that the pathological consequence of many chronic diseases is fibrosis, we here discuss the role of these proteins in organ fibrosis, particularly of the kidney, liver, heart, lung, and skin. We discuss the various roles of these proteins, suggesting that MIF might have pro- and antifibrotic roles in different organs. To date, D-DT has been shown to have only antifibrotic roles. We tackle potential translational considerations and propose future research avenues to better understand the involvement of MIF-family in organ fibrosis.

2. Introduction

Cytokines are small, secreted proteins affecting interactions and communications between cells (1). They are modulators of inflammation that participate in acute and chronic inflammation (2). The macrophage migration inhibitory factor (MIF)-family consists of three cytokines: MIF, D-dopachrome tautomerase (D-DT) and D-dopachrome tautomerase like (D-DTL). MIF was first identified in 1966 (3, 4), followed by D-DT in 1993 (5). The structural homology of D-DT and MIF was revealed in 1999 (6).

MIF and D-DT both bind to the extracellular domain of the HLA class II histocompatibility antigen γ chain (Cluster of differentiation (CD)74) (7, 8). As a receptor, CD74 is localized in the membrane but also exists in soluble form after shedding the CD74 ectodomain (sCD74) (9). It is hypothesized that sCD74 might act as a self-regulatory mechanism, or soluble “ligand trap”, preventing the interaction of MIF and D-DT with the CD74 receptor on the cell surface (10, 11). MIF and D-DT also bind to the CXC motif chemokine receptors (CXCR) 4 and CXCR7, and MIF additionally to CXCR2 (12–16). MIF and D-DT are the only known ligands for CD74, whereas the CXCRs have several other ligands (17). This review will focus on the CD74-mediated effects of MIF/D-DT, given that these effects were more specifically investigated compared to those potentially mediated by the CXCRs.

Both MIF and D-DT play a role in various diseases (18–23). This includes inflammatory and chronic conditions, which often lead to organ fibrosis (24). Organ fibrosis represents a significant challenge in medicine, contributing to 17.8% of all global deaths in 2019 with little available effective and specific treatment options (25, 26). Fibrosis is defined by excessive accumulation of fibrous connective tissue (24). It is a physiologically important repair process in certain situations and might have an evolutionary advantage in survival, e.g., in wound repair (27). If fibrosis occurs as a maladaptive repair after acute or chronic tissue damage, it is considered a pathological process (28). During that process, functional tissue is replaced by non-functional connective extracellular matrix (ECM), e.g. collagen or elastin (29). This leads to permanent scarring and organ dysfunction, eventually leading to organ failure and death (24).

Here, we focus on the roles of the MIF-family in organ fibrosis, including kidney, liver, heart, pulmonary, and systemic sclerosis.

2.1. Properties of the MIF-family members and the CD74 receptor

The cytokines belonging to the MIF-family exhibit a high degree of homology (Figure 1). MIF, D-DT, and D-DTL consist of three exons of almost identical size, and only the non-coding introns have different lengths (30) (Figure 1). At the protein level, the amino acid sequences of D-DT and MIF match 34% and 27% in humans and mice, respectively (30). The most recently reported member, D-DTL, shows a 56% mRNA sequence identity to D-DT and a 21% sequence identity to MIF in humans. Currently, it is not known whether D-DTL is expressed in mice. Human MIF, D-DT, and D-DTL are closely linked on chromosome 22 in humans (31, 32) and MIF and D-DT on chromosome 10 in mice (31) (Figure 1a+f+j)

Figure 1. Overview of the MIF-family.

The overview includes the properties of the proteins macrophage migration inhibitors factor (MIF), D-dopachrome tautomerase (D-DT) and D-DT like (D-DTL) in the category: gene, structure, receptors, and CD74 binding. Due to the limited data available on D-DTL, unknown properties are marked with a question mark. Created in BioRender. Boor, P. (2025) https://BioRender.com/q16n769.

2.1.1. Macrophage-migration-inhibitory-factor (MIF)

MIF is an enzyme having an N-terminal enzymatic site that can tautomerize p-hydroxyphenylpyruvate (33, 34). The tautomerase function is not essential for its biological role and is probably a rudimentary property of MIF that derives from its original function in invertebrate immunity (30, 35). However, the catalytic base proline (Pro-1) also plays a structural role in receptor binding, making it a relevant target for the development of small-molecule MIF inhibitors (36).

MIF was named after the initial observation to inhibit macrophage migration during allergic reactions (3, 19). Despite its early discovery, most studies show an effect in support of directed migration and recruitment of leukocytes (19, 37). Like chemokines, MIF induces integrin-dependent arrest and transmigration of monocytes and T cells (38). Important for the chemokine-like function is its pseudo(E)LR domain (39). Under physiological conditions, three MIF monomers build a homotrimer (40, 41) (Figure 1c). As a homotrimer, MIF binds to its receptors (42, 43) (Figure 1c).

MIF binds with a high affinity to the extracellular domain of CD74 (7). MIF homotrimer is kept in place by three CD74 molecules, resulting in a long-lasting complex with a slow association and a slow dissociation constant (43) (Figure 1d). In addition, MIF binds to the non-cognate CXCR2, CXCR4, and CXCR7 (13, 44) (Figure 1c). This binding does not appear specific to MIF, as other cytokines can also bind to CXCRs (15). It modulates the migration of immune cells through the CXCRs (30). MIF was shown to directly activate extracellular signal-regulated kinase (ERK)/ protein kinase B (AKT) or transforming growth factor-β (TGF-β)/SMADs signaling and indirectly promote the production or expression of a large panel of proinflammatory molecules, including cytokines (Tumor necrosis factor (TNF), interferon-γ, interleukin (IL)-1β, IL-2, IL-6, IL-8), nitric oxide, COX2, and several matrix metalloproteinases and their inhibitors (45–48). It also acts as a key upstream regulator in the inflammatory cascade, and toll-like receptor-4-mediated pathways were shown to be involved in the regulation of MIF expression (48). MIF’s synergy with damage-associated molecular patterns was not explicitly demonstrated with respect to fibrosis.

MIF exists in two immunologically distinct conformational isoforms, reduced (redMIF) and oxidized (oxMIF). RedMIF is present in plasma, cells, and tissue of healthy and diseased patients. It converts into oxMIF in an oxidized, inflammatory environment (49, 50). OxMIF was found in the plasma and tissue of patients with inflammatory diseases and in solid tumors. Therapeutic antibodies have been shown to selectively bind oxMIF but not redMIF. (49) The differentiation between oxMIF and redMIF plays a crucial role in therapy, which we discuss in more detail in the chapter “Interventional clinical trials” (Chapter 2.3.). In the subsequently described studies of MIF in organ fibrosis (Chapter 2.2.), a differentiation between oxMIF and redMIF is not made, as no study distinguishes between both.

2.1.2. D-Dopachrome tautomerase (D-DT)

D-DT shares structural similarities with MIF; therefore, it is also named MIF-2 (6, 51). It has an N-terminal enzymatic site that can tautomerize D-dopachrome to 5,6-dihydroxyindole (52). The tautomerase activity of D-DT is considered a potential evolutionary relic and, like MIF, the catalytic base proline (Pro-1) plays an important role in receptor binding (30).

D-DT accumulates in active form as a homotrimer (43) (Figure 1g+h). The C-terminal region is key in molecular recognition because it can regulate the active site opening, protein-ligand interactions, and conformational flexibility of the catalytic pocket’s environment (53).

D-DT binds to CD74, but with lower affinity compared to MIF (8). Topologically, the location of the three CD74 binding regions of the D-DT homotrimer differs substantially from that of the three MIF binding regions (43) (Figure 1i). This key difference in orientation appears to derive from an eight amino acid sequence insertion in D-DT, that topologically limits the binding of D-DT homotrimer to only one CD74 (43) (Figure 1i), resulting in a weaker interaction with a rapid complex formation but also a rapid dissociation rate (43). Molecular simulations reveal that D-DT contains a hydrogen bond network comparable to that of MIF (51).

D-DT also binds to CXCR4 (23) and CXCR7 (16) (Figure 1h). However, D-DT does not bind to CXCR2 because it lacks the pseudo(E)LR (Arg11, Asp4) domain of MIF (54). In the heart, it has been shown that the lack of interaction with CXCR2 abolishes the negative inotropic effect of MIF, which reduces cardiac contractility (12). The missing domain leads to decreased recruitment of monocytes and leukocytes in contrast to MIF (12).

2.1.3. D-Dopachrome tautomerase like (D-DTL)

D-DTL is predicted to enable phenylpyruvate tautomerase activity and be involved in melanin biosynthetic processes. However, it is not clear if the gene is expressed to yield D-DTL protein in humans, and the biological function of this putative protein is unknown (32) (Figure 1).

2.1.4. Cluster of differentiation 74 (CD74)

CD74, the high-affinity receptor of MIF and D-DT, forms a trimer (7, 8). Three CD74 trimers are bound to three binding sites of MIF, and one CD74 trimer is bound to one binding site of D-DT (43). CD74 lacks an intracellular signaling domain (55). Therefore, a co-receptor, CD44, is required, which provides the necessary intracytoplasmic signaling domain (55). By recruiting the co-receptor CD44, the MIF-family initiates the pathway to stimulate cellular effects (30). For example, stimulation of the membrane-bound CD74, together with CD44, activates the ERK1/2/mitogen-activated protein kinase signaling pathway, the phosphatidylinositol 3-kinase/ AKT signal transduction cascade, nuclear factor-κB (NF-κB), or the AMP-activated protein kinase (AMPK) signaling pathway (56, 57). CD74 is required for MIF-induced activation of the ERK1/2/MAP kinase cascade and cell proliferation (58). The ERK pathway, in general, leads to cell proliferation, including fibroblasts, which could have direct effects on fibrosis (59). Currently, the role of MIF-induced ERK activation in fibrosis is not well known.

CD74 is not only found at the cell membrane, but it also exists in a shed, soluble form of the CD74 ectodomain (sCD74) by regulated intramembrane proteolysis (9, 60) (Figure 1c+h). CD74 undergoes several proteolytic events in the endocytic compartments, removing the bulk of the luminal domain and forming a membrane-bound 1–82 truncated protein (60). The biological role of sCD74 is not fully understood, but it is hypothesized that sCD74 acts as a self-regulatory mechanism that prevents the interaction of MIF and D-DT with the CD74 receptor on the cell surface, i.e., functioning as an endogenous “ligand-trap” known for some other receptors (10, 11). D-DT reduced MIF binding to the sCD74 ectodomain in a dose-dependent manner (39). It exhibits a 3-fold higher association rate to sCD74 but an 11-fold faster dissociation rate than MIF (39). In contrast to MIF, D-DT shows a high flexibility of the C-terminus, which can be triggered, for example, by phosphorylation (61).

2.1.5. Expression of the MIF family

Here, we discuss the expression of MIF, D-DT, and D-DTL in various organs (Figure 2) and individual cell types (Figure 3).

Figure 2. RNA and protein expression comparison of the MIF-family, and CD74.

(A) In a heat map, the RNA expression of MIF, D-DT, D-DTL, and CD74 was screened in different tissues. The RNA-seq tissue data generated as part of the Genotype-Tissue Expression (GTEx) project are indicated as nTPM (normalized protein-coding transcripts per million), corresponding to the mean values of the various individual samples from each tissue. The nTPM values got normalized for each organ. (B) MIF, D-DT, and CD74 protein expression were compared in immunohistochemistry staining of the kidney, liver, heart, lung, and skin. D-DTL was excluded because of insufficient staining. Scalebar: 50 μm. The RNA and protein data are sourced from the Human Protein Atlas (72). Image/gene/data available from v24.0.proteinatlas.org. Created in BioRender. Boor, P. (2025) https://BioRender.com/w32f754.

Figure 3. Expression of the MIF-family and CD74 of the respective cell types in different tissues.

The data originates from single-cell RNA analyses. RNA expression of each cluster is indicated as nTPM (normalized protein-coding transcripts per million) from each cell type from the kidney, liver, heart, lung, and skin. The single-cell RNA sequencing data are sourced from the Human Protein Atlas (72). Data are available from v24.0.proteinatlas.org. The cluster data was summarized by taking the mean value of the same cell types. Created in BioRender. Boor, P. (2025) https://BioRender.com/3h6zrcy.

2.1.5.1. Expression of MIF

MIF is located intracellularly, and it gets secreted (62). MIF lacks a signal sequence and is secreted by an unconventional secretion mediated by Golgi-associated protein p115 (63). MIF RNA is strongly expressed by a broad spectrum of organs (Figure 2A). The highest tissue-specific RNA expression of MIF is found in the kidney (Figure 2A).

Under homeostatic conditions, MIF is constitutively and ubiquitously expressed (64). Stimulation of macrophages with lipopolysaccharide (LPS) leads in vitro to the release of MIF, as the in vivo stimulation of mice resulted in a time-dependent increase of MIF (39). It is expressed by immune cells, but also, for example, by fibroblasts (65), endothelial (66), neuroendocrine (67), mesenchymal cells (68), or epithelial cells (69) (Figure 3).

Some data suggest sexual dimorphism of MIF. Neonatal immune signatures displayed higher levels of MIF in newborn male blood spots compared to females (70). In a clinical trial, MIF was positively correlated with testosterone and negatively with estradiol (71). Higher MIF levels are identified in male multiple sclerosis patients compared to female multiple sclerosis patients (20). Suggesting a sex difference between males and females. Despite these few studies, the sexual dimorphism of MIF represents an important gap in knowledge.

2.1.5.2. Expression of D-DT

The mRNA of D-DT lacks an N-terminal or an internal secretory signal sequence, suggesting that D-DT is secreted via a specialized, non-classical export pathway (39). D-DT is located intracellularly (39). D-DT’s highest tissue-specific RNA expression is detected in the liver (Figure 2A). D-DT RNA is also highly expressed in the pancreas and kidney, while expression in the remaining organs is moderate to low (Figure 2A).

Macrophages stimulated with LPS secrete 20-fold more MIF than D-DT (39). Furthermore, MIF shows a steeper dose response in the measurement of macrophage migration inhibition and glucocorticoid overdrive than D-DT (30). Like MIF, D-DT is expressed not only by immune cells but also by epithelial cells (16, 22). (Figure 3)

Sexual dimorphisms were also detected for D-DT. Similarly to MIF, higher D-DT levels are identified in male multiple sclerosis patients compared to female multiple sclerosis patients (20).

2.1.5.3. Expression of D-DTL

D-DTL was predicted to have an intracellular localization (72). Very little, if any D-DTL RNA is found in various organs (Figure 2A), with the highest expression in the liver (Figure 2A + Figure 3). There is no data available on sexual dimorphism for D-DTL.

2.1.5.4. Expression of CD74

The strongest CD74 expression is observed in cells involved in the antigen-presenting cells, like dendritic cells, B cells, and macrophages (73). CD74 functions as a chaperone for the correct folding of major histocompatibility complex class II and regulates its antigen-binding capacity (74). Non-antigen-presenting cells, such as epithelial cells, express CD74 on their surface in various organs, including the kidney, lung, intestine, heart, liver, and skin (56). The strongest RNA expression is detected in the spleen and lungs (Figure 2A). The function of CD74 in epithelial cells remains incompletely elucidated.

In late-stage pancreatic ductal adenocarcinoma, a unique fibroblast population expressing the major histocompatibility complex class II genes was identified (75). CD74-expressing antigen-presenting fibroblasts have also been identified in multiple cancer types, with diverse roles ranging from promoting immunosuppression to enhancing anti-tumor immunity, depending on the specific cancer type (76).”

2.2. The pro- and antifibrotic effect of the MIF-family in organ fibrosis

CD74 is involved in MIF-family-driven immune cell recruitment and activation of a variety of cellular responses, including cell proliferation (77). The transformation of quiescent fibroblasts into actively proliferating, ECM-producing myofibroblasts plays a pivotal role in the pathogenesis of all fibrotic diseases (78). MIF is known to increase and promote fibroblast proliferation during chronic inflammatory diseases (79). However, MIF also exerts an antifibrotic effect, which demonstrates the pleiotropic effect of MIF (80). D-DT also has pleiotropic functions, including a multifaceted role in organ fibrosis. Here we discuss the relevance of MIF and D-DT in kidney, heart, pulmonary, liver fibrosis, and systemic sclerosis (Table 1). D-DTL is not considered since nothing is known to date.

Table 1. Summary of the expression and functional effects of MIF and D-DT in organ fibrosis.

		Kidney	Liver	Heart	Lung	Skin
Circulating level	MIF	CKD/SLE patients: MIF increased	Decompensated cirrhosis: MIF increased, low sCD74 Autoimmune hepatitis/primary biliary cirrhosis: elevated MIF expression	?	IPF: Serum MIF concentration was associated with a higher 3-month mortality CF: Carrying at least one 5-CATT repeat allele exhibit low MIF expression	SSc: Increased MIF / no difference dSSc: Increased MIF
	D-DT	?	?	?	?	SSc: No difference SSc with FEV-1: D-DT increased
Tissue Expression	MIF	Healthy: MIF mainly in tubular epithelial cells Tubulointerstitial fibrosis: MIF decreased	Healthy: MIF Hepatocytes, hepatic stellate cells Autoimmune hepatitis/primary biliary cirrhosis: elevated MIF expression	Healthy: MIF low levels cardiomyocytes Acute myocardial infarction: MIF increased Ischemia: MIF increased Atrial fibrillation: MIF increased	Healthy: MIF mainly in bronchiolar and alveolar epithelium IPF: increased MIF	SSc: MIF increased in keratinocytes and dermal fibroblasts, decreased in endothelial cells dSSc: MIF and CD74 increased om dermal fibroblasts
	D-DT	Healthy: D-DT mainly in tubular epithelial cells Tubulointerstitial fibrosis: D-DT decreased	?	Failing hearts: Decreased D-DT TAC: Increased D-DT	?	?
Functional role: Investigated models	MIF	UUO, I/R, in vitro	TAA, CCl4, hepatitis B virus-related liver fibrosis, in vitro	Ang-II, in vitro	Bleomycin, in vitro	In vitro
	D-DT	UUO, I/R	CCl4	TAC	?	?
Functional role: Expression in animal models	MIF	UUO or I/R: MIF decreased	Hepatitis B virus- related liver fibrosis: MIF expression correlated with the activation of fibrosis marker	?	Bleomycin: increased MIF in bronchoalveolar lavage and fibrotic area	?
	D-DT	UUO or I/R: D-DT decreased	CCl4: D-DT increased	?	?	?
Functional role: Neutralization/ Downregulation	MIF	In vivo: MIF KO →Increased fibrosis in UUO In vivo: MIF inhibition ISO-1 →Increased fibrosis in UUO	In vivo: MIF KO → Increased fibrosis and lower SAM recruitment in CCl4 In vitro: MIF Inhibition ISO-1 → Decreased fibrosis in hepatitis B virus-related liver fibrosis	In vivo: MIF KO → Increased fibrosis in Ang-II	In vivo: Anti-MIF → reduced mortality, no reduction in fibrosis In vivo: ISO-1 and 31 → Decreased fibrosis In vivo: SIX-1 KO → decreased fibrosis	?
	D-DT	In vivo: D-DT KO → Increased fibrosis in UUO	?	Cardiomyocyte- specific D-DT KO: Increased fibrosis	?	?
Functional role: Upregulation	MIF	In vivo: Administration recombinant MIF → Decreased fibrosis in UUO	In vivo: Administration recombinant MIF → Decreased fibrosis	In vitro: Recombinant MIF → Decreased fibrosis or increased fibrosis	?	In vitro: MIF increased the resistance to apoptosis by inhibiting sodium nitroprusside- induced apoptosis in dermal fibroblasts
	D-DT	In vivo: Administration recombinant D-DT → Decreased fibrosis in UUO	?	In vitro: Recombinant D-DT→ Decreased fibrosis	?	?
Functional role: Mechanism	MIF	Tubulointerstitial fibrosis: MIF abrogates cell cycle arrest of tubular epithelial cells	TAA: Decreasing TGF-α1, PDGF-BB, MMP2, MMP9, TIMP-1 CCl4: CD74/AMPK MIF dependent SAM recruitment through MMP13 In vitro: NASH: CD74/AMPK In vitro: Hepatitis B virus-induced: MIF enhanced the TGF-β/SMAD through CD74	In vitro: Myofibroblast →Activation CD74, TGF-β2, MMP-2 →MIF/CD74: Antifibrotic →MIF/CXCR4: Profibrotic	In vitro: Lung fibroblasts →MIF stimulate proliferation	In vitro: Dermal fibroblasts decrease the apoptosis through activation of ERK, AKT and Bcl-2 signaling
	D-DT	Acute kidney injury (I/R): tubular cell regeneration through SLPI- and ATF4-dependent mechanisms	CCl4: Acceleration of the melanin biosynthesis, leading to the protection of the liver from oxidative stress	In vitro: Myofibroblast →inhibiting TGF-β induced SMAD-2 activation CD74/CaMKK2	?	?
Anti-/Profibrotic	MIF	In vivo: Antifibrotic in tubulointerstitial fibrosis	In vivo: Antifibrotic in liver fibrosis; In vitro: Hepatoprotection in NASH	In vivo: Antifibrotic In vitro: Anti-/Profibrotic	In vivo: Antifibrotic	In vitro: Potentially Profibrotic
	D-DT	In vivo: Antifibrotic in tubulointerstitial fibrosis	In vivo: Potentially antifibrotic	In vivo/In vitro: Antifibrotic	?	?

2.2.1. Kidney fibrosis

Kidney fibrosis is the terminal pathway of almost all kidney diseases (81). It is the key pathological process behind chronic kidney disease (CKD), which is defined as a chronic dysfunction or structural abnormality of the kidney independent of the cause (82). Worldwide, more than 10% of the population is affected by CKD, which is associated with high morbidity and mortality (83). The three main kidney compartments can all be affected by various diseases and lead to fibrosis (81). Fibrosis in each compartment is termed differently, i.e., tubulointerstitial fibrosis in the tubulointerstitium, glomerulosclerosis in the glomeruli, and arteriosclerosis in the vasculature (27). With disease progression, fibrosis can extend to other compartments; for instance, glomerular diseases can initially lead to glomerulosclerosis and subsequently to tubulointerstitial fibrosis (81). We focus on the role of MIF and D-DT in tubulointerstitial fibrosis and glomerulosclerosis, as there are no studies available evaluating MIF and D-DT in renal arteriosclerosis.

In healthy human and murine kidneys, MIF was mainly expressed in the renal tubular epithelial, endothelial, mesangial cells, and podocytes (84, 85). In human kidneys with tubulointerstitial fibrosis, MIF expression was significantly decreased (84). In contrast, MIF was significantly elevated in the serum of patients with CKD compared with healthy individuals (86). Interestingly, the authors found no correlation between glomerular filtration rate and MIF serum concentration but a correlation with markers of oxidative stress and endothelial activation. In patients with focal glomerular sclerosis, levels of MIF in the urine were significantly higher than those of healthy individuals and correlated with the extent of mesangial matrix increase and that of tubulointerstitial fibrosis. (87) Systemic lupus erythematosus (SLE) is a serious autoimmune disease that can often lead to kidney fibrosis. Increased MIF concentration in the serum, kidneys, and urine have been correlated with active systemic disease as well (88).

Various animal models are used to study kidney diseases, and many develop kidney fibrosis (81). For tubulointerstitial fibrosis, animal models like unilateral ureteral obstruction (UUO) or ischemia-reperfusion (I/R) were used. MIF was less expressed in kidney fibrosis induced by UUO or I/R (84). This reflects the expression in humans, as MIF is less expressed in patients’ samples of kidney fibrosis (84). Nothing is known about the expression of MIF or D-DT in glomerulosclerosis. However, there are findings about MIF and D-DT in the context of glomerular diseases like IgA nephropathy or rapidly progressive glomerulonephritis, which can lead to glomerulosclerosis. Here, a higher MIF expression was found in the glomeruli. A murine model of crescentic glomerulonephritis reflected the situation in humans. (86) MIF was secreted from podocytes, parietal epithelial cells, and mesangial cells in this murine model (85).

The functional role of MIF in tubulointerstitial fibrosis was investigated by UUO and I/R in a systemic MIF knockout (KO). Significantly more fibrosis formation was observed in MIF KO compared to wild-type mice. In an in vivo intervention study, the MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) was administered therapeutically three days after fibrosis induction by UUO. The inhibitor significantly increased fibrosis and inflammation. Administration of recombinant MIF resulted in reduced fibrosis and inflammation. In vitro, deletion of MIF in LPS-stimulated tubular epithelial cells leads to increased G2/M cell cycle arrest and increased expression of CDK inhibitor 1B and proinflammatory and profibrotic mediators. Thereby, MIF abrogated cell cycle arrest of tubular epithelial cells, induced apoptosis, and thereby reduced kidney fibrosis. (84)

Autosomal dominant polycystic kidney disease is characterized by renal cyst formation, inflammation, and fibrosis. MIF promotes cystic epithelial cell proliferation by activating ERK, mTOR, and Rb/E2F pathways. MIF also regulated cystic renal epithelial cell apoptosis through p53-dependent signaling. The effect on fibrosis was not directly investigated. (89)

No study investigated the functional role of MIF in glomerulosclerosis. However, one study investigated the connection between MIF and glomerulonephritis, which leads to glomerulosclerosis. Therefore, they used a murine model of crescentic glomerulonephritis, showing that MIF is upregulated and mediates glomerular injury through MIF and its receptor complex CD74/CD44. (85)

Initial data shows that D-DT is mainly located in proximal tubular epithelial cells in healthy human and murine kidneys. In human and murine fibrotic kidneys, D-DT was downregulated. (90) The functional role of D-DT was studied in acute kidney injury. Acute kidney injury can progress to CKD due to maladaptive repair processes, persistent inflammation, and fibrosis (78). D-DT was suggested to mediate proximal tubular cell regeneration via secretory leukocyte protease inhibitor- and activating transcription factor 4-dependent mechanisms in acute kidney injury (22). Initial data indicated increased fibrosis in D-DT KO mice and a potential protective role of D-DT in the UUO model of kidney fibrosis (90).

In the kidney, MIF-family seems to have antifibrotic effects in tubulointerstitial fibrosis. However, many kidney diseases, particularly glomerular diseases, involve complex processes with extensive cellular interactions in different kidney compartments. MIF appears to be disease-promoting in immune and autoimmune-mediated glomerulonephritis and may therefore contribute to secondary glomerulosclerosis, which in turn might drive tertial tubulointerstitial fibrosis. Likely, the role of MIF in kidney fibrosis depends on the specific disease and its stage.

2.2.2. Liver fibrosis

Liver fibrosis is caused by nearly every chronic liver injury or inflammation, e.g., alcohol consumption, non-alcoholic steatohepatitis, chronic viral hepatitis, autoimmune hepatitis, Schistosoma mansoni infection, etc. Mechanistically, activation of hepatic stellate cells, which in turn acquire a myofibroblast-like phenotype, leads to liver fibrosis. In this manner, tissue remodeling in the liver is initiated through the secretion of ECM proteins and MMPs by activated hepatic stellate cells. (91) Compared to other organs, the liver has a high regenerative capacity. With respect to terminology, the most advanced stage of liver fibrosis is termed cirrhosis. Cirrhosis is defined as the histological development of regenerative nodules surrounded by fibrous bands, representing advanced chronic injury and organ failure, associated with high mortality. (92)

MIF was expressed in hepatocytes and hepatic stellate cells (93, 94). Impaired survival was shown in patients with decompensated cirrhosis having high MIF and low sCD74 serum concentrations compared to controls. The increase in MIF serum concentration was independent of liver function but reflected systemic inflammation. It was hypothesized that sCD74 would neutralize MIF in the systemic circulation and specifically antagonize its proinflammatory function. (9) In autoimmune liver diseases such as primary biliary cholangitis and autoimmune hepatitis, serum MIF concentration and hepatic MIF expression were elevated compared to healthy controls (95).

Liver fibrosis can be evaluated using various in vivo models. A distinction was made between chemical, dietary, surgical, transgenic, and immune liver fibrosis models (96). The functional role of MIF in liver fibrosis was investigated in two in vivo chemical, toxin models using carbon tetrachloride (CCl₄) and thioacetamide (80, 93, 97). CCl₄ led to oxidative damage caused by lipid peroxidation (98). Thioacetamide-induced liver injury is mainly caused by reaction metabolites secreted by thioacetamide, which activate hepatic stellate cells and produce fibrinogen and growth factors (96).

In CCl₄-induced liver fibrosis, MIF KO showed increased fibrosis. It was associated with alterations in fibrosis-relevant genes but not by a changed intrahepatic immune cell infiltration. The administration of recombinant MIF displayed decreased fibrosis by CD74/AMPK. (80) Another study focused on the recruitment of scar-associated macrophages by MIF in CCl₄-induced liver fibrosis, since scar-associated macrophages regulate hepatic stellate cell activation and ECM degradation. Recruitment of scar-associated macrophages was lower in MIF KO mice compared with wild-type mice. MIF-dependent recruitment of scar-associated macrophages facilitates ECM degradation through MMP13, emphasizing the crucial role of proper macrophage recruitment and phenotypic characteristics in fibrosis resolution. (93) In a thioacetamide-induced liver fibrosis model, MIF showed an antifibrotic activity by decreasing TGF-β1, platelet-derived growth factor-BB, MMP2, MMP9, and tissue inhibitors of MMP1 (97).

In an in vitro nonalcoholic fatty liver disease model, hepatocytes were stimulated with oleic acid. MIF showed a protective effect in hepatocytes by reducing the triglyceride content through CD74/AMPK signaling. Blockade of CD74 but not CXCR2 or CXCR4 fully reverted the protective effect of MIF, comparable to AMPK inhibition. (94) Since non-alcoholic steatohepatitis is one important cause of liver fibrosis, an antifibrotic and protective role of MIF in this disease could be assumed in hepatocytes.

Chronic hepatitis B and C virus infection is a major risk factor for liver fibrosis, cirrhosis, and hepatocellular carcinoma. In a hepatitis B virus-induced liver fibrosis in vitro model, hepatic stellate cells were stimulated with a hepatitis B virus-containing cell culture supernatant and the hepatitis B virus surface antigen. Hepatitis B virus-induced upregulation of MIF and CD74 in hepatic stellate cells. MIF expression correlated with the activation of fibrosis markers, and MIF inhibition with ISO-1, attenuated fibrosis markers. The authors identified MIF to enhance the TGF-β/SMAD signaling through its receptor CD74, promoting hepatic stellate cell activation. Besides, MIF participates in a positive feedback loop, where TGF-β1 further upregulates MIF expression. (48) In hepatitis C virus-induced liver fibrosis, MIF polymorphisms are associated with disease severity and complications in a stage- and context-dependent manner (99).

Schistosoma mansoni infection invariably results in liver fibrosis (100). In patients with a low burden of these parasites, a positive association was found between plasma concentrations of MIF and markers of liver fibrosis (101).

In summary, this could indicate that MIF has an antifibrotic effect in toxin-induced liver fibrosis, but a profibrotic effect in hepatitis B-induced fibrosis.

In an in vivo CCl₄ rodent model, an increased production of D-DT was detected. D-DT increase led to an acceleration of melanin biosynthesis, leading to the protection of the liver from oxidative stress. They introduced D-DT as a candidate key protein protecting the rodent liver from CCl₄ damage. (102) In a follow-up study, an N-terminal proline glutathionyl carbonylate modification of D-DT was detected, which was formed by condensation of phosgene and reduced glutathione in damaged CCl₄ livers (103). Albeit fibrosis was not analyzed, it could be expected that the improved liver damage will be accompanied by reduced liver fibrosis.

Taken together, the role of MIF can differ in liver fibrosis based on the disease leading to fibrosis, with its signaling pathways showing contrasting pro- or antifibrotic effects (48). Currently, there is no study evaluating the direct effect of D-DT in liver fibrosis. In a liver injury model involving liver fibrosis, a protective mechanism induced by D-DT could be identified.

2.2.3. Heart fibrosis

Cardiovascular diseases are a leading cause of mortality worldwide. Heart fibrosis is an essential component and consequence of cardiovascular diseases. It can be divided into two major forms: replacement and reactive (interstitial) fibrosis. Replacement fibrosis occurs focally after necrosis of cardiomyocytes due to ischemia. (104) Heart fibrosis is a pathological process, but it is important to prevent rupture of the heart after myocardial infarction (105). Interstitial fibrosis occurs diffusely in the myocardium as a result of various diseases, e.g., hypertension or diabetes (104). Both forms of heart fibrosis lead to systolic and diastolic dysfunction as well as arrhythmias (106).

In healthy rats, MIF was constitutively expressed at low levels by cardiomyocytes. MIF expression was rapidly upregulated in acute myocardial infarction by surviving cardiomyocytes in the infarcted area. (107) MIF expression was also increased in cardiac fibroblasts after ischemia or pressure overload due to transverse aortic constriction (108, 109). MIF is expressed by inflammatory cells and cardiomyocytes in pathological foci of experimental autoimmune myocarditis (110).

The functional role of MIF in heart fibrosis was investigated in an angiotensin-II infusion-induced hypertrophy model, which mimics hypertension-related interstitial heart fibrosis. MIF KO aggravated heart fibrosis and caused decreased systolic function and increased heart weight (111). These in vivo findings align with the in vitro findings, where overexpression of MIF inhibits the expression of fibrosis markers in mouse cardiac fibroblasts by activating CD74 and targeting the profibrotic genes of transforming growth factor beta-2 and MMP-2 (111). In contrast, another study claimed that cardiac fibroblasts in vitro treated with recombinant MIF promoted proliferation and upregulation of fibrosis markers (96). However, the specific activated receptor was not examined, leaving the involvement of CD74 unresolved. The receptors activated by MIF could explain the contradictory pro- and antifibrotic functions. CD74 was described as mediating antifibrotic properties, while CXCR4 promotes profibrotic effects in cardiac myofibroblasts (112). Furthermore, as autoimmune myocarditis can lead to heart fibrosis, the role of MIF in this condition will be discussed (113). Anti-MIF-antibody treatment in both the early and late phases of experimental autoimmune myocarditis significantly inhibited the disease in a vascular cell adhesion protein-1-dependent manner, by suppressing the expression of TNF-α and IL-1β in the heart (110).

MIF was investigated in the context of aging-induced cardiac anomalies, examining the cardiac geometry, contractile and intracellular Ca²⁺ properties in young (3-4 months) or old (24 months) wild-type and MIF KO mice. The hearts of aged MIF KO mice had higher heart mass, heart-to-body weight ratio, cardiomyocyte size, and interstitial fibrosis compared to those of aged wild-type mice. (114)

Explanted human hearts had significantly reduced D-DT compared to normal or hypertrophic hearts. No difference was evident in D-DT expression between normal and hypertrophic hearts. In mice, cardiac D-DT was increased due to pressure overload seven weeks after transverse aortic constriction, when left ventricular function was still normal. (109)

The functional role of intrinsic cardiomyocyte-derived D-DT was investigated in modulating the cardiac response to left ventricular pressure overload by transverse aortic constriction. Increased interstitial fibrosis was evident in cardiomyocyte-specific D-DT KO hearts one week after transverse aortic constriction. Seven weeks after transverse aortic constriction, cardiomyocyte-specific D-DT KO mice continued to exhibit a significantly higher degree of fibrosis. Recombinant D-DT in vitro exhibited an antifibrotic effect in cardiac fibroblasts by inhibiting TGF-β-induced SMAD-2 activation. Cardiomyocyte-specific MIF KO demonstrated little susceptibility to heart failure, even when extending the observation period to 10 weeks after transverse aortic constriction. Myocardial D-DT expression was mildly increased in cardiomyocyte-specific MIF KO and might have had a compensatory effect in preventing a phenotype. In contrast, cardiomyocyte-specific D-DT KO mice showed no compensatory increase in MIF after transverse aortic constriction. (109) In a previous study, a protective effect of D-DT required activation of the metabolic stress enzyme AMPK, which was mediated by a CD74/CaMKK2-dependent mechanism after ischemic injury (115).

In summary, an antifibrotic effect of MIF triggered by CD74 was found in heart fibrosis. It is noteworthy that MIF appears crucial for maintaining physiological homeostasis in the heart, as aged MIF KO mice showed heart fibrosis. One study showed a potential profibrotic effect of MIF that could be triggered by CXCR4. D-DT has an antifibrotic and compensatory effect in heart fibrosis, probably mediated by a CD74/CaMKK2-dependent mechanism. All functional studies focused on interstitial heart fibrosis, not replacement fibrosis.

2.2.4. Pulmonary fibrosis

Pulmonary fibrosis is the predominant component of all interstitial lung diseases. Many patients with pulmonary fibrosis can be diagnosed with an autoimmune disorder or hypersensitivity pneumonitis. In patients where no identifiable cause can be determined, the diagnosis of idiopathic pulmonary fibrosis (IPF) is proposed. IPF is a life-threatening disease leading to progressive chronic hypoxemic respiratory failure, and only very limited treatment options are available. It is suggested that a chronic alveolar epithelium injury leads to the activation of fibroblasts, followed by an accumulation of ECM. The injured alveolar epithelium is characterized by cellular senescence and secretion of profibrotic mediators. Immune cells, the microbiome, and alterations in mucin expression are also supposed to be involved in the pathogenesis of IPF. (116) Fibrotic processes are involved in other lung diseases such as cystic fibrosis (CF). CF is an autosomal-recessive, monogenetic disorder caused by mutations in the CF transmembrane conductance regulator gene (117). Because of the reduced mucociliary clearance, patients with CF suffer from recurrent pulmonary bacterial infections (e.g., caused by Pseudomonas aeruginosa or Haemophilus influenzae), causing airway remodeling and finally irreversible loss of lung function. (118)

In the lung, MIF was expressed in the bronchiolar and alveolar epithelium (119). In IPF, MIF was additionally expressed in fibrotic areas, especially in their peripheral regions (119, 120). In bronchoalveolar lavage of patients diagnosed with IPF, a higher MIF concentration was detected compared to healthy individuals (120). In serum, MIF concentration was associated with a higher 3-month mortality in patients with acute exacerbation of IPF (121). CF patients carrying at least one 5-CATT repeat allele exhibit low MIF expression and experience a favorable lung function outcome compared to those without the 5-CATT repeat allele, who are high MIF expressers (122).

Immune cells might be involved in the pathogenesis of IPF. Peripheral lymphocytes from patients with IPF produced more MIF when stimulated with collagen I compared to lymphocytes from healthy individuals (19, 123). Single-cell transcriptomics of lung tissue from patients with IPF revealed that MIF was also involved in the interaction between IPF-associated macrophages and other macrophage subpopulations (124), albeit the functional relevance of MIF in this interaction remains unclear.

A model of pulmonary fibrosis induced by the intratracheal application of bleomycin is widely used (46, 125, 126). As is often the case, this animal model only partially replicates IPF, e.g., it is not inherently idiopathic (116). In this model in mice revealed an increased MIF concentration in the bronchoalveolar lavage, and increased MIF expression in fibrotic areas (46, 125, 126). This is consistent with the findings in patients with IPF as described above.

The functional role of MIF in this animal model has been investigated in several studies. Using a neutralizing anti-MIF antibody, mice showed reduced mortality and infiltration with immune cells but no reduction in fibrosis (125). Using the mouse model and two specific MIF inhibitors ISO-1 and N-(3-hydroxy-4-fluorobenzyl)-5 trifluoromethylbenzoxazol-2-thione 31, another study was able to demonstrate an antifibrotic effect of MIF inhibition (126). The anti-inflammatory effect and a reduction in pulmonary hypertension were also shown (126). A third study using this model in mice confirmed the antifibrotic role of MIF inhibition. They knocked down a MIF transcription factor sine oculis homeobox homolog 1, which is upregulated in alveolar type II cells of lungs with IPF and in the mouse model and drives MIF expression in these cells. The authors further proved in vitro that MIF could stimulate the proliferation of primary human lung fibroblasts. (127) In rats with the bleomycin model, MIF inhibition via a lentivirus reduced lung injury, inflammation, and fibrosis (46, 126). The authors suggested this is driven by the TGF-β1/SMADs signaling pathway (46). An essential process in pulmonary fibrosis is vascular remodeling. MIF inhibition might protect against this process via thrombospondin 2 and serpin family B member 5 signaling (46).

In summary, there is growing evidence suggesting a protective effect of MIF inhibition in pulmonary fibrosis and IPF. So far, nothing is known about the role of D-DT in pulmonary fibrosis.

2.2.5. Systemic sclerosis (Scleroderma)

Systemic sclerosis is a rare autoimmune connective-tissue disease divided into subtypes, such as limited or diffuse cutaneous systemic sclerosis (128, 129). Systemic sclerosis has the highest mortality among all rheumatic diseases (128). It is characterized by progressive fibrosis of the skin and internal organs (129). The most affected organs are the lungs, pericardium, kidneys, skeletal muscle, and gastrointestinal tract (128). The pathogenesis of systemic sclerosis is not completely understood. Several processes are proposed, including microvascular injury, inflammatory cell infiltration, excessive proliferation of fibroblasts, and resistance of fibroblasts to apoptosis. (130) Over time, excessive collagen deposition thickens the skin and restricts joint movement, leading to contractures and disability. As several organs are affected and various complications occur, the treatment is complex, and the progression of the disease can only be slowed down, but not completely stopped. (128) We will focus on the role of MIF in the skin in systemic sclerosis.

Serum levels of MIF were significantly higher in patients with systemic sclerosis than in healthy controls (130–132). However, in another larger study, MIF serum levels showed no difference between systemic sclerosis patients and healthy controls. MIF mRNA expression was significantly lower in peripheral blood mononuclear cells in systemic sclerosis patients compared to controls, while CD74 showed no difference. Immunohistochemistry staining of healthy skin revealed a strong MIF expression in all layers of the epidermis, in endothelium, and in fibroblasts of the papillary dermis. (133) In skin biopsies of systemic sclerosis patients, MIF was enhanced in keratinocytes, dermal fibroblasts, and decreased in endothelial cells (130, 132, 133). MIF expression in diffuse systemic sclerosis patients’ skin was detected in suprabasal keratinocytes, perivascular clusters of infiltrating mononuclear cells, and fibroblast-like cells (132).

To date, the functional role of MIF in systemic sclerosis has not been investigated in vivo. However, in vitro, the functional role of MIF was investigated in systemic sclerosis-derived dermal fibroblasts, which showed a lower degree of apoptosis compared to normal fibroblasts (130, 132). Even when using the apoptosis inducer sodium nitroprusside, systemic sclerosis-derived dermal fibroblasts showed a lower degree of apoptosis than normal dermal fibroblasts. MIF further increased the resistance to apoptosis by inhibiting sodium nitroprusside-induced apoptosis in dermal fibroblasts in a dose-dependent manner. Both ERK inhibition and AKT inhibition almost completely blocked the inhibitory effect of MIF on apoptosis in dermal fibroblasts. (130) These in vitro data indicated that MIF might enhance fibrosis in systemic sclerosis by increasing the resistance to apoptosis of dermal fibroblasts.

The serum concentration of D-DT showed no significant differences between systemic sclerosis patients and control patients. However, this study showed significantly higher D-DT serum concentration in systemic sclerosis patients with a low forced vital capacity of the lungs compared to those without. (131) No further data on D-DT in systemic sclerosis are available.

In summary, several studies described and correlated MIF in serum in systemic sclerosis. However, little functional data exist, overall suggesting disease aggravation by MIF.

2.3. Interventional clinical trials

Several interventional clinical trials using MIF or CD74 inhibition have been conducted, largely in the cancer field. Albeit none of them were performed in fibrotic diseases per se, we provide a brief overview of these trials showing the various available drugs for potential use in patients (based on a search on ClinicalTrials.gov). To date, there was no trial with drugs targeting D-DT or D-DTL.

Imalumab, also known as BAX69, is a recombinant, human monoclonal antibody that specifically binds to a β-sheet structure within oxMIF, including a highly conserved catalytic motif (₅₇Cys-Ala-Leu-Cys₆₀) (134). So far, Imalumab has only been tested in malignant tumors. One study investigated an unspecified anti-MIF antibody in lupus nephritis, but this study was terminated, and no results were published (NCT01541670) (Table 2).

Table 2. Overview of the clinical studies in which MIF, oxMIF, or CD74 was used as a target.

ID	Phase	Target	Drug	Condition	Status
NCT01765790	1	oxMIF	Imalumab (BAX69)	Metastatic adenocarcinoma of the colon or rectum, malignant solid tumors	Completed
NCT02448810	2	oxMIF	Imalumab (BAX69)	Metastatic colorectal cancer	Terminated
NCT02540356	1,2	oxMIF	Imalumab (BAX69)	Refractory ovarian cancer with recurrent symptomatic malignant ascite	Terminated
NCT06212076	1,2	MIF	IPG1094	Solid tumor	Not yet recruiting
NCT05112159	1	MIF	IPG1094	Safety issues	Unknown status
NCT01541670	1	MIF	unknown	Systemic lupus erythematosus	Terminated
NCT03782415	1,2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Glioblastoma, recurrent glioblastoma	Active, not recruiting
NCT03489850	2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Alcohol use disorder	Completed
NCT02025998	1	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Alcohol use disorder	Completed
NCT02714036	1,2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Amyotrophic lateral sclerosis	Completed
NCT01317992	1,2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Medication overuse headache	Unknown status
NCT01860807	2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Methamphetamine dependence	Completed
NCT01389193	1	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Migraine headache	Completed
NCT04429555	2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Pneumonia, viral (covid-19)	Completed
NCT03489850	2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Alcohol use disorder	Completed
NCT03533387	1	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Healthy volunteers	Completed
NCT04054206	1	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Healthy volunteers	Completed
NCT01217970	1	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Methamphetamine-dependence, substance abuse,	Completed
NCT04057898	2, 3	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Amyotrophic lateral sclerosis	Completed
NCT02238626	2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Amyotrophic lateral sclerosis	Completed
NCT05513560	2, 3	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Long COVID	Active, not recruiting
NCT01982942	2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Progressive multiple sclerosis	Completed
NCT03594435	2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Alcohol use disorder	Completed
NCT05414240	2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Alcohol use disorder	Recruiting
NCT03341078	2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Methamphetamine dependence	Recruiting
NCT01740414	2	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Opioid abuse	Completed
NCT04631471	3	MIF, PDE3, 4, 10	Ibudilast (MN-166)	Myelopathy, spinal cord diseases	Recruiting
NCT01663766	1	CD74	Milatuzumab (hLL1, IMMU-115)	GVHD (acute or chronic)	Terminated
NCT00421525	1, 2	CD74	Milatuzumab (hLL1, IMMU-115)	Multiple myeloma	Completed
NCT00504972	1	CD74	Milatuzumab (hLL1, IMMU-115)	Non-hodgkin’s lymphoma, chronic lymphocytic leukemia	Completed
NCT00868478	1, 2	CD74	Milatuzumab (hLL1, IMMU-115)	Chronic lymphocytic leukemia	Unknown status
NCT00989586	1, 2	CD74	Milatuzumab (hLL1, IMMU-115)	Relapsed and refractory B-cell NHL	Completed
NCT01845740	1	CD74	Milatuzumab (hLL1, IMMU-115)	Systemic lupus erythematosus	Completed
NCT00603668	1, 2	CD74	Milatuzumab (hLL1, IMMU-115)	Chronic lymphocytic lymphoma	Completed
NCT01101594	1, 2	ADC (CD74) (Conjugat with Doxorubicin)	hLL1-DOX	Multiple myeloma	Terminated
NCT01585688	1, 2	ADC (CD74) (Conjugat with Doxorubicin)	hLL1-DOX	Non-hodgkin’s lymphoma, chronic lymphocytic leukemia	Terminated
NCT05611853	1,2	ADC (CD74)	BN301	Advanced B-cell malignancies	Terminated
NCT06563804	1, 2	ADC (CD74)	S227928	Relapsed/refractory AML, MDS/AML, CMML	Not recruiting
NCT03424603	1	ADC (CD74)	STRO-001	Advanced B-cell malignancies	Completed

Ibudilast is a small-molecule inhibitor of MIF. It was first discovered as an inhibitor of phosphodiesterase 3, 4, and 10, and secondarily, an MIF-inhibiting function was described (135). Ibudilast binds as a noncompetitive inhibitor to the enzymatically active site of MIF (135). This active site is closely related to the binding site of MIF to CD74 (136). This prevents MIF from binding to CD74 (136). Ibudilast has so far been studied in tumor diseases and neuronal and psychiatric diseases, but not yet in fibrotic diseases (Table 2). IPG1094 is another small-molecule inhibitor of MIF, which is under investigation in two trials in healthy individuals and patients with advanced solid tumors (Table 2).

CD74 has been targeted by the monoclonal antibody Milatuzumab, also known as hLL1 or IMMU-115. Six trials investigated Milatuzumab in hematological neoplasms. Milatuzumab was investigated in patients with systemic lupus erythematosus (SLE), showing no safety concerns and reduced disease activity (NCT01845740) (137). Five trials investigate anti-CD74-antibody-drug-conjugates (ADC) in hematological malignancies (Table 2).

3. Conclusion and future prospects

The MIF-family has emerged as a crucial regulator of tissue remodeling in organ fibrosis. In in vivo fibrosis models, MIF showed predominantly antifibrotic effects, except for lung fibrosis. This is interesting since inflammation is mostly considered to drive fibrosis and MIF is considered pro-inflammatory. D-DT showed antifibrotic effects in fibrosis, albeit much less data is available compared to MIF. The role of D-DTL remains unexplored. Another open question is whether and how the MIF-family members might functionally interact in fibrosis.

MIF and D-DT lead to proliferation through signaling pathways such as ERK signaling. In the kidney, the proliferation of tubular cells exerts regenerative effects and is proposed as the main mechanism of anti-fibrotic effects in the kidneys. In lung fibrosis, the profibrotic effects were related to the proliferation of fibroblasts.

MIF has already been tested in clinical studies for various diseases, with no clear positive effects reported so far (Table 2). Trials in fibrotic diseases are missing. Given the potentially opposing effects of MIF in various diseases and organs, a highly selective patient population might be required for such trials. Also, given the different effects on various cells, future research should focus on specific targeting of such treatments. This is particularly true for fibrosis, which would normally require prolonged therapeutic interventions, thereby elevating the risk of adverse effects. The potential of D-DT as a therapeutic target is currently unclear. Its slightly different receptor-binding profile with potentially lower proinflammatory actions could have beneficial effects on some diseases, but dedicated studies are needed.

While many open questions remain, the MIF-family remains an interesting molecular system involved in the pathophysiology of organ fibrosis. Ultimately, a comprehensive understanding of the MIF-family has the potential to improve the way we diagnose, monitor, and treat fibrotic diseases.

Abbreviations

ADC

Antibody-drug-conjugates

AKI

Acute kidney injury

AKT

Protein kinase B

AMPK

AMP-activated protein kinase

bp

Base pairs

CD

Cluster of differentiation

CKD

Chronic kidney disease

CF

Cystic fibrosis

Col

Collagen

CXCR

CXC motif chemokine receptors

D-DT

D-dopachrome tautomerase

D-DTL

D-dopachrome tautomerase like

ECM

Extracellular matrix

ERK

Extracellular signal-regulated kinase

IL

Interleukin

I/R

Ischemia-reperfusion

IPF

Idiopathic pulmonary fibrosis

kDa

Kilodalton

KO

Knockout

LPS

Lipopolysaccharide

MIF

Macrophage-migration-inhibitory-factor

MMP

Matrix metalloproteinase

NF-κB

Nuclear factor-κB

OxMIF

Oxidized MIF

PDGF

Platelet-derived growth factor

RedMIF

Reduced MIF

sCD74

soluble CD74

TGF-β

Transforming growth factor-β

TNF

Tumor necrosis factor

UUO

Unilateral ureteral obstruction

Grants

PB is supported by the German Research Foundation (DFG, Project IDs 322900939 & 445703531), European Research Council (ERC Consolidator Grant No 101001791), and the Federal Ministry of Education and Research (BMBF, STOP-FSGS-01GM2202C). PD is supported by the START-Program of the Faculty of Medicine RWTH Aachen University (grant number 102/24).