Allogeneic hematopoietic stem cell transplantation (HSCT) has become the treatment of choice for a wide variety of hematologic malignancies and non-malignant disorders (1). Increasing numbers of HSCT are being performed every year and their indications have expanded in the recent years. However, graft-versus-host disease (GVHD) remains a significant challenge to the broader use of allogeneic HSCT. Once established, managing it becomes exceedingly difficult. Although GVHD prophylaxes exist, they come with the tradeoff of increased risk of relapse, rejection, or delayed immune recovery. Consequently, although significant progress has been made, there is no universally effective strategy for preventing or treating GVHD, particularly steroid-refractory disease. This immune-mediated condition is the most serious complication following HSCT, affecting approximately half of all recipients and causing immune dysregulation and organ dysfunction (2–5).

Several risk factors for developing GVHD were identified including: histocompatibility disparity, donor and recipient age, donor and recipient sex disparity (female donor to male recipient), intensity of conditioning regimen, use of total body irradiation (TBI), and stem cells source (2–5). Classically, the pathophysiology of acute GVHD has three phases: The afferent phase, in which an exaggerated inflammatory response leads to activation of antigen presenting cells (APCs). This phase is followed by the efferent phase, which is characterized by donor T-cell trafficking and expansion. Lastly, the effector phase, in which effector cells cause end-organ damage (6, 7).

Systemic high dose Glucocorticoids (GC) is the standard first line therapy for acute and chronic GVHD (aGVHD and cGVHD, respectively). GC have broad anti-inflammatory and regulatory effects on most of the immune cells: They reduce production of pro-inflammatory cytokines, decrease cell migration to sites of inflammation and promote apoptosis of activated cells (8, 9). In addition, GC were shown to promote regulatory T cells and anti-inflammatory macrophages (10, 11). However, therapeutic response ranges from approximately 60% in patients with grade II aGVHD to 30 to 40% in patients with grade IV disease. More than 70% of patients suffering from cGVHD will need further treatment due to the toxicity and limited efficacy of initial therapies. In both patient groups, GC refractoriness encompasses a dismal prognosis (12–16).

Thus, identifying patients that might develop GVHD and might not respond to GC treatment (steroid-refractory (SR)) and initiating early enough alternative treatments might prevent worsening of the disease and spare steroid toxicity.

Macrophage migration inhibitory factor (MIF) is a key mediator of many inflammatory diseases such as septic shock, rheumatoid arthritis, atherosclerosis and multiple sclerosis (17–23). It was reported that MIF primarily drives disease progression by promoting inflammatory cell recruitment, preventing apoptosis of activated cells and amplifying the secretion of pro-inflammatory cytokines (24, 25). MIF is secreted from various cells types such as, dendritic cells, macrophages, T cells and endothelial cells (26, 27). MIF engagement of its receptor, CD74, leads to the recruitment and activation of CD44 and CXCR2/4/7 to initiate signaling pathways necessary for mitogen-activated protein kinase (MAPK) activation, which leads to cell motility, migration and survival (28–30). MIF has a central role in counter-regulating glucocorticoid action. Increased inflammation in turn triggers rapid production of endogenous GC that eventually resolve inflammation and reduce MIF levels (31). However, chronically high MIF levels override the GC effects (32–34).

Interestingly, resistance to GC therapy is also common and occurs in up to 30% of subjects suffering from arthritis, inflammatory bowel disease and asthma. Additional studies have demonstrated that these patients express increased levels of MIF compared with GC responsive patients (35–39).

MIF expression is genetically regulated by two functional polymorphisms located in the MIF gene: alleles of the −794 CATT_5–8 microsatellite repeats and the −173 G/C single nucleotide polymorphism (SNP), which have been reported to modulate MIF promoter activity and to correlate with MIF expression levels. MIF promoter activity is proportional to increased numbers of the CATT repeats at position −794, whereas the −173 C allele may be associated with increased MIF promoter activity by its linkage disequilibrium with the high-expression of the −794 CATT₇ variant (27, 40).

Only a handful of studies have examined the role of MIF in GVHD. Their findings have shown that MIF is increased significantly in serum at the onset of aGVHD compared with levels before allogeneic HSCT. They also demonstrated that mice that received bone marrow (BM) cells from MIF knockout (KO) mice exhibited a milder form of aGVHD compared with mice that received BM cells from wild type mice (41, 42). Furthermore, Chang et al. demonstrated that patients receiving cells from donors who carry the MIF -173C allele (which correlates with high MIF expression) were at higher risk of developing chronic GVHD. However, in the same report it was suggested that recipients carrying the -173C allele have better overall survival rates and lower risk of relapse (43). Taken together, this implies that MIF might have a pivotal role as disease severity modifiers in patients suffering from aGVHD and moreover in those resistant to GC treatment.

In this study, we conducted a comprehensive evaluation of the role of MIF in patients undergoing HSCT, with a specific focus on its involvement in those who developed aGVHD and SR aGVHD. This involved measuring analyzing the frequencies of functional MIF promoter variants as well as measuring MIF serum levels, before and after HSCT.

GVHD is a major complication after HSCT. GC are the first line of treatment for both acute and chronic GVHD. However, failure to respond to GC is associated with a poor outcome with mortality rates reaching up to 80% (6, 7, 51–54). Therefore, early identification of patients who are likely to develop GVHD and those who may not respond to GC treatment [steroid-refractory (SR)] could allow for earlier intervention with alternative therapies, potentially preventing disease progression and sparing steroid toxicity. In the present study, we demonstrated that MIF levels and functional polymorphisms, are associated with GVHD severity and resistance to GC treatment.

MIF, a key mediator of numerous inflammatory diseases, was one of the first cytokines to be described (24, 25). Various studies suggested that MIF mainly contribute to diseases progression by promoting leukocyte recruitment, inhibiting apoptosis of activated monocytes, enhancing secretion of proinflammatory cytokines and counter-regulating GC suppressive effects (23, 31–33, 55).

This study evaluated the functional polymorphisms in patients undergoing HSCT, of MIF promoter and measured circulating MIF levels at several time points before and following allogeneic HSCT. A previous study by Chang et al. demonstrated that patients receiving cells from donors who carry the MIF -173C allele (which is associated with high MIF expression) had a higher risk of developing chronic GVHD, but not acute GVHD (43). In our study, both functional polymorphisms in the MIF promoter, -794 CATT5-8 repeats and -173G/C SNP, were evaluated in HSCT recipients. There was no difference in the frequencies of the -794 CATT7 and the -173 C containing genotypes between patients who did not develop aGVHD or developed only grade I aGVHD compared to those who developed grade II aGVHD. However, these genotypes were significantly associated with grades III-IV aGVHD. This suggests that high genotypic MIF expression acts as a disease modifier. Furthermore, in the multivariate analysis, this effect of MIF on disease severity was found to be superior to the effects of the conditioning regimen and HLA disparity. This role of MIF has been previously demonstrated in other autoimmune and inflammatory conditions, such as multiple sclerosis, rheumatoid arthritis and systemic sclerosis (17, 23, 27, 56). Our results are in agreement with the findings of Chang et al., as we did not observe any significant differences in the frequencies of the -173G/C genotypes between patients who did not develop aGVHD and those who did. In our study, the significant difference between the two groups was observed only for the -794 CATT polymorphism. Moreover, we have assessed MIF polymorphisms in relation to aGVHD severity and found that it was significantly correlated with the frequencies of the high-expression CATT7 containing genotypes (grade III-IV vs grade II vs grade I and non 36.8% vs. 0% vs. 5.8% p=0.0019 OR=10.4, p=0.0080 OR=10.2, respectively). These data might give clinicians an important tool for GVHD severity prediction and may help guide clinical decision-making, such as introducing additional therapeutic interventions earlier in the course of treatment for high-risk patients. Unfortunately, we did not evaluate MIF polymorphisms in donors, since most of the patients received grafts from MUD that were not available for consent. This should be further studied in the future, especially in light of the data from Toubai et al. that showed in a murine model of GVHD that GVHD severity did not differ between mice that received cells from MIF knockout (KO) mice compared with mice that received cells from WT mice. However, they reported that there was an attenuation in disease severity when the transplanted cells were from MIF KO donors compared with WT donor mice (57). Interestingly, it should be noted that in these sets of experiments, the MIF KO mice strain was only BALB/c. Therefore, it would be interesting to perform similar experiments with C57BL/6 MIF KO mice, as donor or recipient mice. This would provide an additional informative comparison where the donor and recipient mice are from the same strain.

Consistent with previous studies (12, 51, 58), approximately 30% of patients suffering from aGVHD in our study had SR-aGVHD. This underscores the need to identify biomarkers for SR GVHD. We suggest that MIF functional genetic polymorphisms could serve as potential biomarkers for this purpose. This novel finding in aGVHD is in-line with studies on asthma and inflammatory bowel disease, which have also described the effect of MIF on resistance to GC therapy (35–37). Moreover, it was demonstrated that T cells from individuals carrying high-expressing genotypes were more resistant glucocorticoid-induced apoptosis compared with low-expressing MIF genotypes (59).

It is important to note that the frequencies of the CATT7 containing genotypes in patients with grade III-IV (36.8%) and in patients with GVHD that are resistant to steroid treatment (46.6%) are higher than the frequency of these genotypes in the general Caucasian population (20-22%) (60).

Lo et al. and Toubai et al. reported that serum MIF levels were significantly elevated before the onset of aGVHD, suggesting its role in this pathology (41, 42). Similarly, we observed that circulating MIF levels were significantly elevated in patients that developed severe aGVHD compared to MIF levels before HSCT and to patients who did not develop aGVHD or developed grade I disease. This significant difference was noted on day-30 post-HSCT, before the median day of aGVHD onset, which occurred on day 36 post-HSCT, pointing to MIF’s potential role in the etiology of acute GVHD. The lack of significance on day 90 post-HSCT could be attributed to the impact of second-line anti-GVHD treatments and to the fact that some patients with severe aGVHD did not survive to this time point. Interestingly, we show that MIF serum levels were significantly elevated in males who developed GVHD but not in females. This observation aligns with previous studies that reported a gender dependent effect of MIF on multiple sclerosis progression (17). We did not observe any gender dependent effect in MIF genetic polymorphisms, which could point to gender dependent post-translational regulation on MIF levels. This effect should be further discerned in a larger study. It is important to note that while some studies on large and clinically homogeneous inflammatory syndromes have observed a concordance between MIF genotype and serum levels, other studies have not (47, 61). In patients undergoing allogeneic HSCT, MIF levels can also be influenced by donor cells, introducing a potentially significant factor that may impact the relationship between genotype and serum levels. It is also important to note that serum MIF levels may not fully represent MIF expression, as its production can be genetically regulated at the site of inflammation.

This study is limited by its relatively small cohort size and the lack of donor samples. Furthermore, since the samples were collected at predefined time points, we did not correlate them with the day of aGVHD onset or the response to treatment.

In conclusion, our findings suggest that MIF acts as a disease modifier in aGVHD. High recipient genotypic MIF expression likely corresponds with increased MIF levels and additional proinflammatory factors that enhance disease progression and resistance to GC treatment. Taken together, we propose potential use of MIF polymorphism as a tool for personalized tailoring of GVHD prophylaxis and treatment. In addition, novel therapies targeting MIF such as, ISO-1 (62), partial MHC class II constructs (63) and Ibudilast (64), could potentially reduce aGVHD severity and counteract the inhibitory effect on GC. This approach might also preserve the graft versus leukemia response and aligns with the role of MIF in inflammation progression and exacerbation rather than initiation.