Lung cancer is the leading cause of cancer-related mortality worldwide, accounting for more deaths than any other malignancy. Despite advances in treatment, it remains highly lethal, with 5-year survival rates showing minimal improvement over the past several decades, highlighting a critical unmet clinical need. Macrophage Migration Inhibitory Factor (MIF) is a multifunctional cytokine that contributes to inflammation and cancer, promoting tumor growth, progression, and metastasis through modulation of the tumor microenvironment, stimulation of angiogenesis, and regulation of immune responses. Polymorphisms in the promoter region of MIF, such as high-expression CATT repeats, influence MIF expression and susceptibility to a range of inflammatory, autoimmune, and malignant disorders, yet their role in lung cancer remains largely unexplored. Therapeutic strategies targeting MIF, including small-molecule inhibitors, antibodies, and peptide-based agents, have shown promise in preclinical models, although their clinical translation is still limited. This review discusses the dual role of MIF in inflammation and oncology, summarizes current therapeutic developments, and emphasizes the potential of MIF-targeted interventions in lung cancer. It discusses the significance of genetic predisposition, particularly high-expression MIF alleles, in guiding personalized treatment strategies for lung cancer and identifying patients who may derive benefit from MIF inhibition.

The isolation of bone marrow-derived macrophages (BMDMs) from the bones of mice provides an excellent tool to study the different activation states of macrophages both in vitro and ex vivo. This chapter utilizes BMDMs derived from wild-type and -794 CATT7 human transgenic mice to investigate the influence of high human MIF expression on macrophage polarization. Real-time qPCR and ELISA can be used to easily distinguish M1 and M2 polarization phenotypes.

Macrophage migration inhibitory factor (MIF) is an upstream regulatory cytokine that is associated with advanced disease and poor outcomes in multiple cancer types, including melanoma. We investigated whether anti-MIF therapy could enhance the antitumor effects of the immune checkpoint inhibitor anti-programmed cell death 1 (anti-PD-1) in 2 murine tumor models. The therapeutic efficacy of anti-MIF, alone or combined with anti-PD-1, was tested in the YUMMER1.7 melanoma and MC38 colorectal cancer models. Tumor growth and survival were assessed in untreated Mif-knockout (KO) and low-expression human MIF allele (CATT5) mice and compared with wild-type (WT) or high-expression MIF allele (CATT7) mice. Tumor-bearing animals underwent cytokine profiling, tumor immunohistochemistry, flow cytometry, and scRNA-Seq. We also correlated functional variant MIF alleles with melanoma incidence and progression in patients. Our results showed that combined anti-MIF and anti-PD-1 significantly reduced tumor growth, improved survival, and promoted tumor regression, accompanied by enhanced TH1 cytokine levels, increased macrophage activation-related cytokines, and increased type 1 conventional dendritic cells. scRNA-Seq analysis revealed an expansion of intratumor Cd74/C1q/Aif1-expressing macrophages, which exhibited an antitumor phenotype, in response to anti-MIF therapy. MIF-KO and CATT5 mice exhibited reduced tumor burdens compared with WT or CATT7 mice alone and in the presence of anti-PD-1. In patients with melanoma, the high-MIF expression genotype (-173C/C) occurred at higher frequencies compared with healthy controls. These findings highlight that the addition of anti-MIF to anti-PD-1 reduces tumor growth, enhances antitumor responses, prolongs survival, and augments key intratumor immune cell populations involved in immune activation against tumors. This approach merits further consideration for clinical trial development.

Enhancing mesenchymal stromal cell (MSC) therapeutic efficacy through licensing with proinflammatory cytokines is now well established. We have previously shown that macrophage migration inhibitory factor (MIF)-licensed MSCs exerted significantly enhanced therapeutic efficacy in reducing inflammation in house dust mite (HDM)-driven allergic asthma. Soluble mediators released into the MSC secretome boast cytoprotective properties equal to those associated with the cell itself. In asthma, epithelial barrier damage caused by the inhalation of allergens like HDM drives goblet cell hyperplasia. Vascular endothelial growth factor (VEGF) plays a pivotal role in the repair and maintenance of airway epithelial integrity. Human bone marrow-derived MSCs expressed the MIF receptors CD74, CXCR2, and CXCR4. Endogenous MIF from high MIF expressing CATT 7 bone marrow-derived macrophages increased MSC production of VEGF through the MIF CXCR4 chemokine receptor, where preincubation with CXCR4 inhibitor mitigated this effect. CATT 7 -MIF licensed MSC conditioned media containing increased levels of VEGF significantly enhanced bronchial epithelial wound healing via migration and proliferation in vitro. Blocking VEGFR2 or the use of mitomycin C abrogated this effect. Furthermore, CATT 7 -MIF MSC CM significantly decreased goblet cell hyperplasia after the HDM challenge in vivo. This was confirmed to be VEGF-dependent, as the use of anti-human VEGF neutralising antibody abrogated this effect. Overall, this study highlights that MIF-licenced MSCs show enhanced production of VEGF, which has the capacity to repair the lung epithelium.

High level expression of the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) has been associated with severe asthma. The role of MIF and its functional promotor polymorphism in innate immune training is currently unknown. Using novel humanized CATT7 MIF mice, this study is the first to investigate the effect of MIF on bone marrow-derived macrophage (BMDM) memory after house dust mite (HDM) challenge. CATT7 BMDMs demonstrated a significant primed increase in M1 markers following HDM and LPS stimulation, compared to naive mice. This M1 signature was found to be MIF-dependent, as administration of a small molecule MIF inhibitor, SCD-19, blocked the induction of this pro-inflammatory M1-like phenotype in BMDMs from CATT7 mice challenged with HDM. Training naive BMDMs in vitro with HDM for 24 h followed by a rest period and subsequent stimulation with LPS led to significantly increased production of the pro-inflammatory cytokine TNFα in BMDMs from CATT7 mice but not WT mice. Addition of the pan methyltransferase inhibitor MTA before HDM training significantly abrogated this effect in BMDMs from CATT7 mice, suggesting that HDM-induced training is associated with epigenetic remodelling. These findings suggest that trained immunity induced by HDM is under genetic control, playing an important role in asthma patients with the high MIF genotypes (CATT6/7/8).

Background: Trained immunity results in long-term immunological memory, provoking a faster and greater immune response when innate immune cells encounter a secondary, often heterologous, stimulus. We have previously shown that house dust mite (HDM)-induced innate training is amplified in mice expressing the human macrophage migration inhibitory factor (MIF) CATT(7) functional polymorphism. Aim: This study investigated the ability of mesenchymal stromal cells (MSCs) to modulate MIF-driven trained immunity both in vitro and in vivo. Methods: Compared with wild-type mice, in vivo HDM-primed bone marrow-derived macrophages (BMDMs) from CATT(7) mice expressed significantly higher levels of M1-associated genes following lipopolysaccharide stimulation ex vivo. Co-cultures of CATT(7) BMDMs with MSCs suppressed this HDM-primed effect, with tumor necrosis factor alpha (TNF-alpha) being significantly decreased in a cyclooxygenase 2 (COX-2)-dependent manner. Interestingly, interleukin 6 (IL-6) was suppressed by MSCs independently of COX-2. In an in vitro training assay, MSCs significantly abrogated the enhanced production of pro-inflammatory cytokines by HDM-trained CATT(7) BMDMs when co-cultured at the time of HDM stimulus on day 0, displaying their therapeutic efficacy in modulating an overzealous human MIF-dependent immune response. Utilizing an in vivo model of HDM-induced trained immunity, MSCs administered systemically on day 10 and day 11 suppressed this trained phenomenon by significantly reducing TNF-alpha and reducing IL-6 and C-C motif chemokine ligand 17 (CCL17) production. Conclusions: This novel study elucidates how MSCs can attenuate an MIF-driven, HDM-trained response in CATT(7) mice in a model of allergic airway inflammation.

Functional variants of the gene for the cytokine macrophage migration inhibitory factor (MIF) are defined by a 4-nucleotide promoter microsatellite (-794 CATT5-8, rs5844572) and confer risk for autoimmune, infectious, and oncologic diseases. We describe herein the discovery of a prototypic, small molecule inhibitor of MIF transcription with selectivity for high microsatellite repeat number and correspondingly high gene expression. Utilizing a high-throughput luminescent proximity screen, we identify 1-carbomethoxy-5-formyl-4,6,8trihydroxyphenazine (CMFT) to inhibit the functional interaction between the transcription factor ICBP90 (namely, UHRF1) and the MIF -794 CATT5-8 promoter microsatellite. CMFT inhibits MIF mRNA expression in a -794 CATT5-8 length-dependent manner with an IC50 of 470 nM, and preferentially reduces ICBP90-dependent MIF mRNA and protein expression in high-genotypic versus low-genotypic MIF- expressing macrophages. RNA expression analysis also showed CMFT to downregulate MIF-dependent, inflammatory gene expression with little evidence of off-target metabolic toxicity. These findings provide proof-of-concept for advancing the pharmacogenomic development of precision-based MIF inhibitors for diverse autoimmune and inflammatory conditions.

Macrophage migration inhibitory factor (MIF) expression is controlled by a functional promoter polymorphism, where the number of tetranucleotide repeats (CATTn ) corresponds to the level of MIF expression. To examine the role of this polymorphism in a pre-clinical model of allergic asthma, novel humanized MIF mice with increasing CATT repeats (CATT5 and CATT7 ) were used to generate a physiologically relevant scale of airway inflammation following house dust mite (HDM) challenge. CATT7 mice expressing high levels of human MIF developed an aggressive asthma phenotype following HDM challenge with significantly elevated levels of immune cell infiltration, production of inflammatory mediators, goblet cell hyperplasia, subepithelial collagen deposition, and airway resistance compared to wild-type controls. Importantly the potent MIF inhibitor SCD-19 significantly mitigated the pathophysiology observed in CATT7 mice after HDM challenge, demonstrating the fundamental role of endogenous human MIF expression in the severity of airway inflammation in vivo. Up to now, there are limited reproducible in vivo models of asthma airway remodeling. Current asthma medications are focused on reducing the acute inflammatory response but have limited effects on airway remodeling. Here, we present a reproducible pre-clinical model that capitulates asthma airway remodeling and suggests that in addition to having pro-inflammatory effects MIF may play a role in driving airway remodeling.

Background Genetic predisposition to coronavirus disease 2019 (COVID-19) may contribute to its morbidity and mortality. Because cytokines play an important role in multiple phases of infection, we examined whether commonly occurring, functional polymorphisms in macrophage migration inhibitory factor (MIF) are associated with COVID-19 infection or disease severity. Aim To determine associations of common functional polymorphisms in MIF with symptomatic COVID-19 or its severity. Methods This retrospective case-control study utilized 1171 patients with COVID-19 from three tertiary medical centers in the USA, Hungary and Spain, together with a group of 637 pre-pandemic, healthy control subjects. Functional MIF promoter alleles (-794 CATT(5-8,)rs5844572), serum MIF and soluble MIF receptor levels, and available clinical characteristics were measured and correlated with COVID-19 diagnosis and hospitalization. Experimental mice genetically engineered to express human high- or low-expression MIF alleles were studied for response to coronavirus infection. Results In patients with COVID-19, there was a lower frequency of the high-expression MIF CATT(7) allele when compared to healthy controls [11% vs. 19%, odds ratio (OR) 0.54 [0.41-0.72], P < 0.0001]. Among inpatients with COVID-19 (n = 805), there was a higher frequency of the MIF CATT(7) allele compared to outpatients (n = 187) (12% vs. 5%, OR 2.87 [1.42-5.78], P = 0.002). Inpatients presented with higher serum MIF levels when compared to outpatients or uninfected healthy controls (87 ng/ml vs. 35 ng/ml vs. 29 ng/ml, P < 0.001, respectively). Among inpatients, circulating MIF concentrations correlated with admission ferritin (r = 0.19, P = 0.01) and maximum CRP (r = 0.16, P = 0.03) levels. Mice with a human high-expression MIF allele showed more severe disease than those with a low-expression MIF allele. Conclusions In this multinational retrospective study of 1171 subjects with COVID-19, the commonly occurring -794 CATT(7)MIF allele is associated with reduced susceptibility to symptomatic SARS-CoV-2 infection but increased disease progression as assessed by hospitalization. These findings affirm the importance of the high-expression CATT(7)MIF allele, which occurs in 19% of the population, in different stages of COVID-19 infection.

Current asthma therapies focus on reducing symptoms but fail to restore existing structural damage. Mesenchymal stromal cell (MSC) administration can ameliorate airway inflammation and reverse airway remodeling. However, differences in patient disease microenvironments seem to influence MSC therapeutic effects. A polymorphic CATT tetranucleotide repeat at position 794 of the human macrophage migration inhibitory factor (hMIF) gene has been associated with increased susceptibility to and severity of asthma. We investigated the efficacy of human MSCs in high- vs. low-hMIF environments and the impact of MIF pre-licensing of MSCs using humanized MIF mice in a clinically relevant house dust mite (HDM) model of allergic asthma. MSCs significantly attenuated airway inflammation and airway remodeling in high-MIF-expressing CATT7 mice but not in CATT5 or wild-type littermates. Differences in efficacy were correlated with increased MSC retention in the lungs of CATT7 mice. MIF licensing potentiated MSC anti-inflammatory effects at a previously ineffective dose. Mechanistically, MIF binding to CD74 expressed on MSCs leads to upregulation of cyclooxygenase 2 (COX-2) expression. Blockade of CD74 or COX-2 function in MSCs prior to administration attenuated the efficacy of MIF-licensed MSCs in vivo. These findings suggest that MSC administration may be more efficacious in severe asthma patients with high MIF genotypes (CATT6/7/8).

Background Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial pneumonia of unknown aetiology with a mean survival rate of less than 3 years. No previous studies have been performed on the role of co-infection (viral and bacterial infection) in the pathogenesis and progression of IPF. In this study, we investigated the role of viral/bacterial infection and coinfection and their possible association with pathogenesis and progression of IPF. Methods We investigated the prevalence and impact of bacterial and viral coinfection in IPF patients (n = 67) in the context of pulmonary function (FVC, FEV 1 and DL CO ), disease status and mortality risk. Using principal component analysis (PCA), we also investigated the relationship between distribution of bacterial and viral co-infection in the IPF cohort. Results Of the 67 samples, 17.9% samples were positive for viral infection, 10.4% samples were positive for bacterial infection and 59.7% samples were positive coinfection. We demonstrated that IPF patients who were co-infected had a significantly increased risk of mortality compared ( p = 0.031) with IPF patients who were non-infected [Hazard ratio: 8.12; 95% CI 1.3–26.9]. Conclusion In this study, we report for the first time that IPF patients who were coinfected with bacterial and viral infection have significantly decreased FVC and DL CO (% predicted). Besides, the results demonstrated the increased AE-IPF, increased incidence of death and risk of mortality in infected/coinfected patients compared to non-infected IPF patients.

Rationale: The Toll-like receptor 3 Leu412Phe (TLR3 L412F) polymorphism attenuates cellular antiviral responses and is associated with accelerated disease progression in idiopathic pulmonary fibrosis (IPF). The role of TLR3 L412F in bacterial infection in IPF or in acute exacerbations (AE) has not been reported. Objectives: To characterize the association between TLR3 L412F and AE-related death in IPF. To determine the effect of TLR3 L412F on the lung microbiome and on antibacterial TLR responses of primary lung fibroblasts from patients with IPF. Methods: TLR-mediated antibacterial and antiviral responses were quantitated in L412F wild-type and 412F-heterozygous primary lung fibroblasts from patients with IPF using ELISA, Western blot analysis, and quantitative PCR. Hierarchical heatmap analysis was employed to establish bacterial and viral clustering in nasopharyngeal lavage samples from patients with AE-IPF. 16S ribosomal RNA quantitative PCR and pyrosequencing were used to determine the effect of TLR3 L412F on the IPF lung microbiome. Measurements and Main Results: A significant increase in AE-related death in patients with 412F-variant IPF was reported. We established that 412F-heterozygous IPF lung fibroblasts have reduced antibacterial TLR responses to LPS (TLR4), Pam3CYSK4 (TLR1/2), flagellin (TLR5), and FSL-1 (TLR6/1) and have reduced responses to live Pseudomonas aeruginosa infection. Using 16S ribosomal RNA sequencing, we demonstrated that 412F-heterozygous patients with IPF have a dysregulated lung microbiome with increased frequencies of Streptococcus and Staphylococcus spp. Conclusions: This study reveals that TLR3 L412F dysregulates the IPF lung microbiome and reduces the responses of IPF lung fibroblasts to bacterial TLR agonists and live bacterial infection. These findings identify a candidate role for TLR3 L412F in viral- and bacterial-mediated AE death.

Aim: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, which has been shown to promote disease severity in cystic fibrosis. Methods: In this study, aerosolized drug-loaded nanoparticles containing SCD-19, an inhibitor of MIF's tautomerase enzymatic activity, were developed and characterized. Results: The aerosolized nanoparticles had an optimal droplet size distribution for deep lung deposition, with a high degree of biocompatibility and significant cellular uptake. Conclusion: For the first time, we have developed an aerosolized nano-formulation against MIF's enzymatic activity that achieved a significant reduction in the inflammatory response of macrophages, and inhibited Pseudomonas aeruginosa biofilm formation on airway epithelial cells. This represents a potential novel adjunctive therapy for the treatment of P. aeruginosa infection in cystic fibrosis.