This study aimed to evaluate the cardioprotective effects of ghrelin in septic mice, focusing on its anti-inflammatory and antioxidant properties. Thirty-five male Swiss mice (8-12 weeks old, 23-33g) were randomly assigned to five groups (n = 7 each): (1) Normal, fed usual diets, (2) Sham, subjected to anesthesia and laparotomy, (3) Sepsis, subjected to cecal ligation and puncture, (4) Vehicle, given an equivalent volume of intraperitoneal saline injections immediately after cecal ligation and puncture, and (5) Ghrelin-treated, administered 80 µg/kg ghrelin intraperitoneal injections immediately following cecal ligation and puncture. Serum levels of tumor necrosis factor-alpha (TNF-α), macrophage migration inhibitory factor (MIF), toll-like receptor 4 (TLR4), and 8-epi-prostaglandin F2 alpha (8-epi-PGF2α) were measured. The extent of cardiac damage was also evaluated histologically. The mean serum levels of TNF-α, MIF, TLR4, and 8-epi-PGF2α levels were significantly higher in the sepsis and vehicle groups than in the normal and sham groups. The levels were significantly lower in the ghrelin-treated group than in the vehicle and sepsis groups. Histological analysis revealed normal myocardial architecture in the normal and sham groups, whereas the sepsis and vehicle groups had severe myocardial injury. The ghrelin-treated group displayed histological features similar to the sham group, indicating reduced myocardial damage. Ghrelin ameliorated sepsis-induced cardiotoxicity in mice by exhibiting strong anti-inflammatory and antioxidant effects. These findings suggest that ghrelin may be a promising therapeutic candidate for the prevention of sepsis-induced cardiotoxicity.

Macrophage Migration Inhibitory Factor (MIF) and its homolog D-dopachrome Tautomerase (DDT) have been implicated as drivers of tumor progression across a variety of cancers. Recent evidence suggests MIF as a therapeutic target in immune checkpoint inhibition (ICI) resistant melanomas, however clinical evidence of MIF and particularly of DDT remain limited. This retrospective study analyzed 97 patients treated at Yale for melanoma between 2002-2020. Bulk-RNA sequencing of patient tumor samples from the Skin Cancer SPORE Biorepository was used to evaluate for differential gene expression of MIF, DDT, CD74, and selected inflammatory markers, and gene expression was correlated with patient survival outcomes. Our findings revealed a strong correlation between MIF and DDT levels, with no statistically significant difference across common melanoma mutations and subtypes. Improved survival was associated with lower MIF and DDT levels and higher CD74:MIF and CD74:DDT levels. High CD74:DDT and CD74:MIF levels were also associated with enrichment of infiltrating inflammatory cell markers. These data suggest DDT as a novel target in immune therapy. Dual MIF and DDT blockade may provide synergistic responses in patients with melanoma, irrespective of common mutations, and may overcome ICI resistance. These markers may also provide prognostic value for further biomarker development.

Colorectal cancer is one of the most common malignant tumors worldwide, with high incidence and mortality rates making it a focus of research. Chemotherapy is a primary treatment modality for colon cancer, but chemotherapy resistance severely impacts treatment efficacy. MIF has been found to promote tumor progression and resistance in various cancers. This study aims to investigate the role of MIF in chemotherapy resistance in colon cancer and its potential mechanisms, particularly through the upregulation of CXCR7 expression, affecting the metabolism and drug sensitivity of colon cancer cells. The expression levels of MIF in colon cancer tissues and its association with patient prognosis were evaluated by analyzing TCGA and HPA data. Subsequently, the expression levels of MIF in colon cancer cell lines and resistant cell lines were detected by qRT-PCR and immunohistochemistry, and the effect of MIF on oxaliplatin sensitivity was assessed. The impact of MIF on the metabolic activity of colon cancer cells was measured using a cellular energy metabolism analyzer. Further experiments explored the mechanism by which MIF affects the metabolic activity of colon cancer cells through the upregulation of CXCR7 expression, and the role of CTCF in regulating CXCR7 transcription was validated by silencing CTCF. Finally, the effect of MIF on drug sensitivity of colon cancer cells was verified in a mouse xenograft tumor model. In this study, we found that the expression of MIF in colon cancer tissues was significantly higher than in normal tissues, and high MIF expression was associated with poor prognosis in patients. The expression levels of MIF in resistant colon cancer cell lines were significantly higher than in parental cell lines, and MIF overexpression significantly increased the resistance of colon cancer cells to oxaliplatin. Conversely, silencing MIF significantly reduced the IC50 value of resistant cells and increased apoptosis. MIF overexpression significantly increased the ECAR and OCR levels of colon cancer cells, while MIF knockdown significantly reduced these metabolic indicators. Further studies indicated that MIF affects the metabolic activity of colon cancer cells by upregulating CXCR7 expression. CTCF binding peaks at the CXCR7 promoter region and luciferase activity assays indicated that CTCF regulates CXCR7 transcription, and silencing CTCF significantly enhanced the sensitivity of colon cancer cells to oxaliplatin. In vivo experiments in mice showed that MIF silencing combined with oxaliplatin treatment significantly inhibited tumor growth and increased the necrotic area of tumor tissues. In conclusion, this study reveals the crucial role of MIF in chemotherapy resistance in colon cancer through the upregulation of CXCR7 expression, with CTCF playing an important regulatory role in this process. Our findings provide new theoretical insights and potential therapeutic targets for overcoming chemotherapy resistance in colon cancer. Future research should further explore the roles of MIF and CXCR7 in other types of cancers and the potential of MIF and CXCR7 as therapeutic targets.

Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with roles in innate and adaptive human immune responses, as well as inflammation. MIF exerts its biological activity by binding to the cell surface receptor CD74 as well as intracellular signalling proteins. MIF also possesses keto-enol tautomerase activity. Inhibition of the tautomerase activity has been associated with loss of biological activity of MIF and a potential anticancer target. Isothiocyanates (ITCs) are a class of compounds present in cruciferous vegetables that inhibit the MIF tautomerase activity via covalent modification of the N-terminal proline. A range of substituted ITCs featuring benzyl, phenethyl and phenyl propyl isothiocyanates were designed, synthesised and tested to determine any structure activity relationship for inhibiting MIF. Crystal structures of covalent compounds 8 and 9 in complex with rhMIF revealed key hydrogen bonding and edge-to-face π stacking interactions. Compound 9 and 11 with sub micromolar activity were tested in the NCI60 cancer cell lines panel. Both compounds showed tissue-specific reduced growth in colon and renal cancer cell lines, while one of these showed potent, dose-dependent inhibition of growth against all seven colon cancer cell lines (GI50 < 2.5 µM) and all eight renal cancer cell lines (GI50 < 2.2 µM).

MIF is a ubiquitous protein involved in proinflammatory processes, which undergoes an oxidation-driven conformational change to oxidized (ox)MIF. We demonstrate that hypochlorous acid, produced by neutrophil-released myeloperoxidase (MPO) under inflammatory conditions, effectively oxidizes MIF into the oxMIF isoform, which is specifically recognized by the anti-oxMIF therapeutic antibody, ON104. NMR investigation of MIF oxidized by the MPO system revealed increased flexibility throughout the MIF structure, including at several catalytic and allosteric sites. Mass spectrometry of MPO-oxMIF revealed methionines as the primary site of oxidation, whereas Pro2 and Tyr99/100 remained almost unmodified. ELISA, SPR and cell-based assays demonstrated that structural changes caused by MPO-driven oxidation promoted binding of oxMIF to its receptor, CD74, which does not occur with native MIF. These data reveal the environment and modifications that facilitate interactions between MIF and its pro-inflammatory receptor, and a route for therapeutic intervention targeting the oxMIF isoform.

BACKGROUND: The role of tumor inflammatory microenvironment in the advancement of cancer, particularly prostate cancer, is widely acknowledged. ELL-associated factor 2 (EAF2), a tumor suppressor that has been identified in the prostate, is often downregulated in prostate cancer. Earlier investigations have shown that mice with EAF2 gene knockout exhibited a substantial infiltration of inflammatory cells into the prostatic stroma.
METHODS: A cohort comprising 38 patients who had been diagnosed with prostate cancer and subsequently undergone radical prostatectomy (RP) was selected. These patients were pathologically graded according to the Gleason scoring system and divided into two groups. The purpose of this selection was to investigate the potential correlation between EAF2 and CD163 using immunohistochemistry (IHC) staining. Additionally, in vitro experimentation was conducted to verify the relationship between EAF2 expression, macrophage migration and polarization.
RESULTS: Our study demonstrated that in specimens of human prostate cancer, the expression of EAF2 was notably downregulated, and this decrease was inversely associated with the number of CD163-positive macrophages that infiltrated the cancerous tissue. Cell co-culture experiments revealed that the chemotactic effect of tumor cells towards macrophages was intensified and that macrophages differentiated into tumor-associated macrophages (TAMs) when EAF2 was knocked out. Additionally, the application of cytokine protein microarray showed that the expression of chemokine macrophage migration inhibitory factor (MIF) increased after EAF2 knockout.
CONCLUSIONS: Our findings suggested that EAF2 was involved in the infiltration of CD163-positive macrophages in prostate cancer via MIF.

Accurate screening of COVID-19 infection status for symptomatic patients is a critical public health task. Although molecular and antigen tests now exist for COVID-19, in resource-limited settings, screening tests are often not available. Furthermore, during the early stages of the pandemic tests were not available in any capacity. We utilized an automated machine learning (ML) approach to train and evaluate thousands of models on a clinical dataset consisting of commonly available clinical and laboratory data, along with cytokine profiles for patients (n = 150). These models were then further tested for generalizability on an out-of-sample secondary dataset (n = 120). We were able to develop a ML model for rapid and reliable screening of patients as COVID-19 positive or negative using three approaches: commonly available clinical and laboratory data, a cytokine profile, and a combination of the common data and cytokine profile. Of the tens of thousands of models automatically tested for the three approaches, all three approaches demonstrated > 92% sensitivity and > 88 specificity while our highest performing model achieved 95.6% sensitivity and 98.1% specificity. These models represent a potential effective deployable solution for COVID-19 status classification for symptomatic patients in resource-limited settings and provide proof-of-concept for rapid development of screening tools for novel emerging infectious diseases.

Macrophages are a rich source of macrophage migration inhibitory factor (MIF). It is well established that macrophages and MIF play a pathogenic role in anti-glomerular basement membrane crescentic glomerulonephritis (anti-GBM CGN). However, whether macrophages mediate anti-GBM CGN via MIF-dependent mechanism remains unexplored, which was investigated in this study by specifically deleting MIF from macrophages in MIFf/f-lysM-cre mice. We found that compared to anti-GBM CGN induced in MIFf/f control mice, conditional ablation of MIF in macrophages significantly suppressed anti-GBM CGN by inhibiting glomerular crescent formation and reducing serum creatinine and proteinuria while improving creatine clearance. Mechanistically, selective MIF depletion in macrophages largely inhibited renal macrophage and T cell recruitment, promoted the polarization of macrophage from M1 towards M2 via the CD74/NF-κB/p38MAPK-dependent mechanism. Unexpectedly, selective depletion of macrophage MIF also significantly promoted Treg while inhibiting Th1 and Th17 immune responses. In summary, MIF produced by macrophages plays a pathogenic role in anti-GBM CGN. Targeting macrophage-derived MIF may represent a novel and promising therapeutic approach for the treatment of immune-mediated kidney diseases.

BACKGROUND: Inflammatory macrophage infiltration plays a critical role in acute kidney disease induced by ischemia-reperfusion (IRI-AKI). Calycosin is a natural flavone with multiple bioactivities. This study aimed to investigate the therapeutic role of calycosin in IRI-AKI and its underlying mechanism.
METHODS: The renoprotective and anti-inflammatory effects of calycosin were analyzed in C57BL/6 mice with IRI-AKI and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA-seq was used for mechanism investigation. The molecular target of calycosin was screened by in silico methods and validated by surface plasmon resonance (SPR). Macrophage chemotaxis was analyzed using Transwell and agarose gel spot assays.
RESULTS: Calycosin treatment significantly reduced serum creatinine and urea nitrogen and attenuated tubular destruction in IRI-AKI mice. Additionally, calycosin markedly suppressed NF-κB signaling activation and the expression of inflammatory mediators IL-1β and TNF-α in IRI-AKI kidneys and LPS-stimulated RAW 264.7 cells. Interestingly, RNA-seq revealed calycosin remarkably downregulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was downregulated in both LPS-stimulated RAW 264.7 cells and IRI-AKI kidneys. Consistently, calycosin treatment attenuated macrophage infiltration in the IRI-AKI kidneys. Importantly, in silico target prediction, molecular docking, and SPR assay demonstrated that calycosin directly binds to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-κB signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells.
CONCLUSIONS: In summary, calycosin attenuates IRI-AKI by inhibiting MIF-mediated macrophage inflammatory chemotaxis, suggesting it could be a promising therapeutic agent for the treatment of IRI-AKI.