D-dopachrome tautomerase in adipose tissue inflammation and wound repair

D-dopachrome tautomerase (D-DT/MIF-2) is a member of the macrophage migration inhibitory factor (MIF) cytokine superfamily, and a close structural homolog of MIF. MIF and D-DT have been reported to be involved in obesity, but there is little known about the regulation of D-DT in adipose tissue inflammation and wound healing. Subcutaneous adipose tissue was collected from 54 healthy donors and 28 donors with acutely inflamed wounds undergoing wound debridement. In addition, epididymal fat pads of mice were injected with lipopolysaccharide to study receptor expression and cell migration in vivo. D-DT protein levels and mRNA expression were significantly decreased in subcutaneous adipose tissue adjacent to acutely inflamed wounds. D-DT improved fibroblast viability and increased proliferation in vitro. While D-DT alone did not have a significant effect on in vitro fibroblast wound healing, simultaneous addition of neutralizing MIF antibody resulted in a significant improvement of fibroblast wound healing. Interestingly, expression of the MIF and D-DT receptor CD74 was down-regulated while the MIF receptors CXCR2 and CXCR4 were up-regulated primarily on macrophages indicating that the MIF-CXCR2/4 axis may promote recruitment of inflammatory cells into adipose tissue. Our results describe a reciprocal role of D-DT to MIF in inflamed adipose tissue, and indicate that D-DT may be beneficial in wound repair by improving fibroblast survival and proliferation

D-Dopachrome tautomerase (D-DT) : functional homologue or cross-regulator of macrophage migration inhibitory factor (MIF)?

Macrophage migration inhibitory factor (MIF) is a pleiotropic immunoregulatory and pro-inflammatory cytokine, which plays a pivotal role in the pathogenesis of various acute and chronic inflammatory diseases. For example, endotoxemia studies in mice reveal that MIF administration decreases the survival rate, whereas MIF neutralization or genetic deletion of Mif improve the chances for a beneficial outcome. Additionally, high MIF levels are measured in a variety of human tumors and functional studies reveal MIF involvement in multiple aspects of tumor progression including control of cell proliferation and prevention of apoptosis. Unlike other cytokines, MIF also is a phenylpyruvate tautomerase that converts two non-physiologic enzymatic substrates, D-dopachrome and p-hydroxyphenylpyruvate (HPP). To date, possible relationships between the tautomerase and inflammatory or tumor-supportive activities of MIF remain unclear. In 1993, D-dopachrome tautomerase (D-DT) was discovered during an investigation of melanogenesis. The tertiary structure of D-DT is remarkably similar to MIF, identifying D-DT as the only known eukaryotic MIF homologue discovered to date. D-DT shares MIF’s tautomerase activity and, thus, both proteins are members of the MIF tautomerase superfamily. Despite D-DT’s intriguing similarities to the very well studied MIF, there have been very few reports on the biological functions and mechanisms of action of D-DT. This thesis aimed at the functional characterization of D-DT in biological and pathophysiologic conditions. Prior to apoptotic studies, D-DT cDNA was cloned into a mammalian expression vector, cells were transfected with the newly generated plasmid and protein overexpression confirmed. Successive data demonstrated that D-DT did not obtain anti-apoptotic effects upon nitric oxide-induced apoptosis in macrophages. For the first time, this suggested contrary effects of D-DT in comparison to MIF. In a subsequent cell survival study of D-DT it was illustrated that D-DT enhanced macrophage viability to a more pronounced extent than MIF. Interestingly, it was further shown that simultaneous treatment of cells with MIF and D-DT increased macrophage viability even more, which suggested additive effects of both proteins. Additionally, D-DT was characterized as a protein that tautomerizes the substrate HPP and, thus, possessed analog enzymatic activity to MIF. However, quantitative differences in the tautomerization rate became evident between different mammalian homologues of D-DT. The covalent MIF inhibitor 4-IPP also inhibited the tautomerase activity of D-DT, whereas D-DT was not susceptible to ISO-1. This thesis also demonstrated first insight into D-DT’s in vivo role in a murine model of endotoxic shock. Neutralization of D-DT had a protective effect in the LPS-shock model. This finding accorded with results reported for MIF and, therefore, suggested analog functions of D-DT in this context. Taken together, this thesis reports on mostly similar, but also contrary biologic functions of MIF and D-DT. As this study is one of the first approaches to determine D-DT’s role in disease, continuative investigations need to be conducted to further validate these observations. Recent studies showed that the deletion of the MIF receptor, CD74, abrogates downstream MIF effects to a greater extend than MIF deficiency alone. My work also indicates the importance of further studies to determine D-DT’s potency as a MIF receptor ligand

D-Dopachrome tautomerase (D-DT) : functional homologue or cross-regulator of macrophage migration inhibitory factor (MIF)?

Macrophage migration inhibitory factor (MIF) is a pleiotropic immunoregulatory and pro-inflammatory cytokine, which plays a pivotal role in the pathogenesis of various acute and chronic inflammatory diseases. For example, endotoxemia studies in mice reveal that MIF administration decreases the survival rate, whereas MIF neutralization or genetic deletion of Mif improve the chances for a beneficial outcome. Additionally, high MIF levels are measured in a variety of human tumors and functional studies reveal MIF involvement in multiple aspects of tumor progression including control of cell proliferation and prevention of apoptosis. Unlike other cytokines, MIF also is a phenylpyruvate tautomerase that converts two non-physiologic enzymatic substrates, D-dopachrome and p-hydroxyphenylpyruvate (HPP). To date, possible relationships between the tautomerase and inflammatory or tumor-supportive activities of MIF remain unclear. In 1993, D-dopachrome tautomerase (D-DT) was discovered during an investigation of melanogenesis. The tertiary structure of D-DT is remarkably similar to MIF, identifying D-DT as the only known eukaryotic MIF homologue discovered to date. D-DT shares MIF’s tautomerase activity and, thus, both proteins are members of the MIF tautomerase superfamily. Despite D-DT’s intriguing similarities to the very well studied MIF, there have been very few reports on the biological functions and mechanisms of action of D-DT. This thesis aimed at the functional characterization of D-DT in biological and pathophysiologic conditions. Prior to apoptotic studies, D-DT cDNA was cloned into a mammalian expression vector, cells were transfected with the newly generated plasmid and protein overexpression confirmed. Successive data demonstrated that D-DT did not obtain anti-apoptotic effects upon nitric oxide-induced apoptosis in macrophages. For the first time, this suggested contrary effects of D-DT in comparison to MIF. In a subsequent cell survival study of D-DT it was illustrated that D-DT enhanced macrophage viability to a more pronounced extent than MIF. Interestingly, it was further shown that simultaneous treatment of cells with MIF and D-DT increased macrophage viability even more, which suggested additive effects of both proteins. Additionally, D-DT was characterized as a protein that tautomerizes the substrate HPP and, thus, possessed analog enzymatic activity to MIF. However, quantitative differences in the tautomerization rate became evident between different mammalian homologues of D-DT. The covalent MIF inhibitor 4-IPP also inhibited the tautomerase activity of D-DT, whereas D-DT was not susceptible to ISO-1. This thesis also demonstrated first insight into D-DT’s in vivo role in a murine model of endotoxic shock. Neutralization of D-DT had a protective effect in the LPS-shock model. This finding accorded with results reported for MIF and, therefore, suggested analog functions of D-DT in this context. Taken together, this thesis reports on mostly similar, but also contrary biologic functions of MIF and D-DT. As this study is one of the first approaches to determine D-DT’s role in disease, continuative investigations need to be conducted to further validate these observations. Recent studies showed that the deletion of the MIF receptor, CD74, abrogates downstream MIF effects to a greater extend than MIF deficiency alone. My work also indicates the importance of further studies to determine D-DT’s potency as a MIF receptor ligand

Cytokines in Sepsis: Potent Immunoregulators and Potential Therapeutic Targets-An Updated View

Sepsis and septic shock are among the leading causes of death in intensive care units worldwide. Numerous studies on their pathophysiology have revealed an imbalance in the inflammatory network leading to tissue damage, organ failure, and ultimately, death. Cytokines are important pleiotropic regulators of the immune response, which have a crucial role in the complex pathophysiology underlying sepsis. They have both pro- and anti-inflammatory functions and are capable of coordinating effective defense mechanisms against invading pathogens. On the other hand, cytokines may dysregulate the immune response and promote tissue-damaging inflammation. In this review, we address the current knowledge of the actions of pro- and anti-inflammatory cytokines in sepsis pathophysiology as well as how these cytokines and other important immunomodulating agents may be therapeutically targeted to improve the clinical outcome of sepsis

MIF but not MIF-2 recruits inflammatory macrophages in an experimental polymicrobial sepsis model

Excessive inflammation drives the progression from sepsis to septic shock. Macrophage migration inhibitory factor (MIF) is of interest because MIF promoter polymorphisms predict mortality in different infections, and anti-MIF antibody improves survival in experimental models when administered 8 hours after infectious insult. The recent description of a second MIF superfamily member, D-dopachrometautomerase (D-DT/MIF-2), prompted closer investigation of MIF-dependent responses. We subjected Mif(-/-) and Mif-2(-/-) mice to polymicrobial sepsis and observed a survival benefit with Mif but not Mif-2 deficiency. Survival was associated with reduced numbers of small peritoneal macrophages (SPMs) that, in contrast to large peritoneal macrophages (LPMs), were recruited into the peritoneal cavity. LPMs produced higher quantities of MIF than SPMs, but SPMs expressed higher levels of inflammatory cytokines and the MIF receptors CD74 and CXCR2. Adoptive transfer of WT SPMs into Mif(-/-) hosts reduced the protective effect of Mif deficiency in polymicrobial sepsis. Notably, MIF-2 lacks the pseudo-(E) LR motif present in MIF that mediates CXCR2 engagement and SPM migration, supporting a specific role for MIF in the recruitment and accumulation of inflammatory SPMs