"Near Cure" treatment of severe acute EAE in MIF-1-deficient female and male mice with a bifunctional MHCII-derived molecular construct.

Our previous studies demonstrated increased serum levels of macrophage migration inhibitory factor (MIF-1) and its homologue, MIF-2, in males during MS progression; and that genetically high-MIF-expressing male subjects with relapsing multiple sclerosis (MS) had a significantly greater risk of conversion to progressive MS than lower-MIF-expressing males and females. However, female MS subjects with severe disease expressed higher levels of CD74, the common MIF-1/MIF-2 receptor, on blood cells. In the murine model of MS, experimental autoimmune encephalomyelitis (EAE), both male and female mice lacking MIF-1 and/or MIF-2 were clinically improved during development of moderately severe disease, thus implicating both homologs as co-pathogenic contributors. The current study using MIF-deficient mice with severe acute EAE revealed a highly significant reduction of EAE scores in MIF-1-deficient females, in contrast to only minor and delayed reduction of clinical signs in MIF-1-deficient males. However, clinical EAE scores and factor expression were strongly suppressed in males and further reduced in females after treatment of WT and MIF-1-, MIF-2- and MIF-1/2-DUAL-deficient female and male mice with a MHCII DRα1-MOG-35-55 molecular construct that competitively inhibits MIF-1 & MIF-2 signaling through CD74 as well as T cell activation. These results suggest sex-dependent differences in which the absence of the MIF-1 and/or MIF-2 genotypes may permit stronger compensatory CD74-dependent EAE-inducing responses in males than in females. However, EAE severity in both sexes could still be reduced nearly to background (a "near cure") with DRα1-MOG-35-55 blockade of compensatory MIF and CD74-dependent factors known to attract peripheral inflammatory cells into the spinal cord tissue.

Brief report: Enhanced DRα1-mMOG-35-55 treatment of severe EAE in MIF-1-deficient male mice.

Macrophage migration inhibitory factor (MIF-1) and its homologue d-dopachrome tautomerase (MIF-2) share the common CD74 receptor and function innately to enhance severity of multiple sclerosis (MS) as well as the experimental autoimmune encephalomyelitis (EAE) model for MS. We previously demonstrated that genetically high-MIF-expressing male subjects with relapsing MS had a significantly greater risk of conversion to progressive MS (PMS) than lower-MIF-expressing males. To expand on this observation, we utilized MIF-1, MIF-2, and MIF-1/2-DUAL-deficient male mice to discern if there would be a greater contribution of these inflammatory factors in EAE mice with severe vs. moderate clinical disease signs. As shown previously, mice deficient in either MIF-1 or MIF-2 each had a ∼25% reduction of moderate EAE compared to WT mice, with significant differences in disease onset and trajectory. However, EAE induction in mice deficient in both MIF-1 and MIF-2 genes did not result in a further reduction in EAE severity. This result suggests that the two MIF homologues were likely affecting the same pathogenic pathways such that each could partially compensate for the other but not in an additive or synergistic manner. However, MIF-1-KO, MIF-2-KO, and MIF-1/2-DUAL-KO mice with severe EAE did not exhibit a significant reduction in cumulative EAE scores compared with WT mice, but the MIF-1-KO and, to a lesser extent, MIF-1/2-DUAL-KO mice did show a significant reduction in daily EAE scores over the last 3 days of observation, and MIF-2-KO mice showed a more modest but still consistent reduction over the same span. Furthermore, deletion of MIF-1 resulted in a massive reduction in the expression of EAE- and Complete Freund's Adjuvant-associated inflammatory factors, suggesting delayed involvement of the MIF/CD74 axis in promoting disease expression. To further explore modulation of MIF-1 and MIF-2 effects on EAE, we treated WT mice with moderate EAE using DRα1-mMOG-35-55, an inhibitor of CD74 that blocks both MIF-1 and MIF-2 action. This treatment reduced ongoing moderate EAE severity in excess of 25%, suggesting efficient blockade of the MIF/CD74 axis in disease-enhancing pathways. Moreover, DRα1-mMOG-35-55 treatment of mice with severe EAE strongly reversed EAE- and CFA-associated expression of inflammatory cytokines and chemokines including Tnf, Ccr7, Ccr6, Ccl8, Cxcr3, and Ccl19 in MIF-deficient mouse genotypes, and also exceeded innate MIF-1 and MIF-2 EAE enhancing effects, especially in MIF-1-KO mice. These results illustrate the therapeutic potential of targeting the disease-enhancing MIF/CD74 pathway in male mice with moderate and severe EAE, with implications for treatment of high-MIF-expressing RRMS human males at risk of conversion to progressive MS as well as those that have already transitioned to PMS.

Sex differences in EAE reveal common and distinct cellular and molecular components.

Experimental autoimmune encephalomyelitis (EAE) is commonly used as an animal model for evaluating clinical, histological and immunological processes potentially relevant to the human disease multiple sclerosis (MS), for which the mode of disease induction remains largely unknown. An important caveat for interpreting EAE processes in mice is the inflammatory effect of immunization with myelin peptides emulsified in Complete Freund's Adjuvant (CFA), often followed by additional injections of pertussis toxin (Ptx) in some strains to induce EAE. The current study evaluated clinical, histological, cellular (spleen), and chemokine-driven processes in spinal cords of male vs. female C57BL/6 mice that were immunized with mouse (m)MOG-35-55/CFA/Ptx to induce EAE; immunized with saline/CFA/Ptx only (CFA, no EAE); or were untreated (Naïve, no EAE). Analysis of response curves utilized a rigorous and sophisticated methodology to parse and characterize the effects of EAE and adjuvant alone vs. the Naive baseline responses. The results demonstrated stronger pro-inflammatory responses of immune cells and their associated cytokines, chemokines, and receptors in male vs. female CFA and EAE mice that appeared to be offset partially by increased percentages of male anti-inflammatory, regulatory and checkpoint T cell, B cell, and monocyte/macrophage subsets. These sex differences in peripheral immune responses may explain the reduced cellular infiltration and differing chemokine profiles in the Central Nervous System (CNS) of male vs. female CFA immunized mice and the reduced CNS infiltration and demyelination observed in male vs. female EAE groups of mice that ultimately resulted in the same clinical EAE disease severity in both sexes. Our findings suggest EAE disease severity is governed not only by the degree of CNS infiltration and demyelination, but also by the balance of pro-inflammatory vs. regulatory cell types and their secreted cytokines and chemokines.

Estrogen-induced compensatory mechanisms protect IL-10-deficient mice from developing EAE.

BACKGROUND: IL-10 knockout (KO) mice are protected from experimental autoimmune encephalomyelitis (EAE) with low-dose estrogen (E2) treatment similar to wild-type (WT) mice. Previous studies have demonstrated a decrease in tumor necrosis factor in all E2-treated groups, which led to the protection of the mice. METHODS: This study used IL-10 KO mice and WT mice treated either with E2 or sham pellets 7 days prior to induction of EAE. Mice were observed for 21 days post-immunization. The spleen, inguinal lymph nodes, and brain were evaluated by flow cytometry. Spinal cords were evaluated using a cytokine/chemokine array, RT-PCR, and histology. RESULTS: This study demonstrates that E2 treatment induced three heightened regulatory mechanisms that potentially protect IL-10 KO mice from EAE: (1) an increase in programmed death-ligands 1 and 2 on monocytes and macrophages in the periphery and within the CNS; (2) an increase in CD73 in the inflamed CNS, which can increase the production of the anti-inflammatory molecule adenosine; and (3) a decrease in CD4+CD25+FoxP3+ regulatory T cells in the spleen. Together, these factors comprise an alternative compensatory mechanism that significantly downregulates key pro-inflammatory cytokine, chemokine, and chemokine receptor genes which are enhanced in the spinal cord of IL-10 KO mice. This group of E2-treated mice remained asymptomatic after EAE challenge similar to E2-treated WT mice, despite their having more T and B lymphocytes in the brain, and modestly increased demyelination in the spinal cord. CONCLUSION: These results indicate that previously unrecognized compensatory mechanisms of EAE protection are stimulated by E2 in the absence of IL-10, which can provide disease protection comparable to the IL-10-dependent effects induced by E2 in WT mice.

Increased CD74 binding and EAE treatment efficacy of a modified DRα1 molecular construct.

Multiple sclerosis (MS) is a demyelinating and degenerative disease of the central nervous system (CNS) with a strong inflammatory component that affects more than 2 million people worldwide (and at least 400,000 in the United States). In MS, macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT) enhance the inflammatory event as a result of their interaction with their cognate receptor CD74. Therefore, the search for new agents aimed at blocking this interaction is critical for therapeutic purposes and will be of paramount importance for the treatment of MS. DRα1-MOG-35-55 constructs have been demonstrated to be effective in the treatment of experimental autoimmune encephalomyelitis (EAE) a mouse model for MS. This effect is directly correlated with the binding to its cell surface receptor, CD74, apparently preventing or blocking the binding of two inflammatory factors, MIF and D-DT. Here we report that a single amino acid substitution (L50Q) in the DRα1 domain of the human and mouse DRα1-MOG-35-55 constructs (notated as DRhQ and DRmQ, respectively) possessed increased affinity for CD74, a greater capacity to block MIF binding, the ability to inhibit pERK1/2 signaling and increased therapeutic activity in mice with EAE. These data suggest that binding affinity for CD74 could serve as an in vitro indicator of biological potency of DRhQ and thus support its possible clinical utility as an effective therapy for MS and perhaps other diseases in which there is an inflammatory reaction driven by MIF and D-DT.

Antibiotics protect against EAE by increasing regulatory and anti-inflammatory cells.

A seven day pretreatment course of an oral antibiotic cocktail (Ampicillin, Metronidazole, Neomycin Sulfate, and Vancomycin) was shown to induce changes in peripheral immune regulation and protect mice from signs of experimental autoimmune encephalomyelitis (EAE). To determine if a shorter course of antibiotic pretreatment could also protect the mice from EAE and induce regulatory immune cells, studies were conducted using the same oral antibiotic cocktail for three days. In addition, the CNS was examined to determine the effects of antibiotic pretreatment on EAE disease course and immune modulation within the affected tissue. The shorter three day pretreatment course was also significantly protective against severe EAE in C57BL/6 mice. Moreover, our study found increased frequencies of regulatory cells and a decrease in the frequency of anti-inflammatory macrophages in the spleen of EAE protected mice. Additionally, a chemokine and chemokine receptor array run on mRNA from spinal cords revealed that genes associated with regulatory T cells and macrophage recruitment were strongly upregulated in the antibiotic pretreated mice. Additional RT-PCR data showed genes associated with anti-inflammatory microglia/macrophages were upregulated and pro-inflammatory genes were downregulated. This suggests the macrophages recruited to the spinal cord by chemokines are subsequently polarized toward an anti-inflammatory phenotype. These results lend strong support to the conclusion that a three day course of antibiotic treatment given prior to the induction of severe EAE profoundly protected the mice by inducing regulatory lymphocytes in the periphery and an anti-inflammatory milieu in the affected spinal cord tissue.

Case Report of Multisystem Inflammatory Syndrome in Adults (MIS-A): A 31-Year-Old Man with Fever, Rash, and Cardiac Symptoms 6 Weeks Following SARS-CoV-2 Infection, Successfully Resuscitated Following Cardiac Arrest.

BACKGROUND Multisystem inflammatory syndrome in adults (MIS-A) is an uncommon condition after a confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, manifesting as multiorgan failure despite apparent resolution of initial symptoms. While this syndrome shares similar characteristics with a syndrome found in children, fewer cases are reported in adults. This report details a 31-year-old man fulfilling the diagnostic criteria of MIS-A, who was successfully resuscitated following cardiac arrest. CASE REPORT A 31-year-old man was admitted to the intensive care unit for 3 days of progressively worsening fever, chills, diaphoresis, exanthematous rash, headache, and neck stiffness. The patient had a history of mild, resolved SARS-CoV-2 infection 6 weeks prior to his presentation, diagnosed by rapid antigen and reverse transcription polymerase chain reaction (RT-PCR) testing. Meningitis and autoimmune pathologies were initially suspected but were ruled out. Given the patient's symptoms, prior SARS-CoV-2 infection, and positive inflammatory markers, findings correlated with the Centers for Disease Control and Prevention's diagnostic criteria for multisystem inflammatory syndrome in adults. On hospital day 1, the patient decompensated into severe respiratory distress requiring intubation. Shortly after, the patient developed cardiac arrest and was successfully resuscitated. He was transferred from our rural hospital to an intensive care unit at a facility with additional resources. He remained critically ill for several weeks while receiving high-dose steroids, intravenous immunoglobulin (IVIG), and hemodialysis until his recovery. CONCLUSIONS Early diagnosis and treatment of MIS-A would significantly improve outcomes in this subset of patients, especially in clinical settings with limited resources.

Pathogenesis of Human Immunodeficiency Virus-Mycobacterium tuberculosis Co-Infection.

Given that infection with Mycobacterium tuberculosis (Mtb) is the leading cause of death amongst individuals living with HIV, understanding the complex mechanisms by which Mtb exacerbates HIV infection may lead to improved treatment options or adjuvant therapies. While it is well-understood how HIV compromises the immune system and leaves the host vulnerable to opportunistic infections such as Mtb, less is known about the interplay of disease once active Mtb is established. This review explores how glutathione (GSH) depletion, T cell exhaustion, granuloma formation, and TNF-α upregulation, as a result of Mtb infection, leads to an increase in HIV disease severity. This review also examines the difficulties of treating coinfected patients and suggests further research on the clinical use of GSH supplementation.