Background and ObjectivesSpinal cord injury (SCI) is associated with severe immunologic changes, such as SCI-induced immune deficiency syndrome, which heightens susceptibility to infections. However, the immune components underlying this immune reorganization remain poorly defined. This study aimed to characterize immune remodeling in patients with SCI across different time points postinjury.MethodsHigh-dimensional flow cytometric profiling was performed on peripheral blood samples from patients with SCI in a cross-sectional observational study to assess immune changes at different postinjury time points. Patients in the subacute phase (22-67 days of postinjury [dpi]) and chronic phase (>= 365 dpi) were compared with healthy, sex-matched, and age-matched controls.ResultsAlterations in the T-cell and natural killer (NK) cell compartments were observed, particularly in the subacute phase postinjury. Memory T cells and NK cells showed elevated expression of the NAD+ metabolizing enzyme CD38 and immune checkpoint molecules, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1), indicating immune activation and possible exhaustion. Coexpression of CD38 and CTLA-4 on T cells was rare, suggesting distinct activation and inhibitory states. In chronic patients, we observed decreased frequencies of NK cells with no substantial changes in T cells and B cells. Notably, changes in CD38, CTLA-4, and PD-1 were no longer found in patients in the chronic phase.DiscussionThese findings reveal noteworthy changes in immune cell activation and exhaustion markers that may contribute to immune vulnerability after SCI, offering novel insights into potential therapeutic targets, such as NAD+ metabolism and immune checkpoint modulation.

STUDY DESIGN:Narrative review and data-based perspective OBJECTIVES: Compare the current literature on poly(ADP-ribose) (PAR) polymerase (PARP) inhibition and NAD+ supplementation in spinal cord injury (SCI) and determine the effectiveness of both treatment paradigms in improving pathophysiological outcomes induced by SCI in mice. METHODS:Using a spinal cord contusion mouse model, a severe SCI was induced at the L1 spinal level in female C57Bl/6 J mice. 25 mg/kg PARP inhibitor (Veliparib), 750 mg/kg NR, or vehicle was administered intraperitoneally starting at 1 h post-injury (n = 13-18 mice per group), followed by daily treatments up to 8 days post-injury, and every other day thereafter until sacrifice (28 days post-injury). Functional recovery (by Basso Mouse Scale, BMS) and tissue-level effects were evaluated. RESULTS:Functional recovery, lesion size (demyelinated (MBP), astrocyte (GFAP), and inflammatory (IBA1) area), and DNA damage load (γH2AX and PAR) did not improve with either Veliparib or NR treatment compared to vehicle-treated animals. Moreover, NR treatment decreased survival and increased astrogliosis in SCI mice compared to the vehicle control group. CONCLUSIONS:Within the experimental paradigm, neither PARP inhibition nor NAD+ supplementation improved major pathophysiological outcomes associated with an SCI. The results are reviewed in the context of previous reports using PARP inhibition or NAD+ supplementation strategies in SCI. At present, caution is recommended when considering these clinical modalities in treating SCI, and priorities to identify the best therapeutic paradigms are presented.

Intestinal immune activation and neuro-immune interactions have been proposed to contribute to the pathophysiology of functional dyspepsia (FD), but advanced characterization of the immune landscape is largely lacking. This study aimed to extensively characterize the duodenal and peripheral immune landscape in FD; to evaluate the potential of the duodenal microenvironment to activate spinal sensory neurons; and to assess immunomodulatory effects of high-dose proton pump inhibitor (PPI, 40 mg pantoprazole 2×/day) intervention in FD. Duodenal tryptase release (P = 0.0041) and CD45RA- CD8+ T cells (P = 0.0079) were elevated in 30 prospectively recruited patients with Rome IV FD compared to 30 healthy controls (HC), and correlated with gastrointestinal (GI) symptoms. In vitro response rate of murine dorsal root ganglion neurons to duodenal biopsy supernatants of FD and HC was similar (P = 0.43), despite associations with GI symptoms but not duodenal mediator release. High-dose PPI improved GI symptoms (P = 0.0002) but increased duodenal mast cells (P = 0.044), systemic monocytes (P = 0.011) and α4β7+ gut-homing T cells (P = 0.0073) in FD. These limited immunological differences in patients with FD not taking PPI suggest an overestimation of the extent of immune activation in previous studies. Neuro-immune interactions can underly visceral pain sensation in FD, although presumably not mediated by mast cell tryptase. ClinicalTrials.gov registration: NCT04713969, 13/01/2021.

Autoimmune diseases like multiple sclerosis (MS) and type 1 diabetes (T1D) lack therapies that induce durable, antigen-specific immune tolerance. We investigated whether mRNA lipid nanoparticles (LNPs) encoding disease-relevant autoantigens could re-establish immune homeostasis in preclinical models. While mRNA-LNP microbial vaccines evoke strong effector immune responses, we show that both systemic and intramuscular delivery of MOG27-63 mRNA-loaded LNPs attenuated disease severity in experimental autoimmune encephalomyelitis (EAE). Antigen-specific protection was similarly observed in a T1D adoptive transfer model. Therapeutic efficacy achieved using immunostimulatory LNPs challenges the current assumption that tolerogenic mRNA vaccines require immune-silent LNPs. Furthermore, divergent outcomes between autoantigens and irrelevant antigens suggest that antigen identity determines whether mRNA-LNPs promote immune tolerance or activation. Mechanistically, optimized LNPs efficiently targeted antigen-presenting cells (APCs) in the liver and spleen. This promoted a homeostatic APC phenotype and a hyporesponsive CD4+ T cell phenotype without inducing regulatory T cells (Tregs). Therefore, autoantigen mRNA was co-delivered with "immunoregulatory" mRNAs encoding cytokines (IL-2 mutein) or chemokines (CCL1) known to enhance Treg expansion and recruitment. This co-delivery further improved clinical outcomes in EAE. Together, these findings demonstrate that systemic and intramuscular treatment with mRNA-LNPs encoding autoantigens alongside immunoregulatory molecules represents a promising strategy for antigen-specific immunotherapy in autoimmune diseases.

Background Genetic and preclinical data highlight CD6 as a promising target for multiple sclerosis (MS), yet the impact of clinically available CD6-targeting treatments on MS immunopathogenesis remains insufficiently defined. Itolizumab, a humanized anti-CD6 antibody with established safety and clinical efficacy in other autoimmune disorders, represents a potential candidate to interrogate this pathway in MS. Methods CD6 expression was quantified by flow cytometry on circulating lymphocytes from MS patients and healthy donors. Functional consequences of CD6 blockade using itolizumab were evaluated using human in vitro models of brain barriers, including T cell diapedesis, barrier integrity, and inflammatory responses. T cell co-cultures with an oligodendrocyte cell line were performed to reveal the impact on survival and differentiation. Results Circulating lymphocytes from MS patients displayed increased CD6 levels, associated with heightened activation and proliferation features in CD4+ memory T cells. In brain barrier assays, migrated T cells exhibited higher CD6 expression than non-migrated cells. CD6 blockade with itolizumab selectively reduced memory and cytotoxic T cell diapedesis across hCMEC/D3 monolayers, while maintaining naïve and regulatory T cell migration, by disrupting interaction with ALCAM but not CD318, attenuating cytokine-driven upregulation of endothelial adhesion molecules, and strengthening BBB integrity. Itolizumab limited the retention and acquisition of a disease-promoting CD69+TRM phenotype in migrated CD4+ T cells, while attenuating their activity in the CNS, thereby promoting oligodendrocyte survival and differentiation. Conclusion These findings provide mechanistic support for targeting CD6 in MS with itolizumab as a promising therapeutic strategy to reduce neuroinflammation and pathogenic T cell accumulation in the CNS.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a critical regulator of extracellular matrix remodelling and an important mediator of remyelination in demyelinating disorders such as multiple sclerosis. In addition, TIMP-1 has emerged as a promising therapeutic target in cancer due to its interaction with CD63, which promotes tumorigenic signalling and carcinogenesis. Although several structures of TIMP-1 bound to matrix metalloproteinases have been reported, no unbound structure with all druggable sites available has previously been reported. Here, we present the first unbound crystal structure of human TIMP-1, resolved at 1.95 Å resolution. Comparison with the MMP-bound complex reveals localized conformational changes and altered intramolecular hydrogen bonding in the unbound structure, indicating increased structural plasticity in the absence of the protease. Crystals were obtained in multiple conditions, but only two diffracted to high resolution. Although optimization and seeding did not significantly improve the morphology, the additive screen enhanced both the morphology and reproducibility and provided intrinsic cryoprotection. The resulting crystal form proved compatible with soaking-based screening campaigns, providing a robust structural basis for the discovery of TIMP-1 ligands with clinical potential.

Background and ObjectivesChronic immune activation is a hallmark of latent viral infections and autoimmune disorders, profoundly shaping immune cell phenotypes, including CD4+ cytotoxic T lymphocytes (CD4 CTL). The mechanisms underlying CD4 CTL development remain elusive, although antigenic triggers and the local microenvironment are thought to influence their phenotype. In this study, it was investigated if CD4 CTL induced under different circumstances exhibit phenotypic differences.MethodsUsing single cell multiomics, we analyzed CD4 CTL from healthy cytomegalovirus (CMV)-seropositive donors, patients with CMV-seronegative relapsing-remitting multiple sclerosis (RR-MS) (autoimmune trigger), and patients with CMV-seropositive RR-MS (combination of viral and autoimmune trigger).ResultsOur findings reveal that the heterogeneous pool of CD4 CTL encompasses distinct subsets with divergent expression of proinflammatory, cytotoxic, and migratory markers. Moreover, we identified a pathogenic CD4 CTL subset coexpressing the MS-associated transcription factor eomesodermin (EOMES) and the migratory receptor class I-restricted T-cell-associated molecule, which accumulates in MS lesions and demonstrates resistance to natalizumab treatment.DiscussionCMV was implicated as a dominant driver of development of highly cytotoxic CD4+ T cells, as these cells were markedly enriched in CMV-seropositive individuals. This comprehensive phenotypic atlas of CD4 CTL advances our understanding of their development and highlights potential targets for diagnosing, treating, and preventing MS progression.

BACKGROUND AND OBJECTIVES:Spinal cord injury (SCI) is associated with severe immunologic changes, such as SCI-induced immune deficiency syndrome, which heightens susceptibility to infections. However, the immune components underlying this immune reorganization remain poorly defined. This study aimed to characterize immune remodeling in patients with SCI across different time points postinjury. METHODS:High-dimensional flow cytometric profiling was performed on peripheral blood samples from patients with SCI in a cross-sectional observational study to assess immune changes at different postinjury time points. Patients in the subacute phase (22-67 days of postinjury [dpi]) and chronic phase (≥365 dpi) were compared with healthy, sex-matched, and age-matched controls. RESULTS:Alterations in the T-cell and natural killer (NK) cell compartments were observed, particularly in the subacute phase postinjury. Memory T cells and NK cells showed elevated expression of the NAD+ metabolizing enzyme CD38 and immune checkpoint molecules, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1), indicating immune activation and possible exhaustion. Coexpression of CD38 and CTLA-4 on T cells was rare, suggesting distinct activation and inhibitory states. In chronic patients, we observed decreased frequencies of NK cells with no substantial changes in T cells and B cells. Notably, changes in CD38, CTLA-4, and PD-1 were no longer found in patients in the chronic phase. DISCUSSION:These findings reveal noteworthy changes in immune cell activation and exhaustion markers that may contribute to immune vulnerability after SCI, offering novel insights into potential therapeutic targets, such as NAD+ metabolism and immune checkpoint modulation.

Progressive neurodegeneration in the central nervous system (CNS) in multiple sclerosis (MS) is driven by chronic inflammatory demyelination. Neutrophils are increasingly recognized as versatile innate immune cells with potentially underappreciated roles in CNS inflammation, but their contribution to MS pathology remains poorly understood. Interestingly, we observed foamy neutrophils in active CNS lesions of MS patients. Therefore, we investigated the ability of human neutrophils to internalize myelin debris and assessed how this impacts their functional phenotype. Neutrophils exhibited efficient myelin uptake, peaking between 3 and 6 hours, predominantly through complement opsonization and internalization via complement receptor 3. Prolonged exposure to high concentrations of myelin induced a pro-inflammatory phenotype, marked by increased production of reactive oxygen species, neutrophil extracellular traps, and inflammatory mediators such as CXCL8 and CCL3. Gene expression analysis revealed a dose-dependent inflammatory signature after myelin uptake, characterized by gradual upregulation of CXCL8 and decreased ARG1 expression, suggesting a shift toward a pro-inflammatory neutrophil state. These findings provide novel insights into the role of neutrophils in myelin clearance and inflammation in the CNS, highlighting complement receptor 3-mediated uptake and downstream pro-inflammatory activation as key mechanisms.

Background/Objectives: The ongoing evolution of SARS-CoV-2 has highlighted the limitations of parenteral vaccines in preventing viral transmission, largely due to their failure to elicit robust mucosal immunity. Methods: Here, we evaluated an intranasal (IN) vaccine formulation consisting of recombinant receptor-binding domain (RBD) adsorbed onto human probiotic Bacillus subtilis DG101 spores. Results: In BALB/c mice, IN spore-RBD immunization induced strong systemic and mucosal humoral responses, including elevated specific IgG, IgM, and IgA levels in serum, bronchoalveolar lavage fluid (BALF), nasal-associated lymphoid tissue (NALT), and saliva. It further promoted mucosal B cell and T cell memory, along with a Th1/Tc1-skewed T cell response, characterized by increased IFN-γ-expressing CD4+ and CD8+ T cells in the lungs. Conclusions: All in all, these findings highlight the potential of intranasal vaccines adjuvanted with probiotic B. subtilis spores in inducing sterilizing immunity and limiting SARS-CoV-2 transmission.

Modulating the excitatory amino acid transporter 3 (EAAT3) can be considered a novel approach for the treatment of multiple sclerosis (MS). EAAT3 plays a crucial role in regulating oxidative stress and oligodendrocyte function through its ability to transport cysteine, the rate-limiting building block in the synthesis of the antioxidant glutathione. Therefore, EAAT3 activation is hypothesised to improve oligodendrocyte health and relieve its differentiation block in MS, improving remyelination capacity. Using a cuprizone-induced demyelination model, the effects of EAAT3 overexpression by viral transduction of oligodendrocytes and pharmacological inhibition of EAAT3 were examined. Surprisingly, EAAT3 overexpression significantly hampered remyelination, while EAAT3 inhibition prevented demyelination and improved functional remyelination as assessed by visual evoked potentials and post mortem myelin basic protein fluorescent staining. Next, cellular mechanisms underlying these results were investigated. Consistent with the in vivo findings, post mortem gene expression analysis of the corpus callosum of cuprizone treated animals revealed a trend towards upregulation of oligodendrocyte lineage genes in response to EAAT3 inhibition, supporting its role in oligodendrocyte health and myelination processes. In vitro studies using the human oligodendroglioma (HOG) cell line demonstrated the beneficial effects of EAAT3 inhibition on cellular morphology, indicating potential roles in promoting oligodendrocyte maturation and myelination. In contrast, EAAT3 overexpression appears to hamper these processes. These findings suggest that, contrary to our initial hypothesis, EAAT3 inhibition could improve oligodendrocyte function and myelination processes, highlighting its potential as a therapeutic target for demyelinating disorders. Future studies should address the exact molecular mechanism through which this effect is obtained.

>Oncostatin M and multiple sclerosis:Every 5minutes,someone in the world is diagnosed with multiple sclerosis(MS),a chronic inflammatory and degenerative disease of the central nervous system(CNS).MS appears in unpredictable episodes of symptoms,which are highly patient-dependent,but often include visual impairment,muscle weakness/spasms,fatigue,cognitive difficulties,and bladder.bowel.or sexual dysfunction.

Spinal cord injury (SCI) is a life-changing event that severely impacts the patient's quality of life. Modulating neuroinflammation, which exacerbates the primary injury, and stimulating neuro-regenerative repair mechanisms are key strategies to improve functional recovery. Cyclic adenosine monophosphate (cAMP) is a second messenger crucially involved in both processes. Following SCI, intracellular levels of cAMP are known to decrease over time. Therefore, preventing cAMP degradation represents a promising strategy to suppress inflammation while stimulating regeneration. Intracellular cAMP levels are controlled by its hydrolyzing enzymes phosphodiesterases (PDEs). The PDE4 family is most abundantly expressed in the central nervous system (CNS) and its inhibition has been shown to be therapeutically relevant for managing SCI pathology. Unfortunately, the use of full PDE4 inhibitors at therapeutic doses is associated with severe emetic side effects, hampering their translation toward clinical applications. Therefore, in this study, we evaluated the effect of inhibiting specific PDE4 subtypes (PDE4B and PDE4D) on inflammatory and regenerative processes following SCI, as inhibitors selective for these subtypes have been demonstrated to be well-tolerated. We reveal that administration of the PDE4D inhibitor Gebr32a, even when starting 2 dpi, but not the PDE4B inhibitor A33, improved functional as well as histopathological outcomes after SCI, comparable to results obtained with the full PDE4 inhibitor roflumilast. Furthermore, using a luminescent human iPSC-derived neurospheroid model, we show that PDE4D inhibition stabilizes neural viability by preventing apoptosis and stimulating neuronal differentiation. These findings strongly suggest that specific PDE4D inhibition offers a novel therapeutic approach for SCI.

Sphingosine-1-phosphate receptor (S1PR) modulators are clinically used to treat relapse-remitting multiple sclerosis (MS) and the early phase of progressive MS when inflammation still prevails. In the periphery, S1PR modulators prevent lymphocyte egress from lymph nodes, hence hampering neuroinflammation. Recent findings suggest a role for S1PR modulation in remyelination. As the Gi alpha-coupled S1P1 subtype is the most prominently expressed S1PR in oligodendrocyte precursor cells (OPCs), selective modulation (functional antagonism) of S1P1 may have direct effects on OPC functionality. We hypothesized that functional antagonism of S1P1 by ponesimod induces remyelination by boosting OPC differentiation. In the cuprizone mouse model of demyelination, we found ponesimod to decrease the latency time of visual evoked potentials compared to vehicle conditions, which is indicative of functional remyelination. In addition, the Y maze spontaneous alternations test revealed that ponesimod reversed cuprizone-induced working memory deficits. Myelin basic protein (MBP) immunohistochemistry and transmission electron microscopy of the corpus callosum revealed an increase in myelination upon ponesimod treatment. Moreover, treatment with ponesimod alone or in combination with A971432, an S1P5 monoselective modulator, significantly increased primary mouse OPC differentiation based on O4 immunocytochemistry. In conclusion, S1P1 functional antagonism by ponesimod increases remyelination in the cuprizone model of demyelination and significantly increases OPC differentiation in vitro. Ponesimod reverses a cuprizone-induced working memory deficit, restores the cuprizone-induced delay in latency time of the optic pathway, and enhances remyelination after cuprizone intoxication in vivo. Furthermore, ponesimod enhances differentiation of oligodendrocyte precursor cells into mature oligodendrocytes in vitro.image

BACKGROUND:Magnetic resonance imaging (MRI) is the gold standard for imaging disease activity in multiple sclerosis (MS) patients. However, recent studies indicate that positron emission tomography (PET) may provide added value in visualizing MS disease in the future. OBJECTIVE:This study aims to investigate the barriers to implementing PET for MS patients and its potential added value in the context of MS. METHODS:11 semi-structured in-depth interviews with neurologists specialized in MS were conducted. The neurologists were selectively recruited from six medical centers in Belgium and the Netherlands. Inductive thematic analysis was used to analyze the data. RESULTS:The interviews revealed several hurdles that play a role in using PET for MS, including financial and scientific considerations. Potential clinical applications of PET were also identified, such as understanding unexplained symptoms, making a more accurate prognosis, evaluating the nature and seriousness of a lesion, and assessing disease activity. In addition, research applications were highlighted, including unraveling the pathophysiology of MS and developing new treatment options for MS. CONCLUSION:Using PET is advancing our understanding of MS and can accelerate the development of novel therapies to combat its progression. However, its integration into routine clinical practice for MS remains a future prospect, contingent upon further technological advancements and supportive healthcare frameworks.

Bruton's tyrosine kinase (BTK) is a cytoplasmic, non-receptor signal transducer, initially identified as an essential signaling molecule for B cells, with genetic mutations resulting in a disorder characterized by disturbed B cell and antibody development. Subsequent research revealed the critical role of BTK in the functionality of monocytes, macrophages and neutrophils. Various immune cells, among which B cells and neutrophils, rely on BTK activity for diverse signaling pathways downstream of multiple receptors, which makes this kinase an ideal target to treat hematological malignancies and autoimmune diseases. First-generation BTK inhibitors are already on the market to treat hematological disorders. It has been demonstrated that B cells and myeloid cells play a significant role in the pathogenesis of different autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren's syndrome. Consequently, second-generation BTK inhibitors are currently being developed to treat these disorders. Despite the acknowledged involvement of BTK in various cell types, the focus on B cells often overshadows its impact on innate immune cells. Among these cell types, neutrophils are often underestimated in the pathogenesis of autoimmune diseases. In this narrative review, the function of BTK in different immune cell subsets is discussed, after which an overview is provided of different upcoming BTK inhibitors tested for treatment of autoimmune disease. Special attention is paid to BTK inhibition and its effect on neutrophil biology.

In autoimmunity, FOXP3+ Tregs skew toward a proinflammatory, nonsuppressive phenotype and are, therefore, unable to control the exaggerated autoimmune response. This largely affects the success of autologous Treg therapy, which is currently under investigation for autoimmune diseases, including multiple sclerosis (MS). There is a need to ensure in vivo Treg stability before successful application of Treg therapy. Using genetic fate-mapping mice, we demonstrate that inflammatory, cytokine-expressing exFOXP3 T cells accumulate in the CNS during experimental autoimmune encephalomyelitis. In a human in vitro model, we discovered that interaction with inflamed blood-brain barrier endothelial cells (BBB-ECs) induces loss of function by Tregs. Transcriptome and cytokine analysis revealed that in vitro migrated Tregs have disrupted regenerative potential and a proinflammatory Th1/17 signature, and they upregulate the mTORC1 signaling pathway. In vitro treatment of migrated human Tregs with the clinically approved mTORC1 inhibitor rapamycin restored suppression. Finally, flow cytometric analysis indicated an enrichment of inflammatory, less-suppressive CD49d+ Tregs in the cerebrospinal fluid of people with MS. In summary, interaction with BBB-ECs is sufficient to affect Treg function, and transmigration triggers an additive proinflammatory phenotype switch. These insights help improve the efficacy of autologous Treg therapy of MS.