Rodriguez, Moses

Background: Pediatric patients with multiple sclerosis (POMS) and related disorders, clinically isolated syndrome (CIS), myelin oligodendrocyte glycoprotein antibody disorder (MOGAD), and neuromyelitis optica spectrum disorder (NMOSD), are commonly treated with immunosuppressants. Understanding the impact of SARS-CoV-2 infection in patients may inform treatment decisions. Objective: Characterize SARS-CoV-2 infection prevalence and severity among a cohort of patients with POMS and related disorders, as well as the impact of disease-modifying therapies (DMTs). Methods: POMS and related disorders patients enrolled in a large, prospective registry were screened for COVID-19 during standard-of-care neurology visits. If confirmed positive of having infection, further analysis was undertaken. Results: Six hundred and sixty-nine patients were surveyed between March 2020 and August 2021. There were 73 confirmed COVID-19 infections. Eight of nine hospitalized patients (89%), and all patients admitted to the ICU were treated with B cell depleting therapy. The unadjusted odds ratio of hospitalization among those who tested positive of having had COVID-19 was 15.27 among those on B-cell-depleting therapy ( p = 0.016). Conclusions: B-cell-depleting treatment was associated with a higher risk of COVID-19, higher rates of hospitalization, and ICU admission, suggesting this therapy carries a higher risk of severe infection in POMS and related disorders.

Background: Rare genetic variants are emerging as important contributors to the heritability of multiple sclerosis (MS). Whether rare variants also contribute to pediatric-onset multiple sclerosis (POMS) is unknown. Objective: To test whether genes harboring rare variants associated with adult-onset MS risk ( PRF1, PRKRA, NLRP8 , and HDAC7 ) and 52 major histocompatibility complex (MHC) genes are associated with POMS. Methods: We analyzed DNA samples from 330 POMS cases and 306 controls from the US Network of Pediatric MS Centers and Kaiser Permanente Northern California for which Illumina ExomeChip genotypes were available. Using the gene-based method “SKAT-O,” we tested the association between candidate genes and POMS risk. Results: After correction for multiple comparisons, one adult-onset MS gene ( PRF1, p = 2.70 × 10 −3 ) and two MHC genes ( BRD2, p = 5.89 × 10 −5 and AGER, p = 7.96 × 10 −5 ) were significantly associated with POMS. Results suggest these are independent of HLA-DRB1*1501. Conclusion: Findings support a role for rare coding variants in POMS susceptibility. In particular, rare minor alleles within PRF1 were more common among individuals with POMS compared to controls while the opposite was true for rare variants within significant MHC genes, BRD2 and AGER . These genes would not have been identified by common variant studies, emphasizing the merits of investigating rare genetic variation in complex diseases.

Abstract The HLA class II region on chromosome 6p21 accounts for majority of familial clustering in MS and is by far the major susceptibility locus. However due to multiple class II genes, high polymorphism and linkage disequilibrium, it has been difficult to understand the role of individual class II genes and interaction between class II genes in the disease process. Previously, we have shown that PLP91-110 induced classical EAE in DR3.Abo but not in DQ6.Abo transgenic (tg) mice, suggesting that DR3 is a disease susceptible gene in the context of PLP. In this study, we have generated double (tg) mice expressing HLA-DR3/DQ6 to simulate heterozygous haplotype of MS patients. Introduction of DQ6 onto DR3 tg mice led to decrease in disease incidence on immunization with PLP91-110 suggesting a protective role of DQ6. We further characterized that this protective effect was due to expression of high level of IFNγ by DQ6 restricted T cells, which suppressed proliferation of encephalitogenic DR3-restricted T cells by inducing apoptosis in antigen specific T cells. Our IFNγ blocking experiments confirmed an anti-inflammatory role of IFNγ. Our study suggests that DQ6 modifies the PLP91-110 specific T cell response in DR3 through IFNγ mediated activation induced cell death. Thus our double tg mouse model provides a novel tool to study linkage disequilibrium and explain how HLA-DQ molecule might modulate disease in MS-susceptible individual.

Abstract Our laboratory generated a transgenic mouse that ubiquitously expresses 3D, a picorno viral RNA-dependent RNA polymerase. 3D transgenic mice display an extreme antiviral phenotype when challenged with viruses from distinct families. To probe this protective molecular mechanism, we assayed differential gene expression in tissues of uninfected 3D transgenic mice. Microarray analysis revealed increased levels of 78 genes >4-fold, including a distinct upregulation of critical innate antiviral effectors across tissues. RT-PCR analysis of liver, lung, kidney, CNS and heart tissue from uninfected 3D mice showed a significant upregulation of antiviral genes (up to 300-fold). Following infection with EMCV, 3D transgenic mice exhibited significantly decreased viral tissue titers and survived lethal viral infection. This effect is specific to the 3D transgene since other mice expressing the picorno viral transgenes VP1 or VP2 did not display an antiviral phenotype. Studies of 3D-Ifnar1-/- or 3D-Ifngr1-/- mice, supported the conclusion that type I IFN signaling is necessary for the antiviral state in 3D mice, whereas IFNg signaling is dispensable. Maintenance of a viral-resistant state in uninfected 3D mice is independent of adaptive immunity since uninfected 3D-Rag1-/- mice are able to highly upregulate antiviral factors. Collectively, our data indicate that intrinsic upregulation of critical innate immune effectors confers broad-spectrum viral resistance in 3D mice.

Abstract Among all the genetic factors linked with multiple sclerosis (MS), MHC class-II molecules show the strongest association. Generation of HLA class-II transgenic mice has helped to elucidate the role of HLA class-II gene in MS. We have shown that the human HLA-DR3 (DRβ1*0301) gene predisposes to proteolipid protein (PLP)-induced EAE, whereas HLA-DQβ1*0601 (DQ6) was resistant. We also showed that the DQ6 molecule protects from EAE in DR3DQ6 double transgenic mice by producing anti-inflammatory interferon gamma (IFNγ). To further confirm the role of IFNγ in protection, we generated DR3DQ8 mice lacking IFNγ (DR3DQ8.IFNγ-/-). Immunization with PLP91-110 peptide caused atypical EAE in DR3DQ8.IFNγ-/- mice characterized by ataxia and spasticity, hallmarks of brain-specific disease. Severe brain specific inflammation and demyelination in DR3DQ8.IFNγ-/- mice with minimal spinal cord pathology further confirmed brain-specific pathology. Severe disease in DR3DQ8.IFNγ-/- mice was due to increased encephalitogenicity of CD4 T cells and its ability to produce higher levels of IL-17 and GM-CSF compared to DR3DQ8 mice. Further, areas with demyelination showed increased presence of CD68+ inflammatory cells suggesting an important role for monocytes/microglia in causing brain pathology. Thus, our study supports a protective role for IFNγ in inflammatory and demyelinating disease through down regulation of IL-17 and induction of neuro-protective factors in the brain by monocytes/microglial cells.

Abstract Multiple sclerosis is a chronic inflammatory and demyelinating disease of the CNS. Although, MS is well characterized in terms of the role played by immune cells, cytokines and CNS pathology, nothing is known about the metabolic alterations that occur during the disease process in circulation. Here, we profiled the plasma metabolites of B6 with EAE, a known model of chronic MS using untargeted global metabolomics. Using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, a total of 326 metabolites were identified, with significant changes observed in 105 metabolites (13 up- and 92 down-regulated), that mapped to lipid and amino acid pathways, followed by the peptide, xenobiotic, carbohydrate, nucleotide and energy pathways (p<0.05). To understand the functional role of these alterations, the KEGG metabolic library was analyzed using Metaboanalyst. The top nine most significant pathways in terms of GlobeTest p-value and impact are 1) fatty acid biosynthesis, 2) lysine degradation, 3) histidine metabolism, 4) beta-alanine metabolism, 5) pentose phosphate pathway, 6) arachidonic acid metabolism, 7) linoleic acid, 8) D-arginine and ornithine. Overall, these metabolic changes could be exploited as biomarkers for EAE/MS disease and to design new treatment paradigms where metabolic interventions could be combined with present and experimental therapeutics to achieve better treatment.

Abstract In search of metabolites signature as biomarkers during EAE disease, we profiled urine from B6 EAE using global untargeted metabolomics. Evaluation of metabolomic profiling of urine from EAE and healthy B6 group by using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, we found that 132 out of 322 (41%) metabolites were differentially altered (P<0.05) indicating robust alteration in the urine metabolomics profile during disease. Among the perturbed metabolites, 11 were up regulated in EAE urine whereas 121 were down regulated. We conducted pathway analysis of the biochemical pathways of the KEGG and considered both concerted changes in metabolite intensity within the pathway (GlobalTest) and alterations of high impact, and found that a number of pathways were significantly altered including glyoxylate and dicarboxylate, phenylanine metabolism, porphyrin & chlorophyll metabolism, primary bile acid biosynthesis, cysteine & methione metabolism, taurine and hypotaurine metabolism, glycine, serine & threonine metabolism, and beta-alanine metabolism. Alteration in these pathways during EAE disease suggesting that perturbation of certain central metabolites could have impact on multiple metabolic pathways. While some of these metabolite changes could easily be developed as biomarkers, the key to translating metabolomics into therapeutics would require figuring out the central altered metabolic pathway(s), once studied in detail.

Abstract AMPK is an energy-sensing metabolic switch implicating in various metabolic disorders. Here we report its new role in controlling the immunopathology of experimental autoimmune encephalitis by polarizing pro-inflammatory phenotype of Macrophage. AMPK alpha1 knockout (alpha1KO) developed severe EAE due to higher expression of IL17 with reduced IFNy and IL4. AMPK alpha1KO also exhibited increased number of infiltrated mononuclear cells in CNS due to impaired blood brain barrier integrity. Macrophage from alpha1KO mice exhibited M1 type phenotype (IL10lowIL12/23high) with stronger capacity to induce allogenic and antigen-specific T-cell response and also heightened the encephalitogenic property of MOG35-55-primed CD4 T cells in B6 mice. Collectively, our data shows AMPK controls the clinical outcome of disease by regulating M1 phenotype-Th17 axis in animal model of MS.