Efficacy and safety of mycophenolate mofetil for steroid reduction in neuromyelitis optica spectrum disorder: a prospective cohort study

Neuromyelitis optica spectrum disorder (NMOSD) is a rare autoimmune inflammatory disease that can affect multiple generations and cause complications with long-term prednisolone treatment. This study aimed to evaluate the efficacy and safety of mycophenolate mofetil (MMF) in preventing NMOSD relapse while reducing prednisolone dosage. The trial involved nine patients with NMOSD who received MMF along with prednisolone dose reduction. MMF was effective in achieving prednisolone dose reduction without relapse in 77.8% of patients, with a significant decrease in mean annualized relapse rate. All adverse events were mild. The findings suggest that MMF could be a viable treatment option for middle-aged and older patients who require steroid reduction. Clinical trial registration number: jRCT, jRCTs051180080. Registered February 27th, 2019-retrospectively registered, https://jrct.niph.go.jp/en-latest-detail/jRCTs051180080.</p

DataSheet_1_Circulating plasmablasts and follicular helper T-cell subsets are associated with antibody-positive autoimmune epilepsy.docx

Autoimmune epilepsy (AE) is an inflammatory disease of the central nervous system with symptoms that have seizures that are refractory to antiepileptic drugs. Since the diagnosis of AE tends to rely on a limited number of anti-neuronal antibody tests, a more comprehensive analysis of the immune background could achieve better diagnostic accuracy. This study aimed to compare the characteristics of anti-neuronal antibody-positive autoimmune epilepsy (AE/Ab(+)) and antibody-negative suspected autoimmune epilepsy (AE/Ab(-)) groups. A total of 23 patients who met the diagnostic criteria for autoimmune encephalitis with seizures and 11 healthy controls (HC) were enrolled. All patients were comprehensively analyzed for anti-neuronal antibodies; 13 patients were identified in the AE/Ab(+) group and 10 in the AE/Ab(-) group. Differences in clinical characteristics, including laboratory and imaging findings, were evaluated between the groups. In addition, the immunophenotype of peripheral blood mononuclear cells (PBMCs) and CSF mononuclear cells, particularly B cells and circulating Tfh (cTfh) subsets, and multiplex assays of serum and CSF were analyzed using flow cytometry. Patients with AE/Ab(+) did not show any differences in clinical parameters compared to patients with AE/Ab(-). However, the frequency of plasmablasts within PBMCs and CSF in patients with AE/Ab(+) was higher than that in patients with AE/Ab(-) and HC, and the frequency of cTfh17 cells and inducible T-cell co-stimulator (ICOS) expressing cTfh17 cells within cTfh subsets was higher than that in patients with AE/Ab(-). Furthermore, the frequency of ICOShighcTfh17 cells was positively correlated with that of the unswitched memory B cells. We also found that IL-12, IL-23, IL-6, IL-17A, and IFN-γ levels were elevated in the serum and IL-17A and IL-6 levels were elevated in the CSF of patients with AE/Ab(+). Our findings indicate that patients with AE/Ab(+) showed increased differentiation of B cells and cTfh subsets associated with antibody production. The elevated frequency of plasmablasts and ICOS expressing cTfh17 shift in PBMCs may be indicative of the presence of antibodies in patients with AE.</p

Plasmablasts as Migratory IgG-Producing Cells in the Pathogenesis of Neuromyelitis Optica

<div><p>Neuromyelitis optica (NMO) is an inflammatory disease characterized by recurrent attacks of optic neuritis and myelitis. It is generally accepted that autoantibodies against aquaporin 4 water channel protein play a pathogenic role in neuromyelitis optica. We have recently reported that plasmablasts are increased in the peripheral blood of this autoimmune disease, and are capable of producing autoantibodies against aquaporin 4. Here, we demonstrate that CD138⁺HLA-DR⁺ plasmablasts, a subset of IgG-producing cells, are increased in the peripheral blood and are enriched among the cerebrospinal fluid (CSF) lymphocytes during the relapse of neuromyelitis optica. Notably, these CD138⁺HLA-DR⁺ plasmablasts overexpress CXCR3, whose ligands are present in the cerebrospinal fluid during the relapse of neuromyelitis optica. These results led us to speculate that plasmablasts producing anti-aquaporin 4 autoantibodies might traffic toward the central nervous system (CNS). Furthermore, we performed single-cell sorting of plasmablasts from peripheral blood and CSF samples from NMO and sequenced the complementarity-determining regions (CDRs) of the IgG heavy chain expressed by the sorted plasmablast clones. There were high frequencies of mutations in the CDRs compared with framework regions, indicating that these plasmablast clones would represent a post-germinal center B-cell lineage. Consistent with the preceding results, the plasmablast clones from the peripheral blood shared the same CDR sequences with the clones from the CSF. These results indicate that IgG-producing plasmablasts, which are guided by helper T-cells, may migrate from the peripheral blood preferentially to the CSF. Since migratory plasmablasts could be involved in the inflammatory pathology of NMO, the B-cell subset and their migration might be an attractive therapeutic target.</p> </div

UniFrac Principal Coordinate (PCoA) and UniFrac distance analyses for HC40 and MS20 subjects.

<p>(a, c) Open and closed circles indicate individual subjects from HC40 and MS20, respectively. (a) The two components of the unweighted PCoA plot explained 6.96% and 4.30% of the variance. ANOSIM statistic, <i>R</i> = 0.239, <i>P</i> ≤ 0.0009. (b) Mean unweighted UniFrac distances for HC-HC, HC-MS, and MS-MS subjects. (c) The two components of the weighted PCoA plot explained 18.44% and 9.86% of the variance. ANOSIM statistic, <i>R</i> = 0.208, <i>P</i> ≤ 0.002. (d) Mean weighted UniFrac distances for HC-HC, HC-MS, and MS-MS subjects. (b, d) Error bars represent standard deviations of the UniFrac distances between samples. *<i>P</i> ≤ 0.05.</p

Dysbiosis in the Gut Microbiota of Patients with Multiple Sclerosis, with a Striking Depletion of Species Belonging to <i>Clostridia</i> XIVa and IV Clusters

<div><p>The pathogenesis of multiple sclerosis (MS), an autoimmune disease affecting the brain and spinal cord, remains poorly understood. Patients with MS typically present with recurrent episodes of neurological dysfunctions such as blindness, paresis, and sensory disturbances. Studies on experimental autoimmune encephalomyelitis (EAE) animal models have led to a number of testable hypotheses including a hypothetical role of altered gut microbiota in the development of MS. To investigate whether gut microbiota in patients with MS is altered, we compared the gut microbiota of 20 Japanese patients with relapsing-remitting (RR) MS (MS20) with that of 40 healthy Japanese subjects (HC40) and an additional 18 healthy subjects (HC18). All the HC18 subjects repeatedly provided fecal samples over the course of months (158 samples in total). Analysis of the bacterial 16S ribosomal RNA (rRNA) gene by using a high-throughput culture-independent pyrosequencing method provided evidence of a moderate dysbiosis in the structure of gut microbiota in patients with MS. Furthermore, we found 21 species that showed significant differences in relative abundance between the MS20 and HC40 samples. On comparing MS samples to the 158 longitudinal HC18 samples, the differences were found to be reproducibly significant for most of the species. These taxa comprised primarily of clostridial species belonging to <i>Clostridia</i> clusters XIVa and IV and <i>Bacteroidetes</i>. The phylogenetic tree analysis revealed that none of the clostridial species that were significantly reduced in the gut microbiota of patients with MS overlapped with other spore-forming clostridial species capable of inducing colonic regulatory T cells (Treg), which prevent autoimmunity and allergies; this suggests that many of the clostridial species associated with MS might be distinct from those broadly associated with autoimmune conditions. Correcting the dysbiosis and altered gut microbiota might deserve consideration as a potential strategy for the prevention and treatment of MS.</p></div

Bacterial composition at the phylum level in gut microbiota samples obtained from HC40 and MS20 subjects.

<p>For phylum-level assignment of 16S reads (16S rRNA gene V1-V2 region) mapped to the known FL-16S sequences and unmapped OTUs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137429#sec010" target="_blank">Results</a>), 70% sequence identity threshold was applied. The vertical axis represents the relative abundance of each phylum in the microbiota of HC40 (open bar) and MS20 (grey bar) subjects. Each box plot represents median, interquartile range, minimum, and maximum values.</p

Fold-change in the abundance of 21 species using longitudinal HC18 samples.

<p>The vertical axis indicates the log value of fold-change in the abundance of 22 species for the comparison between MS20 and HC18 (nine longitudinal samples per individual) samples. Open and closed circles indicate log values of fold-change > 0 (increased in MS) and < 0 (decreased in MS), respectively. The average fold-change in the abundance of each species for the comparison between MS20 and HC40 samples was connected by a line.</p

OTU/species diversity and richness in gut microbiota of HC40 and MS20 subjects.

<p>(a) Number of operational taxonomic units (OTUs) generated by clustering of 3,000 16S reads of gut microbiota samples from 40 healthy control subjects (HC40) and 20 patients with multiple sclerosis (MS20). (b) Estimated OTU numbers obtained from Chao1 extrapolation of the observed OTU numbers shown in (a). (c) Shannon index calculated from the observed OTU numbers. The vertical axes indicate the numbers of OTUs (a, b) and the Shannon index (c). Each box plot represents median, interquartile range, minimum, and maximum values.</p

Bacterial composition at the genus level in gut microbiota samples obtained from HC40 and MS20 subjects.

<p>For genus-level assignment of 16S reads (16S rRNA gene V1-V2 region) mapped to the known FL-16S sequences and unmapped OTUs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137429#sec010" target="_blank">Results</a>), 94% sequence identity threshold was applied. The vertical axis represents the relative abundance (%) of each genus in the microbiota of HC40 (open bar) and MS20 (grey bar) subjects. Error bars represent standard error of the mean. Asterisks indicate statistical significance determined by Welch’s <i>t</i> test (*P < 0.05).</p

Phylogenetic tree of 14 clostridial species exhibiting significant differences among groups and several known species.

<p>The neighbor-joining method was used to construct the phylogenetic tree. Numbers at each node indicate the bootstrap support (1,000 replicates). Those written in red letters are 14 clostridial species having a significant difference in relative abundance between HC40 and MS20 samples. Treg-inducing strains are indicated by “St” and are written in blue letters [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137429#pone.0137429.ref013" target="_blank">13</a>].</p