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

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 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

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

Kinetics of CD138⁺HLA-DR⁺ PB during relapse of NMO.

<div><p>(A) Analysis of pooled peripheral blood mononuclear cells (PBMC) data from neuromyelitis optica (NMO) in remission and relapse. The plasmablasts (PBs) were subdivided into four subpopulations by considering the expression of CD138 and HLA-DR. The individual data show the percentages of each PB subpopulation among the total PB [*p < 0.05 by Mann–Whitney test; each error bar represents the median ± interquartile range (IQR)]. </p> <p>(B) Enrichment of CD138⁺HLA-DR⁺ PBs in the CSF. The PBMC and CSF cells were obtained from NMO during relapse. The values indicate the percentages of CD138⁺HLA-DR⁺ PBs among the total PB. The expression level of CD138 was assessed by mean fluorescence intensity (MFI). Representative data of one out of three different cases are shown.</p></div

CXCR3 expression on plasmablasts (PBs) correlates with the disease state.

<div><p>(A) CXCR3 and CXCR4 on PB in neuromyelitis optica (NMO) relapse and remission. Here, we compared the mean fluorescence intensity (MFI) of CXCR3 and CXCR4 expression in the peripheral blood PBs during remission and relapse of NMO. MFI of CXCR3 and CXCR4 expressions on CD138⁺HLA-DR⁺ PBs were also analyzed [**p < 0.01 and *p < 0.05 by Mann–Whitney test; each error bar represents the median ± interquartile range (IQR]].</p> <p>(B) B-cell subpopulations derived from peripheral blood mononuclear cells (PBMC) during disease relapse and remission were analyzed by flow cytometry to investigate the expression of CXCR3 and CXCR4. The values represent the percentages of CXCR3⁺ or CXCR4⁺ cells within each B-cell subpopulation. Unstained control of PBMC is indicated by Ctrl. Representative data of at least five patients in each disease state are shown.</p></div