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. 2025 Jan 8:15:1494842.
doi: 10.3389/fimmu.2024.1494842. eCollection 2024.

Blood immunophenotyping of multiple sclerosis patients at diagnosis identifies a classical monocyte subset associated to disease evolution

Stéphane Rodriguez  1   2 , Laura Couloume  1 , Juliette Ferrant  1   2 , Nicolas Vince  3 , Marion Mandon  1   2 , Rachel Jean  1   2 , Celine Monvoisin  1 , Simon Leonard  1 , Simon Le Gallou  1   2 , Nayane S B Silva  3   4 , Sonia Bourguiba-Hachemi  3 , David Laplaud  3   5 , Alexandra Garcia  3 , Romain Casey  6   7   8   9 , Helene Zephir  10 , Anne Kerbrat  11 , Gilles Edan  11 , Emmanuelle Lepage  11 , Eric Thouvenot  12   13 , Aurelie Ruet  14   15 , Guillaume Mathey  16   17 , Pierre-Antoine Gourraud  3   5 , Karin Tarte  1   2 , Celine Delaloy  1 , Patricia Amé  1   2 , Mikael Roussel  1   2 , Laure Michel  1   2   8
Affiliations

Blood immunophenotyping of multiple sclerosis patients at diagnosis identifies a classical monocyte subset associated to disease evolution

Stéphane Rodriguez et al. Front Immunol. .

Abstract

Introduction: Myeloid cells trafficking from the periphery to the central nervous system are key players in multiple sclerosis (MS) through antigen presentation, cytokine secretion and repair processes.

Methods: Combination of mass cytometry on blood cells from 60 MS patients at diagnosis and 29 healthy controls, along with single cell RNA sequencing on paired blood and cerebrospinal fluid (CSF) samples from 5 MS patients were used for myeloid cells detailing.

Results: Myeloid compartment study demonstrated an enrichment of a peculiar classical monocyte population in 22% of MS patients at the time of diagnosis. Notably, this patients' subgroup exhibited a more aggressive disease phenotype two years post-diagnosis. This monocytic population, detected in both the CSF and blood, was characterized by CD206, CD209, CCR5 and CCR2 expression, and was found to be more frequent in MS patients carrying the HLA-DRB1*15:01 allele. Furthermore, pathways analysis predicted that these cells had antigen presentation capabilities coupled with pro-inflammatory phenotype.

Discussion: Altogether, these results point toward the amplification of a specific and pathogenic myeloid cell subset in MS patients with genetic susceptibilities.

Keywords: antigen presentation; cerebrospinal fluid; classical monocyte; disability; multiple sclerosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
(A) UMAPs illustrating the myeloid clusters retrieved following FlowSOM unsupervised analysis in HC (left) and MS donors (right). (B) Heatmap summarizing markers expression scaled by row among clusters defined through unsupervised analysis. (C) Dotplots illustrating myeloid subsets frequencies among myeloid cells. Mann-Whitney test was used to determine statistical differences with ns: not significant, **p < 0.01.
Figure 2
Figure 2
Enriched myeloid cells display characteristics of activated and tissue resident classical monocyte. (A) Heatmap figuring cluster differential abundance between HC and MS donors, with each bar within a cluster representing a donor and color is depending on differential enrichment. Differential abundance significance was tested through generalized linear mixed model and p-values for each cluster are indicated on the right. (B) Correlation matrix depicting correlation between myeloid subsets frequencies among HC donors (left) and MS patients (right).
Figure 3
Figure 3
MS patients w CD206hi CD209hi Mo have a peculiar profile. (A) Histogram plot illustrating radio-clinic activity in patients displaying low (MS wo CD206hi CD209hi Mo) or high (MS w CD206hi CD209hi Mo) CD206hi CD209hi Mo cells frequency. Significance is based on Mann and Whitney test with **p < 0.01. (B) percentage of patients with EDSS ≥2 at 2 years (left). Significance is based on Mann and Whitney test with *p <0.05 and **p < 0.01. Histogram plot depicting ARMSS score ≥5 frequency among MS wo CD206hi CD209hi Mo and MS w CD206hi CD209hi Mo, 2 years following diagnosis (right). Significance is based on Fisher’s exact test with p = 0.0178. (C) Boxplot indicating MSGB patients’ score. Mann and Whitney test demonstrated no significant differences between groups (ns) (left). Histogram plot illustrating the frequency of patients HLA-DRB1*15:01 and HLA-DQB1*06:02 genes (right). Fisher’s exact test demonstrated highly significant difference with ***p < 0.001.
Figure 4
Figure 4
CD206hi CD209hi Mo-like cells can be found in RRMS CSF at diagnosis. (A) UMAPs representing immune cells retrieved and analyzed from scRNA-seq cohort in blood and CSF of MS patients following integration and unidentified cells removal. Clusters are labeled with cell identities according to genes expression. (B) UMAPS illustrating myeloid cell compartment in both CSF and blood with cluster labeling according to genes signatures. (C) Circle diagram displaying cell subset proportions among myeloid cells in CSF and blood with bars as median. (D) UMAPs depicting classical monocytes major clusters (upper panels). UMAPs illustrating MRC1/DC-SIGN co-expressing events in CSF and blood (middle panels). UMAPs figuring classical monocytes events expression of MRC1/CD206 (red) and DC-SIGN/CD209 (blue) transcripts in CSF and blood (lower panels). (E) Circle diagram displaying CD206hi CD209hi Mo-like cell proximity to the related myeloid subset gene signatures through GSEA. Circle color depicts normalized enrichment score and circle size: CD206hi CD209hi Mo-like cells/associated subset signatures overlap. (F) (left panels) UMAPs figuring CD206hi CD209hi Mo signature scoring (CCR5, MRC1, DC-SIGN) among classical monocyte events from our dataset (up), Esaulova dataset (middle), Ramesh dataset (lower). (Right panels) UMAP associated histogram plots depicting CD206hi CD209hi Mo signature positive cells frequency among classical monocytes in CSF and PB. Significance is based on Mann and Whitney test with **p < 0.01, ***p < 0.001, ****p < 0.001.
Figure 5
Figure 5
CD206hi CD209hi classical monocyte-like cells phenotypic characterization at the gene level. (A) Heatmap depicting differentially expressed genes characterizing classical monocytes major clusters defined in Figure 4D upper panels. (B) UMAPs illustrating cells enriched in the genes signature belonging to the antigen processing and presentation pathway from the Kegg database. Enriched cells are figured as red dots in CSF (left) and blood (right). (C) Circle diagram illustrating CSF cMo3 cluster comparison to its peripheral blood counterpart through GSEA analysis. Enriched pathways are depicted, with signature overlap with cMo3 CSF versus blood differentially upregulated genes illustrated through circle size and p-value as color gradient. Pathways related to immune cells activation are labeled in red. (D) Heatmap displaying differentially expressed genes between CSF cMo3 cluster and its peripheral blood counterpart. (E) Heatmap displaying top regulated genes among CD206hi CD209hi Mo-like cells with CD206hi CD209hi Mo-like cells defined by MRC1/CD209 expression in comparison to previously defined classical monocyte clusters cMo1, cMo2, and cMo3. (F) UMAPs illustrating CCR2 expression among classical monocytic cells.
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