CHIT1 at diagnosis predicts faster disability progression and reflects early microglial activation in multiple sclerosis

Beliën, Jarne; Swinnen, Stijn; D’hondt, Robbe; de Juan, Laia Verdú; Dedoncker, Nina; Matthys, Patrick; Bauer, Jan; Vens, Celine; Moylett, Sinéad; Dubois, Bénédicte

doi:10.1038/s41467-024-49312-y

CHIT1 at diagnosis predicts faster disability progression and reflects early microglial activation in multiple sclerosis

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Published: 12 June 2024

Volume 15, article number 5013, (2024)
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CHIT1 at diagnosis predicts faster disability progression and reflects early microglial activation in multiple sclerosis

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Abstract

Multiple sclerosis (MS) is characterized by heterogeneity in disease course and prediction of long-term outcome remains a major challenge. Here, we investigate five myeloid markers – CHIT1, CHI3L1, sTREM2, GPNMB and CCL18 – in the cerebrospinal fluid (CSF) at diagnostic lumbar puncture in a longitudinal cohort of 192 MS patients. Through mixed-effects and machine learning models, we show that CHIT1 is a robust predictor for faster disability progression. Integrative analysis of 11 CSF and 26 central nervous system (CNS) parenchyma single-cell/nucleus RNA sequencing samples reveals CHIT1 to be predominantly expressed by microglia located in active MS lesions and enriched for lipid metabolism pathways. Furthermore, we find CHIT1 expression to accompany the transition from a homeostatic towards a more activated, MS-associated cell state in microglia. Neuropathological evaluation in post-mortem tissue from 12 MS patients confirms CHIT1 production by lipid-laden phagocytes in actively demyelinating lesions, already in early disease stages. Altogether, we provide a rationale for CHIT1 as an early biomarker for faster disability progression in MS.

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Introduction

Multiple sclerosis (MS) is a chronic autoimmune disorder of the central nervous system (CNS) characterized by considerable interindividual heterogeneity in terms of disease onset and severity¹. Despite continuous scientific advancement, accurately predicting long-term outcomes and effectively stratifying treatment regimens based on uncertain prognosis remain major challenges in MS care. Clinicians thus face an unmet need for disease activity measures with proven prognostic value. In particular, very few blood and cerebrospinal fluid (CSF) biomarkers have yet found translation into clinical practice².

Over the past few years, the role of innate immune cells such as CNS-resident microglia and monocyte-derived macrophages in MS pathogenesis has gained increased attention^3,4,5. Microglia are anticipated to provide novel strategies in the development of biomarkers for MS disease activity⁶. Previous work from our group showed that among several myeloid markers, CSF concentrations of chitotriosidase or chitinase 1 (CHIT1) at diagnostic lumbar puncture best reflected disability status at a median of five years later⁷. Only few other studies have evaluated CHIT1 concentrations in relation to MS prognosis^8,9. However, these studies – as our previous work – were limited to single-time-point disability assessments. In addition, CHIT1 has been found to be upregulated in chronic active MS lesions at the bulk RNA level, whereas its expression was largely undetectable in chronic inactive lesions and in brain tissue from non-neurological controls^7,10. Nevertheless, since variations in biomarker concentrations must ultimately be coupled to disease processes to be clinically relevant, unraveling cell-type specificity of CHIT1 and the functional state of CHIT1-expressing cells in MS remains a prerequisite¹¹. This is emphasized by the functional diversity and cellular heterogeneity of microglia and CNS-associated macrophages uncovered in recent work^12,13,14.

In this work, we investigate five microglia/macrophage-related proteins – CHIT1, chitinase-3-like protein 1 (CHI3L1, also known as YKL-40), soluble triggering receptor expressed on myeloid cells 2 (sTREM2), glycoprotein non-metastatic melanoma protein B (GPNMB) and C-C motif chemokine ligand 18 (CCL18) in the CSF at diagnostic lumbar puncture in a large cohort of 192 MS patients. Our longitudinal study design allows for multi-time-point disability assessments. Using mixed-effects models and machine learning algorithms, we identify CHIT1 at diagnosis as a strong predictor for faster disability progression. We consequently perform single-cell RNA sequencing (scRNA-seq) on the CSF of 11 MS patients and integrate this data with single-cell profiles from four published datasets of MS CNS tissue to elucidate the phenotype and localization of CHIT1-expressing cells. Lastly, we validate our results at the protein level using immunohistochemistry on post-mortem MS brain tissue in early as well as late disease stages.

Results

Study population for biomarker analysis

We investigated the association between five microglia/macrophage biomarkers in the CSF at diagnostic lumbar puncture and clinical disease activity in our longitudinal MS cohort. These five biomarkers were selected based on their role in myeloid activation and their potential diagnostic or prognostic value, as indicated in prior research on MS and other neurological disorders^10,15,16,17. In this study, we included retrospective data of 196 MS patients. In contrast to our previous work in which we correlated CHIT1 concentrations in a subset of these patients (n = 143) with single-time-point disability assessments⁷, we now examined additional biomarkers and included multi-time-point longitudinal disability outcomes in the extended MS cohort. None of these patients were on disease-modifying therapy at the time of CSF sample collection. After quality control of the CSF protein measurements, samples from 192 MS patients remained for analysis (Table 1, Supplementary Table 1, Supplementary Data 1). Of these 192 MS patients, we had disability assessments for 178 patients and information on relapses for 181 patients. Our study cohort was representative of the general MS patient population; 60.9% of patients were female, 80.7% of patients presented with relapsing-remitting MS (RRMS) and the median age at onset was 33.9 years. The median disease duration at diagnostic lumbar puncture was 0.9 years.

Table 1 Characteristics of study population for biomarker analysis

Full size table

CHIT1 at diagnosis correlates with disability years later: single-time-point analysis

We observed some significant correlations between the five selected CSF biomarkers (CHIT1, CHI3L1, sTREM2, GPNMB and CCL18) (Supplementary Table 2), which is not unexpected since these are all implicated in microglia/macrophage biology. However, as not every correlation was significant, nor very strong, these biomarkers might reflect different aspects of microglia/macrophage biology and thus capture slightly distinct aspects of the disease processes in MS. Furthermore, CHIT1, CHI3L1 and GPNMB were associated with two of the CSF hallmarks currently used in MS clinical practice: immunoglobulin G (IgG) index and oligoclonal bands (OCBs; Supplementary Table 3). Notably, only CHIT1 correlated significantly with CSF neurofilament light chain (NfL; r = 0.31, P < 0.0001; Fig. 1A), an established marker of neuronal damage in MS¹⁸.

Next, we examined how these microglia/macrophage CSF biomarkers measured at diagnosis correlated with single-time-point clinical disease activity parameters at follow-up years later. Disease parameters were assessed at a median of 5.4 years for disability and a median of 2 years for relapse activity before treatment. After correction for multiple testing, only CSF CHIT1 at diagnostic lumbar puncture showed a significant association with disability as measured by Age-Related Multiple Sclerosis Severity (ARMSS), Multiple Sclerosis Severity Score (MSSS) and Expanded Disability Status Scale (EDSS) 5.4 years later (Bonferroni-corrected P ≤ 0.0025; Supplementary Table 4). Correlation of CHIT1 with disability was shown to be independent of CHI3L1 and NfL in our previous work⁷. Given the significant correlations found here between CHIT1, GPNMB and CCL18 (Supplementary Table 2), multiple linear regression analyses including these biomarkers were conducted for the disease activity outcomes. After Bonferroni correction for multiple testing, CHIT1 correlated with future disability independently of GPNMB and CCL18 (Supplementary Table 5). CSF CHIT1 concentrations explained 9.6% – and with known clinical covariates of disability (age at onset, sex and disease course) included 30.3% – of variance in ARMSS scores across MS patients. Analogous to our previous work, CHIT1 concentrations were significantly different between MS patients with high and low disability accumulation (ARMSS ≥ 5 and ARMSS < 5; logistic regression analysis, P = 0.00026; Fig. 1B) and the area under the curve (AUC) reached 70.1% (95% confidence interval [CI] = 59.4–80.7%) and 79.3% (95% CI = 69.8–88.7%) without and with known clinical covariates, respectively (Fig. 1C).

CHIT1 at diagnosis correlates with disability progression: multi-time-point analysis

Given the above-mentioned associations between CSF CHIT1 concentrations at diagnostic lumbar puncture and single-time-point disability parameters, we expanded our investigations to multi-time-point EDSS assessments up to 15 years (median of 5.4 years) after MS diagnosis.

Likelihood ratio tests indicated that a mixed-effects model which accounted for EDSS variance across MS patients and across time – along with an interaction effect between CHIT1 and time – provided the best fit (χ² = 9.94, P = 0.0016; model 3, Supplementary Table 6). Over 50% of MS patients (n = 104) had at least 2 EDSS assessments (Supplementary Table 7). In this model, MS patients with higher CSF CHIT1 concentrations at diagnosis experienced more disability progression over time (β = 0.27, P = 2.63 ×10⁻⁵; Fig. 2). As expected, there was a positive relation between EDSS and time (β = 0.20, P = 5.56 ×10⁻⁸) as well as between EDSS and age at diagnosis (β = 0.42, P = 1.72 ×10⁻¹¹). There was no significant effect for sex, which was consistent across all models tested. Under random effects, we observed a positive correlation between by-patient random EDSS intercepts and by-time random EDSS slopes (r = 0.30). That is, MS patients with a higher EDSS at diagnosis tended to continue to have higher disability scores over time. After correction for fixed effects, we noticed that the interindividual EDSS differences accounted for a much greater proportion of the EDSS variance (77.8%) than the intraindividual disability progression over time (4.8%), as would be expected.

**Fig. 2: CHIT1 at diagnosis correlates with disability progression: multi-time-point analysis.**

After clinical prognostic factors, CHIT1 at diagnosis shows highest predictive value for disability progression

Next, we applied the machine learning approach we developed in D’hondt et al.¹⁹ to model EDSS trajectories within our MS cohort. In short, we used a regressor chain model to analyze the predictive power of several MS patient characteristics for disability progression, in particular the CSF biomarkers under study. To this end, we calculated permutation importance scores²⁰, which identify how much the random shuffle of a characteristic or variable affects the model’s performance. As expected, we found disease course, baseline EDSS, age at diagnosis and disease duration at diagnosis to be important variables for the prediction of disability progression (Fig. 3A). However, after these, CSF CHIT1 concentrations at diagnosis emerged as the most important predictor. Our machine learning model thus demonstrated the prognostic superiority of CHIT1 over the other CSF biomarkers. This was even more apparent when higher-order CSF biomarker interactions were considered (Fig. 3B).

CHIT1 is expressed by a distinct microglia subset in active MS lesions

In order to identify the cellular source of CHIT1 in the CSF of MS patients, we performed scRNA-seq on the CSF of 11 RRMS patients for whom we also had CSF CHIT1 measurements at diagnostic lumbar puncture. As our previous work showed CHIT1 to be expressed in the brain of MS patients using bulk RNA analysis, we expanded our investigation by including scRNA-seq and single-nucleus (sn)RNA-seq datasets of CNS tissue from four published studies (Fig. 4A). Based on our earlier findings, we only included samples taken from lesions or periplaque white matter of MS patients⁷. With the exception of one patient, all patients in the published datasets were diagnosed as progressive MS (either primary, secondary or undefined). For that one patient, the MS subtype was not further specified (Fig. 4A, Supplementary Data 2). All five datasets were subjected to stringent quality control. Ambient RNA contamination, doublets, genes present in less than five cells, cells with less than 200 genes detected and cells with a mitochondrial RNA content higher than 15% (for scRNA-seq data) or 5% (for snRNA-seq data) were all removed. In total, we retained 179,389 high-quality cells: 22,506 from CSF and 156,883 from CNS tissue (Supplementary Data 2). After confirming that CHIT1 expression was indeed predominant in myeloid cells (Supplementary Fig. 1), we isolated a total of 29,676 myeloid cells: 1858 from CSF and 27,818 from CNS tissue. High-quality myeloid cells from all datasets were subsequently integrated, effectively correcting for batch effects and dataset-specific artefacts (Supplementary Fig. 2) prior to downstream analyses (Fig. 4A).

The myeloid cells were subclustered and annotation was performed based on canonical marker genes from the literature (Supplementary Data 3). We identified nine distinct microglia (MG1-9) clusters, two CNS-associated macrophage (CAM1-2) clusters and one monocyte (MON) cluster. One cluster portrayed expression of markers associated with both myeloid dendritic cells (mDC) as well as microglia and was termed ‘mixed myeloid cells’ (MMC; Fig. 4B). To confirm our annotation, we used unbiased module scores of gene sets for microglia, CNS-associated macrophages and monocytes from three recent reference works^12,21,22. Most microglia clusters, and in particular MG4-7, were enriched for the ‘disease-associated microglia’ (DAM) signature, which was expected as every sample in the different datasets was obtained from the disease-specific compartment (CSF and CNS) of MS patients (Supplementary Fig. 3A, B).

We found expression of CHIT1 to be strongest in cluster MG4 (2625 cells; Fig. 4B) and more than half of CHIT1+ cells were indeed located in this cluster (200/386 CHIT1+ cells; Fig. 4C). CHIT1+ cells were almost exclusively found in CNS tissue (spinal cord and brain; Fig. 4D). Based on the metadata available for the CNS datasets, we found samples taken from the lesion edge as well as from chronic active lesions to be most enriched for CHIT1+ cells (Fig. 4D). We verified that CHIT1+ cells were not a dataset-specific feature as all five datasets contributed to the total CHIT1+ cell pool (Supplementary Fig. 4). Module scoring of all CHIT1+ cells indicated that these cells portrayed a high transcriptional similarity to DAMs (Supplementary Fig. 3C).

CHIT1+ microglia are associated with MS and foam cell differentiation

Differential gene expression (DGE) analysis identified marker genes for each cluster (Fig. 5A). The top five enriched markers of cluster MG4 were GPNMB, CPM, NHSL1, NUPR1 and APOC1 (Supplementary Data 4). CHIT1 did not come out as significantly upregulated in MG4 or in any other cluster. However, this is probably due to the overall low expression of CHIT1 we observed in the data. Interestingly, GPNMB – one of the other biomarkers we measured in the CSF and which correlated significantly with CHIT1 – was the most differentially expressed gene in MG4 (1.91-fold change; Supplementary Data 4).

Next, we compiled gene modules for specific microglia/macrophage subsets previously described in MS^{14,23,24,25,26} and once again calculated module scores (Fig. 5B). Overall, clusters MG1-3 were most enriched for ‘homeostatic’ signatures, MG4-7 for ‘MS-associated’, ‘phagocytic’ and ‘MIMS-foamy’ (or ‘microglia inflamed in MS - foamy’) signatures and MG8-9 for ‘pre-activated’ and ‘pro-inflammatory’ signatures. The two predominant modules for MG4 were ‘MS-associated’²³ and ‘MIMS-foamy’²⁵ (Fig. 5C). ‘MS-associated’ microglia were reported by Masuda and colleagues to express high levels of CTSD, APOC1, GPNMB, ANXA2 and LGALS1 and to be involved in de- and remyelination²³. Absinta and colleagues found ‘MIMS-foamy’ to be enriched for pathways such as foam cell differentiation, lipid storage, response to lipoprotein particles and lysosome, which argues for a role in myelin phagocytosis and clearance²⁵.

To more specifically characterize CHIT1+ cells, we performed DGE analysis of all CHIT1+ versus CHIT1- cells (Fig. 5D, Supplementary Data 5). Many of the genes upregulated in CHIT1+ cells were found in the pathways as described for Absinta’s ‘MIMS-foamy’ (e.g. LPL, ABCA1 and NUPR1). Indeed, our own pathway analysis showed CHIT1+ cells to be significantly enriched for several pathways related to foam cell differentiation, lipid homeostasis and clearance (Fig. 5E). Altogether, our analysis showed a considerable overlap in upregulated genes of cluster MG4 and CHIT1+ cells with genes reported in ‘MS-associated’ and ‘MIMS-foamy’ signatures.

CHIT1 expression accompanies the transition from a homeostatic towards a more activated, MS-associated cell state in microglia

Based on our module scores, cluster MG4 – containing most of the CHIT1+ cells – seemed to be positioned on the interface between homeostatic microglia and disease-/MS-associated microglia (Fig. 5B, Supplementary Fig. 3A, B). To investigate these apparent cell state dynamics within the microglia clusters, we performed pseudotime trajectory analysis on clusters MG1-9 (Fig. 6, Supplementary Fig. 5). We identified four distinct lineages (Supplementary Fig. 5A), setting homeostatic cluster MG1 as the starting point. In lineages 1-3, cells belonging to cluster MG4 were positioned around the middle of the trajectories (at pseudotime ±7.5; Fig. 6A, Supplementary Fig. 5A). Global lineage structure showed MG4 to be an intermediate cluster, after which the trajectories diverge towards the ‘MS-associated’ clusters (MG5 and 6) on the one hand and the ‘pre-activated’/‘pro-inflammatory’ clusters (MG8 and 9) on the other hand (Supplementary Fig. 5B). Indeed, when plotting the lineages, CHIT1+ microglia were primarily located in-between the ‘homeostatic’ microglia at the start of the lineages and the ‘MS-associated’ and ‘pre-activated’/‘pro-inflammatory’ microglia at the end of the lineages (Fig. 6B). Next, we performed DGE analysis over the course of the trajectories to validate this observation (Supplementary Data 6). CHIT1 expression was found to be higher at the end point as compared to the starting point across all lineages (two-sided Wald test = 26.77, uncorrected P = 2.21 ×10⁻⁵). However, statistical significance did not hold after correction for multiple testing (Benjamini-Hochberg-corrected P = 0.33). Nevertheless, we did find CHIT1 to be significantly enriched in cells positioned at pseudotime 7.5 (two-sided Wald test = 40.54, Benjamini-Hochberg-corrected P = 5.01 ×10⁻⁴). Moreover, CHIT1 expression was significantly associated with pseudotime in lineage 3 (two-sided Wald test = 123.29, Benjamini-Hochberg-corrected P = 5.92 ×10⁻¹⁷; Fig. 6C, Supplementary Data 6) and peaked as pseudotime passed through cluster MG4 (Fig. 6). To sum up, pseudotime trajectory analysis indicated that CHIT1 expression accompanies the transition of microglia towards a more activated, MS-associated cell state.

Neuropathological evaluation shows phagocytic CHIT1+ cells in actively demyelinating lesions

To validate our transcriptomic results at the protein level, we performed neuropathological evaluation of brain tissue from 5 acute monophasic MS, 1 RRMS, 3 primary progressive (PPMS) and 3 secondary progressive (SPMS) MS patients. A total of 8 inactive and 21 active (including 2 chronic active or smoldering) MS lesions were analyzed (Supplementary Table 8).

Immunohistochemical staining of MS brains showed that CHIT1 was absent in normal-appearing white matter (NAWM, Supplementary Fig. 6). Where 95.2% of actively demyelinating lesions contained CHIT1+ cells (20/21), none of the inactive lesions contained any CHIT1+ cells (0/8). We did not only observe CHIT1+ cells in active lesions of more late-stage MS patients (RRMS, PPMS and SPMS with a disease duration up to ±34 years), but also in acute (monophasic) MS patients (disease duration of 0.2-5 months) – confirming the presence of CHIT1 already early in the disease course. Of the two smoldering lesions, that only have an active lesion edge, one contained few CHIT1+ cells, whereas the other lacked CHIT1 immunoreactivity.

Immunofluorescent multiplex labeling confirmed that ionized calcium-binding adapter molecule 1-positive (Iba1+) and transmembrane protein 119-positive (TMEM119+) microglia do not express CHIT1 in NAWM (Fig. 7A). In active lesions however, small Iba1+ TMEM119+ cells with no or little uptake of proteolipid protein-positive (PLP+) degradation products were found to be CHIT1+ (Fig. 7B, C). Larger, foamy (PLP-laden) CHIT1+ cells were mostly TMEM119- and some also Iba1- (Fig. 7C–E). This was in line with our transcriptomic analyses where we found that AIF1 (the gene encoding for Iba1) was significantly downregulated in CHIT1+ cells as compared to CHIT1- cells (Supplementary Data 5) as well as along the pseudotime trajectories from homeostatic towards more activated, MS-associated microglia (Supplementary Data 6). Likewise, the loss of homeostatic microglia marker TMEM119 with the acquirement of CHIT1 expression corroborated our findings at the transcript level that CHIT1 expression in microglia accompanies the transition into a disease-/MS-associated phenotype. Interestingly, small CHIT1+ Iba1+ TMEM119+ cells (Fig. 7E) seemed to be present more towards the lesion edge, whereas larger, foamy CHIT1+ Iba1- TMEM119- cells were mostly found further away from the lesion edge (Fig. 7B, C). Double staining for CHIT1 and glial fibrillary acidic protein (GFAP) confirmed that none of the CHIT1+ cells were GFAP+ astrocytes (Fig. 7F). In addition, triple staining for CHIT1 together with Iba1 and CD68 illustrated that CHIT1+ Iba1- cells were still phagocytic CD68+ cells (Fig. 7G).

**Fig. 7: Neuropathological evaluation shows phagocytic CHIT1+ cells in actively demyelinating lesions.**

Discussion

We demonstrated that CHIT1 concentration in CSF at diagnosis is a robust predictor for faster disability progression in MS patients and reflects early microglial activation. In comparison to several other microglia/macrophage-related biomarkers, only CHIT1 was found to correlate with future disability in our single-time-point analysis. By means of multi-time-point EDSS assessments and mixed-effects models, we observed that MS patients with higher CSF CHIT1 concentrations at diagnosis experienced more disability progression over time. Our machine learning models showed that CHIT1 had the highest predictive power for faster disability progression, after established clinical prognostic factors such as baseline EDSS. The consistency of our results across methodologies emphasizes the robustness of CSF CHIT1 as a biomarker. Through the analysis of single-cell transcriptomic profiles from CSF as well as CNS tissue of MS patients, we found CHIT1 to be primarily expressed by a distinct microglia subset (MG4). These CHIT1+ cells were most abundant within active MS lesions. We discovered that the CHIT1+ cell state was enriched for pathways related to foam cell differentiation, lipid homeostasis and clearance, which suggests a role in the de- and/or remyelination processes in MS. Moreover, our pseudotime trajectory analysis illustrated that CHIT1 expression in microglia accompanies the transition from a homeostatic towards a more activated, MS-associated phenotype. Neuropathological evaluation of both early- and late-stage MS patients showed that CHIT1+ cells were indeed present in almost all actively demyelinating lesions, whereas completely absent in inactive lesions and normal-appearing white matter. CHIT1+ cells were consistently CD68+ and PLP+, confirming their involvement in phagocytosis of myelin degradation products.

CHIT1 has been investigated as a putative biomarker in several other neurological disorders^27,28. The most extensive research has been conducted in amyotrophic lateral sclerosis (ALS), a prototypic neurodegenerative disease. In parallel to our results, CSF CHIT1 has repeatedly been shown to correlate with faster disease progression in ALS^16,29. CHIT1 has also been implicated in lipid storage disorders such as Gaucher, Niemann-Pick and Fabry disease. Particularly in Gaucher disease, plasma CHIT1 has been commonly used in clinical practice for over a decade to monitor disease severity and effectiveness of treatment^30,31. In these lysosomal disorders, CHIT1 was found to be a marker for specialized lipid-laden macrophages or foam cells^30,32,33. Interestingly, CHIT1 was suggested to reflect a particular activation or differentiation state of such lipid-laden macrophages rather than their absolute cell number³⁴. In accordance with these findings, our pathway and trajectory analyses as well as neuropathological evaluation implied that CHIT1 in MS also marks a subset of activated microglia enriched for processes related to foam cell differentiation and lipid storage. Of note is that most of the studies on lipid storage disorders have looked at the enzymatic activity of CHIT1 rather than its concentration in plasma. In our study, we specifically chose to focus on the concentration of CHIT1 and the other biomarkers for two reasons. First, literature on enzymatic activity of CHIT1 in CSF or plasma of MS patients is inconsistent with regard to correlation with disease course^35,36,37,38. Second, compared to the assay for assessing CHIT1 enzymatic activity, measuring CHIT1 concentration is relatively quick, easy and inexpensive, facilitating its use in routine clinical practice.

A well-known constraint for the use of CHIT1 as a biomarker is the occurrence of a frequent 24-bp duplication in the CHIT1 gene which leads to reduced expression levels and enzymatic activity³⁹. As around 35% of individuals of European ancestry carry the variant⁴⁰, we have previously shown that CHIT1 genotype did not substantially alter the correlation between CSF CHIT1 concentrations and disability⁷. Similar results have been reported in the context of ALS⁴¹. This suggests that variation in CSF CHIT1 concentrations does not mechanistically drive disease activity, but rather mirrors the contribution of microglia to disease. Since homozygous 24-bp duplication carriers – about 6% of individuals of European ancestry – have no or near-absent CHIT1, recommendations in Gaucher disease propose complementary CHIT1 genotyping in case of non-elevated CHIT1 levels^42,43.

CHIT1 is a chitinase with as natural substrate chitin, an N-acetyl-glucosamine polymer and a structural component of fungi, nematodes and arthropods²⁸. As chitin is not expressed in humans, chitinases are produced as a line of defense against these chitin-containing pathogens. In addition, CHIT1 has a role in the homeostasis of the innate immune system, where its precise mechanism of action remains unresolved³⁴. In Alzheimer’s disease, chitin-like glucosamine polymers due to impaired glucose utilization have been found within β-amyloid plaques⁴⁴ and CHIT1 secretion has been implicated in the clearance of these chitin-like polymers⁴⁵. Although glucose metabolism is elevated in inflammatory conditions, Sotgiu and colleagues did not detect such chitin-like deposits in MS. The authors proposed that CHIT1 secretion by microglia/macrophages in MS effectively counteracts the deposition of chitin-like compounds, whereas in severe Alzheimer’s disease this clearance is more imbalanced⁴⁶. Alternatively, CHIT1 production in the absence of chitin might also represent an ancient remnant of microglia/macrophage activation as genes for chitinases are highly evolutionary conserved^34,46.

One of the principal challenges for the development of glial biomarkers in MS has been cellular specificity. Several CSF and serum biomarkers of myeloid cell activity in MS are under investigation (e.g. CHI3L1, soluble CD163 and osteopontin). However, these biomarkers do not sufficiently distinguish between CNS-resident microglia and monocyte-derived macrophages, which complicates their applicability since microglia and macrophages might effectuate different roles in MS¹¹. Here, our transcriptomics showed that CHIT1 expression is largely restricted to microglia and accompanies their transition towards a more activated, MS-associated cell state. After the CHIT1+ cluster MG4, our microglial trajectories diverged towards the ‘MS-associated’ clusters (MG5 and 6) on the one hand and the ‘pre-activated’/‘pro-inflammatory’ clusters (MG8 and 9) on the other hand. This trajectory pattern suggests that homeostatic microglia upregulate CHIT1 when they start to get activated and involved in lipid clearance. However, CHIT1 expression appeared to wane again in most microglia when they start to develop a disease-/MS-associated phenotype, possibly instigated by unrelenting inflammatory cues and/or persistent lipid phagocytosis and accumulation. The second direction towards ‘pre-activated’/‘pro-inflammatory’ clusters might reflect microglia present in the MS brain that obtain a more general activated, inflammatory profile, possibly as a form of bystander activation, although more in-depth functional analyses of these cells are needed to confirm such hypotheses. Neuropathological evaluation showed that small CHIT1+ cells with limited PLP uptake were Iba1+ and TMEM119+ and were mostly situated towards the lesion edge, whereas this Iba1 and TMEM119 immunoreactivity was absent on larger, foamy CHIT1+ cells located more away from the lesion edge, suggesting that CHIT1+ microglia start to lose certain surface markers in the process of PLP accumulation. TMEM119 is one of the main homeostatic microglia markers that is not expressed on monocyte-derived macrophages and has been shown to be downregulated in active MS lesions and other inflammatory conditions of the brain^47,48,49. Moreover, decreased Iba1 expression and even Iba1- microglia have previously been described in neurodegenerative disorders such as Huntington’s and Alzheimer’s disease⁵⁰. Keren-Shaul and colleagues showed that DAMs found in a mouse model of Alzheimer’s disease were characterized by downregulation of AIF1 (the gene encoding for Iba1) as well as homeostatic genes such as TMEM119 and P2RY12⁵¹.

Although relapsing and progressive MS are classified as distinct clinical phenotypes, the field now increasingly recognizes that the clinical course of MS is a continuum, where both inflammatory and neurodegenerative processes co-exist in a spectrum⁵². Moreover, it is known that activation of CNS-resident microglia already occurs early in the disease⁵³. Indeed, most phagocytes initially present in active MS lesions are microglia, and with lesion maturation, more macrophages will infiltrate from the blood⁴⁷. As MS shifts towards a more progressive phase, microglia are once more involved in the slow expansion of chronic active lesions⁵⁴. Our work suggests that already early in the MS disease course CHIT1 is secreted into the CNS parenchyma by activated microglia who lost their homeostatic signature as these cells start to accumulate myelin debris. Conceivably, interstitial CHIT1 consequently ends up in the CSF via the glia-lymphatic or ‘glymphatic’ system in which CSF mixes with the interstitial fluid to wash away extracellular waste, as has been described for β-amyloid^55,56. Elevated CSF CHIT1 concentrations at diagnosis thus likely identify MS patients who already at the start of their disease present with significant CNS pathology – i.e. more extensive microglial activation – and are at greater risk for faster disability progression. As of now, apart from some clinical parameters such as baseline EDSS, older age at diagnosis and spinal cord lesions, MS clinicians are equipped with few predictors for disability progression at time of diagnosis⁵⁷. Therefore, a fast-progressive disease course is now often only recognized retrospectively, when damage has already been acquired. In particular with the advent of CNS-penetrant therapies that modulate microglial activity such as Bruton’s tyrosine kinase inhibitors⁵⁸, the use of microglia biomarkers such as CHIT1 to inform the benefit of these treatments might significantly improve MS outcome.

We note some limitations of our study. First, the clinical data in our MS cohort comes from a real-world clinical environment. Although real-world data achieves high external validity, it introduces non-standardized time-points for disability assessment. To mitigate this limitation, we included covariates that reflect biological differences where relevant and chose analytical methods suitable for unbalanced datasets. With regard to the transcriptomics, the absolute number of CHIT1+ cells was small and within these cells expression of CHIT1 was relatively low, which might have hampered the power of downstream analyses. However, as reported, most statistical tests on CHIT1+ cells yielded highly significant results (P ≤ 0.001) after correction for multiple testing. Furthermore, as we worked with publicly available datasets, some of the transcriptomic analyses were limited by the metadata available in these studies. In particular, evaluation of the enrichment of CHIT1 transcripts within certain lesion types or lesion edge versus core might have been impeded by inconsistent labeling of samples across studies.

Further validation is indispensable before employment of CSF CHIT1 concentration as a prognostic biomarker in MS clinical practice. First and foremost, the prognostic value of CHIT1 should be re-evaluated in a large, prospective and independent MS cohort, which will also be instrumental to define clinically relevant thresholds of CSF CHIT1 concentrations for patient stratification. Secondly, as our primary outcome for the mixed-effects and machine learning models was EDSS, we were not able to dissect the relative contribution of relapse-associated worsening to the global disability progression in our MS cohort. Although CSF CHIT1 concentration was not significantly correlated with annualized relapse rate in this study, an alternative primary endpoint to use in future studies to address this would be ‘progression independent of relapse activity’ or PIRA⁵⁹. Lastly, as we here provided initial biological insights into the prognostic potential of CHIT1 in MS, now more in-depth neuropathology as well as functional cellular studies are needed to 1) further determine the exact location of CHIT1 cells within different lesion types and 2) infer their exact phenotype and role in the de- and/or remyelination processes in MS.

In summary, we combined various complementary methods to propose CSF CHIT1 concentration at diagnosis as a putative biomarker to predict faster disability progression and reflect early microglial activation in MS. Our work provides a rationale for further validation with a prospect to clinical implementation. We believe CHIT1 has potential to cater to the unmet need for tools to aid MS clinicians in patient stratification and therapy selection, which might enable a more personalized approach in the treatment of MS patients.

Methods

All MS patients provided written informed consent for CSF sample collection and data analysis. This study was approved by the Ethics Committee of the University Hospitals Leuven (S60222 and S50354) and performed in accordance with the declaration of Helsinki. The use of post-mortem brain samples from the archives of the Center for Brain Research was approved by the Ethics Committee of the Medical University of Vienna (EK. Nr.: 535-2004).

Study design

In this study, we aimed to identify a strong predictive biomarker for disability progression in MS amongst five microglia/macrophage-related proteins measured in the CSF at diagnostic lumbar puncture – CHIT1, CHI3L1, sTREM2, GPNMB and CCL18. To this end, we extended our previously described cohort (n = 143)⁷ to 196 MS patients. Protein concentrations were measured in duplicate and blinded to clinical data. After rigorous quality control (QC), 192 MS patients were retained for further analyses. Primary and secondary endpoints were defined in advance. For single-time-point analysis, primary outcomes were ARMSS and annualized relapse rate (ARR) before treatment and secondary outcomes were MSSS and EDSS. For multi-time-point analysis (mixed-effects models and machine learning algorithms), we assessed EDSS as the only outcome. Next, we addressed the cellular source of CHIT1 – our most robust CSF biomarker – to provide a biological rationale for its prognostic value. We performed scRNA-seq on CSF of 11 MS patients and supplemented these data with single-cell profiles of MS CNS tissue from four publicly available sc/snRNA-seq datasets. Samples of interest were included based on prior knowledge as described below. All CSF and CNS transcriptomes that failed stringent QC were removed from downstream analyses. Finally, we corroborated our observed CHIT1 expression patterns at the protein level with immunohistochemistry on post-mortem brain tissue of MS patients, both in early and late disease stages. Tissue selection was informed by results from transcriptomics.

Biomarker analysis

Study population

All MS patients were diagnosed according to the 2017 revised McDonald criteria⁶⁰ at the Department of Neurology of the University Hospitals Leuven (Belgium). We distinguished disease course based on a relapsing-remitting or primary progressive MS onset (RRMS or PPMS). Clinical data were collected by the same expert neurologist (BD) at diagnosis and follow-up visits up to 15 years (median of 5.4 years) after diagnosis, effectively minimizing information bias. All EDSS assessments were done when MS patients were not in active relapse. To control for the effect of age and disease duration on our disability measures, individual EDSS scores were converted to ARMSS and MSSS values, respectively^61,62. The ARR was calculated over a minimum period of 90 days before the start of treatment, as reported earlier⁶³. During disability follow-up (EDSS, ARMSS and MSSS), 62% of MS patients were on disease-modifying therapies.

CSF protein measurements

CSF of MS patients was collected during diagnostic lumbar puncture as part of standard clinical care. None of these patients were on disease-modifying therapy at the time of sample collection. Polypropylene tubes were centrifuged for 10 min at 3000× g at 4 °C and CSF supernatant was stored at −80 °C until protein measurements. We assessed CHIT1, CHI3L1, sTREM2, GPNMB and CCL18. NfL was also included as canonical biomarker for neuronal damage. CHIT1, GPNMB, CCL18 and NfL were measured within the entire MS cohort (n = 196). For CHI3L1 and sTREM2, measurements were limited to the original cohort (n = 143) as previously described⁷.

CSF protein concentrations were measured in duplicate and blinded to clinical data. For CHIT1, the CircuLex Human Chitotriosidase ELISA Kit (#CY-8074, MBL Life Science) was used. GPNMB and CCL18 concentrations were quantified with U-PLEX technology (#F21ZH-3 and #F212H-3, MSD). CHI3L1, sTREM2 and NfL measurements were obtained as previously described⁷. We performed all assays conforming to the manufacturer’s instructions.

QC was carried out as follows: we checked for consistency of the standard curves across plates, with 0.74 ˂ r ˂ 1 (P ≤ 0.05) for all assays. Concerning plate-to-plate variability, we calculated the coefficient of variation (CV) based on an internal control sample measured on all plates. Samples with a CV more than 20% or CSF biomarker concentrations below the detection limit were excluded. Samples above the upper detection limit were remeasured using a different dilution factor where possible or were eliminated otherwise. Within-plate variability was assessed based on the CV across duplicates (Supplementary Table 1). Mean values across duplicates were used for analysis.

Single-cell transcriptomics

Datasets

In-house CSF cohort: All patients (n = 11) were diagnosed with RRMS according to the 2017 revised McDonald criteria⁶⁰ by the same expert neurologist (BD) at the Department of Neurology of the University Hospitals Leuven (Belgium) (Supplementary Data 2). None of these patients were on disease-modifying therapy at the time of sample collection.

Previously published CNS datasets: All previously published CNS datasets were downloaded as FASTQ files from the Sequence Read Archive [SRA, National Center for Biotechnology Information (NCBI)] under BioProject accession numbers PRJNA544731 [Schirmer et al.²⁴], PRJNA749443 [Absinta et al.²⁵], PRJNA743676 [Miedema et al.¹⁴] and PRJNA726991 [Trobisch et al.⁶⁴]. The data from Miedema et al.¹⁴ was acquired via scRNA-seq, whereas the others performed snRNA-seq. From all studies, only samples of interest were used, i.e. CNS lesions or periplaque white matter of MS patients (Supplementary Data 2). CNS data was processed in parallel to our in-house CSF data from the Cell Ranger count pipeline onwards.

CSF sample collection and sequencing

CSF samples were obtained during diagnostic lumbar puncture. 5 ml of CSF was collected into round-bottom polypropylene tubes (Sarstedt) and processed within 1 hour after lumbar puncture to ensure optimal sample quality. CSF samples were centrifuged for 15 min at 400× g at 4 °C. We resuspended the CSF cell pellets in 100 µl of phosphate-buffered saline (PBS, Gibco) and counted the cells on a LUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems). CSF cells were centrifuged again (15 min, 400× g, 4 °C) and resuspended in 1.5 ml of RPMI 1640 Medium (Gibco) containing 20% heat-inactivated fetal bovine serum (FBS, Merck) and 10% dimethyl sulfoxide (DMSO, Merck). CSF cells were transferred to Nunc CryoTubes (Thermo Scientific) and stored in a CoolCell container (Corning) at −80 °C. After 24 h, cells were relocated to liquid nitrogen for cryopreservation until further processing. Cryopreservation allowed us to limit batch effects without a substantial effect on gene expression patterns^65,66.

Library preparation and scRNA-seq of our in-house CSF cohort was performed in three separate batches. The average latency between cryopreservation and library construction was 4.3 months (Supplementary Data 2). For every batch, we resuscitated the cryopreserved CSF cells in warmed RPMI 1640 Medium (Gibco) containing 20% heat-inactivated FBS (Merck). Cells were centrifuged for 10 min at 400× g at room temperature (RT) and resuspended in PBS (Gibco) containing 0.04% bovine serum albumin (BSA, Merck). We consistently obtained a high cell viability (mean: 94.7% ± 5.8% SD). Single-cell suspensions were loaded on a Chromium Next GEM Chip K for single-cell partitioning and barcoding with the Chromium Controller according to the Chromium Next GEM Single Cell 5′ Reagent Kit v2 (Dual Index) (#1000286 and #1000263, 10x Genomics). We performed further library preparation conforming to the manufacturer’s instructions. Libraries were sequenced on an Illumina NovaSeq 6000 with a target sequencing depth of 50,000 reads per cell.

Preprocessing of sequencing data

Processing of the sequencing data from both our in-house CSF cohort as well as the four previously published CNS datasets was performed with Cell Ranger v7.1.0 software (10x Genomics). Read alignment and transcript count were done individually for each sample via the Cell Ranger count pipeline with default parameters. Human reference GRCh38 (GENCODE v32/Ensembl 98) version 2020-A (July, 2020) was used for gene mapping.

Quality control

Before integration, all samples from all CSF and CNS datasets went separately through our QC pipeline in R v4.2.2. Ambient RNA contamination was removed using the SoupX v1.6.2 package⁶⁷ with default parameters, except for samples from Miedema et al.¹⁴ where tfidfMin was adjusted to 0.8 (default 1) due to the lower cellular complexity of the dataset (prior FACS). Doublets were identified via scDblFinder v1.12.0⁶⁸ with default parameters. The corrected count matrices and relevant metadata were used to create Seurat objects for further analysis with Seurat v4.3.0⁶⁹. Features (genes) present in less than five cells were removed and only high-quality cells were retained, i.e. singlets with more than 200 features and a maximal percentage of mitochondrial RNA content of 15% for scRNA-seq data⁷⁰ and 5% for snRNA-seq data^25,64 (Supplementary Data 2). We normalized and scaled the count matrices via SCTransform v0.3.5⁷¹ with default parameters. Mitochondrial and ribosomal content as well as the difference in expression between S and G2M cell cycle genes were regressed out.

Data integration, batch effect removal and myeloid clustering

In order to harmonize the different samples and avoid subject-specific clusters, we integrated the individual samples of the five datasets (our in-house CSF dataset and four previously published CNS datasets) into one dataset using the Harmony v0.1.1 package⁷² with default parameters. We assessed different integration strategies with various (combinations of) covariates to correct for. Best integration was obtained when each sample was treated as a batch and sequencing technique (scRNA-seq or snRNA-seq) was included as a variable. To cluster the cells, FindNeighbours() was run with Harmony-corrected principal components, followed by FindClusters(). We visualized the clusters on a Uniform Manifold Approximation and Projection (UMAP) plot. Optimal cluster resolution was determined with guidance of the clustree v0.5.0 package⁷³. To identify myeloid clusters, we checked the expression of canonical myeloid marker genes (Supplementary Data 3). Furthermore, we used SingleR v2.0.0⁷⁴ for automated annotation based on the Novershtern immune reference dataset⁷⁵ from the celldex v1.8.0 package⁷⁴. Non-myeloid clusters were removed.

Annotation of myeloid clustering

DGE analysis was performed using FindAllmarkers() with default parameters, except for min.pct = 0.25 (default 0.10), to investigate the most differentially expressed genes (DEGs) in each cluster. We subdivided the myeloid clusters into monocytes, macrophages and microglia based on canonical marker genes (Supplementary Data 3). For more in-depth annotation of these clusters, we compiled gene sets of interest (or modules) from previously published articles (Supplementary Data 3) and scored their expression levels in our integrated dataset via AddModuleScore()⁷⁶.

Pathway analysis

CHIT1+ cells were identified via WhichCells() as cells with an expression value > 0 in the RNA assay. We used FindMarkers() to get DEGs between CHIT1+ and CHIT1- cells. Only genes with an adjusted p-value < 0.05 were retained and used as input for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set enrichment analysis via the clusterProfiler v4.6.2 package⁷⁷.

Pseudotime trajectory analysis

To estimate cell state dynamics, we performed pseudotime trajectory analysis using the slingshot v2.6.0⁷⁸ and tradeSeq v1.12.0⁷⁹ packages. Homeostatic cluster “Microglia 1” (MG1) was defined as the starting point. Since the inclusion of non-microglia clusters [“Monocytes” (MON), “CNS-associated macrophages 1 and 2” (CAM1 and CAM2) and “Mixed myeloid cells” (MMC)] might lead to nonsensical trajectories, we subsetted our data to only retain microglia clusters for pseudotime trajectory analysis to gauge where the CHIT1+ microglia were positioned among the different microglia cell states. Moreover, we carried out DGE analysis along the inferred trajectories with fitGAM(), assoRes() and startVsEndTest().

Neuropathological evaluation

Patients

For neuropathological evaluation, formalin-fixed paraffin-embedded (FFPE) autopsy material from the archives of the Center for Brain Research (Medical University of Vienna) was used. We assessed samples from acute monophasic MS [n = 5, described before⁸⁰], RRMS (n = 1), PPMS (n = 3) and SPMS (n = 3) patients. From these MS patients, we analyzed a total of 21 active (2 smoldering) and 8 inactive lesions (Supplementary Table 8).

Luxol Fast Blue-Periodic acid Schiff staining for myelin

5 µm thick paraffin sections of MS brain were stained with Luxol Fast Blue-Periodic acid Schiff (LFB-PAS) according to standard procedure to detect demyelinated lesions and macrophages with myelin degradation products.

Immune light microscopical staining

3-5 µm FFPE sections were deparaffinized in xylene (2× 15 min) and rinsed with 96% ethanol. To block endogenous peroxidase activity, samples were incubated in H2O2-methanol (30 min). Next, we gradually rehydrated the samples with ethanol (96%, 70%, 50%) and finally deionized water. Heat-induced epitope retrieval (HIER) was performed with ethylenediaminetetraacetic acid (EDTA, pH 9.0) in a household food steamer (Braun, 1 h). Afterwards, we rinsed the sections with tris-buffered saline solution (TBS) 3-5 times. Non-specific background reactions were blocked with 10% fetal calf serum (FCS) in DAKO buffer (Agilent) for 15 min. Hereafter, we incubated the slides with primary antibody CHIT1 (#HPA010575, Sigma, 1:100, 4 °C overnight) or CD68 (#M0814, Dakocytomation, 1:100, 4 °C overnight) and rinsed them with TBS before incubating them with a secondary biotin anti-rabbit-conjugated antibody (1:200, #711-065-152, Jackson, 1 h at RT) or biotin-anti-mouse-conjugated antibody (#715-065-150, Jackson, 1:500, 1 h at RT). Next, the slides were rinsed with TBS and incubated with peroxidase-conjugated streptavidin (1:500) in 10% FCS-DAKO buffer (1 h at RT). Finally, the sections were rinsed with TBS, developed with 3,3’-Diaminobenzidine (DAB) and counterstained in hematoxylin (15-20 s) before dehydration and mounting with cover slips using EUKITT (ORSAtec).

Immunofluorescent multiplex labeling

Immunofluorescent multiplex labeling was performed with Akoya’s Fluorescent Multiplex reagents according to the PerkinElmer´s application note ‘Automated multiplex Biomarker Staining and Imaging’ for CHIT1 (#HPA010575, Sigma, 1:1000), Iba1 (#019-19741, Wako, 1:10,000), PLP (#MCA839G, Bio-Rad, 1:5000), GFAP (#MS-1376, Thermo Scientific, 1:1000), TMEM119 (#HPA051870, Sigma, 1:1000) and CD68 (#M0814, Dakocytomation, 1:1000). In short, after deparaffination, the sections were fixed with 4% paraformaldehyde (PFA, 20 min), rinsed with deionized water and steamed in Antigen Retrieval buffer (pH 9.0, Akoya, AR900250ML) in a household food steamer (Braun, 1 h). The sections were rinsed with TBS-Tween 2.0 (TBST, pH 7.5) followed by a blocking step with Opal Antibody Diluent/Block solution (Akoya, ARD1001EA, 10 min). Next, we incubated the slides with a first primary antibody (4 °C, overnight). Then, the slides were rinsed with TBST and incubated with Akoya’s secondary antibodies (Opal Polymer horseradish peroxidase conjugated antibodies, ARH1001EA, 10 min at RT). The sections were rinsed with TBST and incubated with a fluorophore (Opal dye 480, 520, 570, 620, 690 or 780, 1:100, 10 min) in 1X Plus Amplification Diluent (Akoya, FP1497). Next, the slides were rinsed with TBST, fixed with 4% PFA (10 min) and steamed with Antigen Retrieval buffer (AR6, Akoya, AR600250ML, 30 min). From here, the above-mentioned steps were repeated for each primary antibody. Finally, we counterstained with 4’,6-Diamidino-2-phenylindole (DAPI) and the slides were mounted with Fluoromount-G (SouthernBiotech). The slides were scanned with a Vectra Polaris scanner (PerkinElmer) at 20× magnification or on a laser scanning microscope equipped with lasers for 504, 488, 543 and 633 nm excitation (Leica SP5, Leica, Mannheim, Germany).

Statistical analysis

For single-time-point analysis and mixed-effects models, all statistical tests were performed with R v4.0.1. Normal distribution of the data was assessed with Shapiro-Wilk tests. Biomarker concentrations and clinical variables were normally distributed after a logarithmic transformation with base 10 and an inverse rank normal transformation, respectively.

Single-time-point analysis

To evaluate the relationships between the five microglia/macrophage CSF biomarkers – CHIT1, CHI3L1, sTREM2, GPNMB and CCL18 – and CSF NfL concentrations, we calculated pairwise Pearson correlation coefficients and tested their significance with two-sided Pearson correlation tests. Next, we tested the association between these biomarker concentrations and several MS disease activity parameters: ARMSS and ARR before treatment (primary outcomes); MSSS and EDSS (secondary outcomes). These associations were assessed in a linear regression model which included age at diagnosis and sex as covariates as well as the rs150192398 genotype for CHIT1 as in our previous work⁷. Based on the results of the linear regression model, we focused on the correlations between CSF CHIT1 concentrations and future disability (ARMSS, MSSS and EDSS). The percentage of variance explained (r²) with and without clinical covariates, the division between MS patients with high (ARMSS ≥ 5) and low (ARMSS < 5) disability accumulation as well as the ROC curve were determined as previously described^7,61. We applied Bonferroni corrections for multiple testing as stated in the table legends.

Multi-time-point analysis – mixed-effects models

The correlation between CSF CHIT1 concentrations at diagnosis and disability progression (EDSS) over time was assessed using both mixed-effects models and artificial intelligence algorithms (see Machine learning algorithms). Mixed-effects models were chosen due to their robustness and power to handle unbalanced data given the gradual loss of EDSS data over time within our MS cohort. Despite the unbalanced EDSS data, the mixed-effects models allowed us to examine the impact of CHIT1 on disability progression over time while also considering variability within and across MS patients and time as well as controlling for relevant variables⁸¹. Within our model development, we tested models with and without the following considerations: controlling for 1) interindividual variance in EDSS scores; 2) intraindividual variance in EDSS scores across time; and 3) interaction effects between CHIT1 and time from MS diagnosis to EDSS score. Mixed-effects models were constructed and assessed with packages lme4 v1.1-31, afex v1.2-1 (convergence checks) and ggplot v3.4.2. Included continuous predictors were scaled to assist with convergence and result interpretation. Model fits were assessed using likelihood-ratio tests.

Multi-time-point analysis – machine learning algorithms

In D’hondt et al.¹⁹, we developed machine learning algorithms to model EDSS trajectories of MS patients. In short, this approach transformed real-world EDSS assessments for each patient to yearly average disability scores up to the 10th year after diagnosis. These 10 disability averages then formed the outcomes of a machine learning model based on random forests. For each of these 10 outcomes, a random forest was trained. In a regressor chain fashion⁸², the disability predictions of previous years were added as extra input to the random forests of the next years. These trained models could then be used to predict the disability of an individual MS patient for up to 10 years after diagnosis.

In this work, we used this regressor chain model to analyze the prognostic importance of input variables, in particular the CSF biomarkers under study. For this, we used permutation importance scores²⁰, which reflect the effect on model performance if one of the input variables is randomly shuffled (simulating the removal of that variable). Model performance (mean absolute error and Pearson correlation) was measured using 5-fold cross-validation (to avoid overfitting) and the variable shuffling was repeated multiple times to counter variability. In order to control for statistical interactions between CSF biomarkers, we conducted a separate analysis where we made a correction on the permutation importance scores for possible CSF biomarker interactions. To this end, in addition to shuffling each biomarker separately, we also shuffled all possible pairs, triplets, quartets and quintets of biomarkers (simulating the removal of several biomarkers simultaneously). In each of these shuffles, the performance drop was calculated and divided by the size of the shuffled subset of biomarkers (1, 2, 3, 4 or 5) and by the number of times each biomarker appears in a subset of that size (1, 5, 15, 5, 1). For each biomarker, a final score was then obtained by summing all performance drops in which that biomarker was shuffled. The machine learning methodology was developed in Python 3.8.10.

Single-cell transcriptomics

With regard to the transcriptomics, all statistical tests were performed in R v4.2.2. DGE results were consistently assessed for statistical significance using a two-sided unpaired Wilcoxon rank-sum test with Bonferroni correction for multiple testing. Differential expression of modules across clusters was demonstrated using a Kruskal-Wallis test followed by a two-sided Dunn’s test for pairwise multiple comparisons with Benjamini-Hochberg correction for multiple testing. For pathway analysis, p-values were calculated by a one-sided Fisher’s exact test with Benjamini-Hochberg correction. For the DGE analyses across trajectories, statistical significance was evaluated by means of a two-sided Wald test with Benjamini-Hochberg correction for multiple testing. The exact method is also always stated in the legends. For histology, no statistical analyses were performed.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data that support the main findings in this study are available in the manuscript or the supplementary materials. Raw biomarker measurements are available in Supplementary Data 1. Source data are provided with this paper. The previously published CNS scRNA-seq and snRNA-seq datasets were downloaded from the Sequence Read Archive [SRA, National Center for Biotechnology Information (NCBI)] under BioProject accession numbers PRJNA544731 (Schirmer et al.²⁴), PRJNA749443 (Absinta et al.²⁵), PRJNA743676 (Miedema et al.¹⁴) and PRJNA726991 (Trobisch et al.⁶⁴). Our previously unpublished CSF scRNA-seq cohort has been uploaded to the SRA under BioProject accession number PRJNA996357. Source data are provided with this paper.

Code availability

The code scripts to recreate the main figures and results are accessible in Zenodo under https://doi.org/10.5281/zenodo.11235175.

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Acknowledgements

This study was funded by the National Multiple Sclerosis Society (International Progressive MS Alliance, PA-2002-36277, A. Goris), the Dutch MS Research Foundation (Monique Blom - de Wagt grant, 22-1146 MS, SM), MS-Liga Vlaanderen (A. Goris) and the Flemish government (through the AI Research Program, CV). J.Be and R.D. hold PhD Fellowships of the Research Foundation-Flanders (FWO-Vlaanderen, 11A0523N and 1S38023N). B.D. holds a research mandate of KU Leuven (BOF-FKO, Bijzonder Onderzoeksfonds – Fundamenteel Klinisch Onderzoeker). Some of the resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation - Flanders (FWO) and the Flemish Government. Figure 4A was partially created with BioRender.com. This study made use of publicly available datasets and we wish to sincerely thank the authors for sharing their data. In particular, we would like to thank S.M. Kooistra for providing us with additional metadata on their samples. We acknowledge all developers who contributed to the excellent tools we were able to use in our analyses. Finally, the authors thank A. Goris for help with conceptualizing this study, L. Yshii, M. David and D. De Wit for fruitful discussions, K. Clysters and C. Thys for their contribution in sample collection, as well as all patients and their families for their willingness to participate in this study.

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These authors contributed equally: Jarne Beliën, Stijn Swinnen.
These authors jointly supervised this work: Sinéad Moylett, Bénédicte Dubois.

Authors and Affiliations

Laboratory for Neuroimmunology, Department of Neurosciences, Leuven Brain Institute, KU Leuven, Leuven, Belgium
Jarne Beliën, Stijn Swinnen, Nina Dedoncker, Sinéad Moylett & Bénédicte Dubois
Department of Neurology, University Hospitals Leuven, Leuven, Belgium
Stijn Swinnen & Bénédicte Dubois
Department of Public Health and Primary Care, KU Leuven, Kortrijk, Belgium
Robbe D’hondt & Celine Vens
Imec research group itec, KU Leuven, Kortrijk, Belgium
Robbe D’hondt & Celine Vens
Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Vienna, Austria
Laia Verdú de Juan & Jan Bauer
Laboratory of Immunobiology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium
Patrick Matthys

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Jarne Beliën
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Stijn Swinnen
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Robbe D’hondt
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Laia Verdú de Juan
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Patrick Matthys
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Jan Bauer
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Celine Vens
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Sinéad Moylett
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Bénédicte Dubois
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Contributions

Conceptualization: J.Be, S.S., R.D., C.V., S.M., B.D. Methodology: J.Be, S.S., R.D., L.V., JBa, S.M. Investigation: J.Be, S.S., R.D., L.V., N.D., J.Ba, S.M. Visualization: J.Be, S.S., R.D., L.V., J.Ba, S.M. Funding acquisition: C.V., S.M., B.D. Supervision: P.M., J.Ba, C.V., S.M., B.D. Writing – original draft: J.Be, S.S., R.D., S.M. Writing – review & editing: J.Be, S.S., R.D., L.V., N.D., P.M., J.Ba, C.V., S.M., B.D.

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Correspondence to Bénédicte Dubois.

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B.D. has received consulting fees from Bristol Myers Squibb, Almirall, Roche, Janssen, Novartis and Sanofi. All other authors declare that they have no competing interests.

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Beliën, J., Swinnen, S., D’hondt, R. et al. CHIT1 at diagnosis predicts faster disability progression and reflects early microglial activation in multiple sclerosis. Nat Commun 15, 5013 (2024). https://doi.org/10.1038/s41467-024-49312-y

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Received: 09 November 2023
Accepted: 30 May 2024
Published: 12 June 2024
DOI: https://doi.org/10.1038/s41467-024-49312-y
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Clinical research in neurological disorders

Collection 14 September 2023

CHIT1 at diagnosis predicts faster disability progression and reflects early microglial activation in multiple sclerosis

Abstract

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Introduction

Results

Study population for biomarker analysis

CHIT1 at diagnosis correlates with disability years later: single-time-point analysis

CHIT1 at diagnosis correlates with disability progression: multi-time-point analysis

After clinical prognostic factors, CHIT1 at diagnosis shows highest predictive value for disability progression

CHIT1 is expressed by a distinct microglia subset in active MS lesions

CHIT1+ microglia are associated with MS and foam cell differentiation

CHIT1 expression accompanies the transition from a homeostatic towards a more activated, MS-associated cell state in microglia

Neuropathological evaluation shows phagocytic CHIT1+ cells in actively demyelinating lesions

Discussion

Methods

Study design

Biomarker analysis

Study population

CSF protein measurements

Single-cell transcriptomics

Datasets

CSF sample collection and sequencing

Preprocessing of sequencing data

Quality control

Data integration, batch effect removal and myeloid clustering

Annotation of myeloid clustering

Pathway analysis

Pseudotime trajectory analysis

Neuropathological evaluation

Patients

Luxol Fast Blue-Periodic acid Schiff staining for myelin

Immune light microscopical staining

Immunofluorescent multiplex labeling

Statistical analysis

Single-time-point analysis

Multi-time-point analysis – mixed-effects models

Multi-time-point analysis – machine learning algorithms

Single-cell transcriptomics

Reporting summary

Data availability

Code availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Supplementary information

Source data

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