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SIGLEC1 (CD169): a marker of active neuroinflammation in the brain but not in the blood of MS patients

Objective: We aimed to evaluate SIGLEC1 (CD169) as a biomarker in Multiple Sclerosis (MS) and Neuromyelitis optica spectrum disorder (NMOSD) and to evaluate the specificity of SIGLEC1+ myeloid cells for demyelinating diseases Methods: We performed flow cytometry-based measurements of SIGLEC1 expression on monocytes in 86 MS patients, 41 NMOSD patients and 31 healthy controls. Additionally, we histologically evaluated the presence of SIGLEC1+ myeloid cells in acute and chronic MS brain lesions as well as other neurological diseases. Results: We found elevated SIGLEC1 expression in 16/86 (18.6%) MS patients and 4/41 (9.8%) NMOSD patients. Almost all MS patients with high SIGLEC1 levels received exogenous interferon beta as an immunomodulatory treatment and only a small fraction of MS patients without interferon treatment had increased SIGLEC1 expression. SIGLEC1+ myeloid cells were abundantly present in active MS lesions as well as in a range of acute infectious and malignant diseases of the central nervous system, but not chronic MS lesions. Conclusion: In our cohort, SIGLEC1 expression on monocytes was - apart from those patients receiving interferon treatment - not significantly increased in patients with MS and NMOSD, nor were levels associated with more severe disease. The presence of SIGLEC1+ myeloid cells in brain lesions could be used to investigate the activity in an inflammatory CNS lesion.


Background
Multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) are both chronic, demyelinating inflammatory diseases of the central nervous system that are a significant cause of disability in the young. While certain aspects of the pathophysiology of both diseases have been uncovered, many aspects towards a complete understanding remain to be elucidated. 1 SIGLEC1 (Sialic acid-binding immunoglobulin-type lectins-1, CD169) is a sialic acid binding cell-surface protein, exclusively expressed on monocytes and macrophages. 2 Its expression is upregulated upon contact with type I interferons and to a lesser degree, other activatory stimuli such as LPS. 3,4 As such, SIGLEC1 expression has been used as a surrogate marker for type I interferon activity in autoimmune diseases like Systemic lupus erythematosus 5 or primary Sjögren syndrome 6 as well as interferonopathies 7 and viral infections 8,9 . In the healthy brain, SIGLEC1 is only expressed by some perivascular and choroid plexus macrophages, but not by microglia. 10 In mice, mechanical insult to the brain led to an accumulation of SIGLEC1+ myeloid cells in the damaged area. 10 The cause of this accumulation, however is unclear -either the contact of serum components or inflammatory signals with resident myeloid cells induces SIGLEC1 expression or blood-derived SIGLEC1+ myeloid cells infiltrate the CNS.
The role of type I interferons and SIGLEC1 in the pathophysiology of MS and NMOSD is not yet clear.
In relapsing-remitting MS (RRMS), type I interferons are used as an immunomodulatory treatment that reduces the rate of relapses. 11 It does, however, not protect against clinical progression in the progressive forms of MS (primary progressive MS (PPMS) and secondary progressive MS (SPMS)). 11 In a recent report, SIGLEC1 positive myeloid cells were found within MS lesions and specific ablation of SIGLEC1 expressing cells in the experimental autoimmune encephalitis (EAE) model of MS led to an amelioration of disease. 12 Additionally, plasmacytoid dendritic cells, the main type I interferonproducing cells, were found in increased frequencies in the cerebrospinal fluid of MS patients during a flare. 13 Two studies found SIGLEC1 expression to be increased on blood monocytes of MS patients, especially those with a progressive type of MS. 12,14 Taken together, there is conflicting evidence on the role of type I interferons and the interferon-induced expression of SIGLEC1 in MS as being protective or pathogenic. This is likely due to the heterogeneity of patients and disease stages and potentially also the confounding effect of interferon therapy.
In NMOSD, little is known about the role of type I interferons; however, many patients with NMOSD have an overlap with additional, type I interferon-dependent diseases, such as SLE 15 and a recent report describes a series of patients with increased levels of endogenous or exogenous interferon α who went on to develop a seropositive NMOSD. 16 Interferon treatment does not prevent NMOSD relapses 17 and anecdotally even increases the disease activity. 18 Additionally, we recently described the presence of low-density granulocytes in MS and NMOSD patients, 19 a subset of granulocytes that produce high levels of type I interferons in SLE. 20 Thus, we hypothesized that type I interferons could also play a role in the pathogenesis of NMOSD.
In this work we investigated expression of SIGLEC1 on monocytes of patients with MS and NMOSD and correlated the expression with clinical parameters. In addition, we analysed human brain tissue sections of patients with both active and inactive MS as well as other neurological and systemic diseases. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

PBMC isolation and flow cytometry
Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA anticoagulated blood. Blood

Immunohistology of human brain tissue
All stains were performed on 8μm cryomicrotome sections, cut on a NX80 cryotome (ThermoFisher, Waltham, Massachusetts, USA) according to standard procedures. Stains included Hematoxylin and is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

SIGLEC1 expression is increased in a subset of patients with multiple sclerosis
We investigated the SIGLEC1 expression on CD14 + monocytes in the peripheral blood of MS and NMOSD patients as well as in healthy controls. In most of the samples, monocyte SIGLEC1 expression was barely above the negative (fluorescence minus one) control (Fig. 1a, b). We defined a physiologic range of SIGLEC1 expression as two standard deviations from the mean SIGLEC1 expression in healthy controls (normal MFI range 0 -564). Accordingly, 16/86 (18.6%) MS patients, 4/41 (9.8%) NMOSD patients and 1/31 (3.2%) healthy controls had increased SIGLEC1 levels. Almost all individuals with available longitudinal samples remained in their respective SIGLEC1 high or low category over a followup period of up to 1965 days. (Fig. 1c)

Correlation with clinical parameters
Next, we aimed to identify parameters that were associated with increased SIGLEC1 in MS patients.
One obvious explanation would be exogenous type 1 interferon as treatment. 11/16 MS patients (68.6%) with increased SIGLEC1 expression levels received interferon beta treatment at the time of measurement (Fig. 1d). While a previous report found increased SIGLEC1 levels to be more prevalent in MS patients with a progressive form of MS, we found only 2/20 patients primary or secondary progressive MS with increased SIGLEC1 levels that were not explained by interferon treatment (Fig.   1e). SIGLEC1 expression on monocytes did not correlate with the Expanded Disability Status Scale is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.18.20248500 doi: medRxiv preprint (EDSS) (Fig. 1f), nor had it a temporal association with relapses.

SIGLEC1 expression on brain-infiltrating myeloid cells
To investigate the presence and specificity of SIGLEC1+ myeloid cells in inflammatory MS lesion, we analysed brain tissue sections from 4 patients with active relapsing-remitting MS (RRMS), 5 patients with secondary-progressive MS (SPMS) and 6 patients who died of cardiovascular or multi-organ failure. In all control samples, SIGLEC1 positivity was limited to cells in the leptomeninges as well as perivascular macrophages, as previously reported. 10 In all four samples from patients with active relapsing-remitting MS (RRMS) as well as in one patient with the Marburg variant of MS, we found a dense infiltrate of CD68+HLA-DR+ myeloid cells that stained predominantly positive for SIGLEC1 (Fig.   2a). In five samples from patients with secondary-progressive MS (SPMS) however, we found CD68+HLA-DR+ infiltrates of varying density, but almost no SIGLEC1 expression (Fig. 2a). This indicates that SIGLEC1 expression on myeloid cells could be used to distinguish active inflammatory lesions from chronic lesions.
To corroborate this hypothesis, we studied the SIGLEC1 expression in the brain tissue of 8 patients with other inflammatory neurological diseases. SIGLEC1+ myeloid infiltrates were present in patients with glioblastoma (n=2), herpes simplex encephalitis (n=2) and cerebral infarction (n=2), as well as toxoplasmosis (n=1), cerebral abscess (n=2) and post-transplant lymphoproliferative disorders (n =1, PTLD, Fig. 2b). No increase in SIGLEC1+ cells was noted in the brain of a patient who died of amyotrophic lateral sclerosis (ALS).
In summary, SIGLEC1+ is expressed on brain-infiltrating myeloid cells in a broad range of active inflammatory lesions, but not in chronic MS lesions.

Conclusion
Here, we report that, after accounting for interferon treatment, patients with multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) did not have increased expression of SIGLEC1 on monocytes in the peripheral blood.
We identified SIGLEC1+ on CD68+HLA-DR+ myeloid cells in active inflammatory MS lesions and a range of other inflammatory, infectious or malignant brain lesions. SIGLEC1+ expression was low on myeloid cells in chronic MS lesions of SPMS patients, indicating that SIGLEC1+ myeloid cells could serve as a marker of inflammatory activity. These findings are in line with a previous report that found SIGLEC1+MHC-II+ myeloid cells to be present in mice after retinal transplantation, but not in the healthy retina or retinal degeneration. 26 Future studies will be needed to investigate, whether the is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.18.20248500 doi: medRxiv preprint increase in SIGLEC1+ cells in inflammatory brain lesions is due to the infiltration of SIGLEC1+ cells from the peripheral blood or an upregulation of SIGLEC1 in tissue-resident microglia, as well as define the inflammatory signals leading to such an upregulation. We propose that SIGLEC1+ can serve as a marker to differentiate active from inactive MS lesions, which will require additional validation for use in routine histopathological diagnostics.
Our results on SIGLEC1 expression on blood monocytes are in contrast to two previous reports which identified increased levels of SIGLEC1 in smaller cohorts of MS patients. 12,14 The study by Malhotra et al 14 focussed on untreated MS patients and identified modest increases in SIGLEC1 expression in MS patients and especially those with progressive disease. A second study by Bogie et al 12 again identified small increases in SIGLEC1 expression on monocytes in MS patients and found that these increases were independent of progressive disease and also of interferon treatment, an observation that is at odds with our own findings and biological plausibility.
This disparity might be due to different reasons: it could be that the authors mostly observed small differences between individuals that would have all been classified as "SIGLEC1 low expressors" in our study, as the fold-differences they describe are significantly smaller compared to the 5-10-fold increases in "SIGLEC1 high expressors" in our study. Different studies investigated the SIGLEC1 expression using different antibody clones which could also explain some of the differences; in this study, we used the same clone as previous studies that investigated SIGLEC1 in rheumatologic diseases. 5,6 One significant weakness of our own study is the relative clinical quiescence of the patients enrolled; most did not have a relapse within the last six months before the measurement and most did not have a change in EDSS in longitudinal sampling. Additionally, most patients were under immunosuppressive treatment. Future studies in MS and NMOSD patients during a clinical relapse could thus still discover the presence of SIGLEC1 expressing monocytes in the peripheral blood.
In summary, our data indicate that type I interferons or SIGLEC1 expressing cells in the peripheral blood do not play a major role in the pathogenesis of most patients with stable NMOSD or MS, however SIGLEC1 + myeloid cells in the brain are present in inflammatory MS lesions as well as in other inflammatory neurological diseases of the CNS.

Declarations
Ethics approval and consent to participate These studies were approved by the ethics committee of the Charité -Universitätsmedizin Berlin (references: EA1/163/12, EA1/041/14, EA1/107/13) and informed consent was obtained from all participants in the study.

Consent for publication
. CC-BY-NC 4.0 International license It is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 22, 2020. ; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 22, 2020. ; https://doi.org/10.1101/2020.12.18.20248500 doi: medRxiv preprint  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 22, 2020. ; with higher levels of SIGLEC1 expression: Kruskal-Wallis test with Dunn's correction, **: p<0.01, ****: p<0.0001. (e) Comparison of SIGLEC1 expression in patients with RRMS (n = 63), SPMS (n = 15) and PPMS (n = 5). Red symbols indicate interferon treatment. (f) Scatter plot of the EDSS disability score against SIGLEC1 expression, no significant correlation was detected.  is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint