A role for DICAM+ mononuclear phagocytes in controlling neuroinflammation in multiple sclerosis

A role for DICAM+ mononuclear phagocytes in controlling neuroinflammation in multiple sclerosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A role for DICAM+ mononuclear phagocytes in controlling neuroinflammation in multiple sclerosis Marina Rode von Essen, Marie Mathilde Hansen, Sahla El Mahdaoui, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5403219/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). In MS, CNS-infiltrating monocytes differentiate to tissue resident macrophages which are found in large numbers within the injured areas of the brain where they play a central role in driving disease progression through demyelination and tissue destruction. However, infiltrating monocytes and their derivative macrophages can also serve protective functions. In this study we investigated a possible role of intrathecal mononuclear phagocytes (infiltrating monocytes and macrophages) expressing dual immunoglobulin domain-containing cell adhesion molecule (DICAM) in neuroinflammation. Compared to symptomatic controls, treatment-naïve patients with relapsing-remitting MS had a reduced prevalence of DICAM + mononuclear phagocytes in CSF. When patients were treated with natalizumab, an antibody blocking migration of blood leukocytes to the CNS, we observed that DICAM + monocytes were still recruited to the CSF and that the level of soluble DICAM (sDICAM) in CSF was significantly increased compared to untreated patients. sDICAM and the prevalence of DICAM + mononuclear phagocytes in CSF furthermore correlated negatively with concentrations of various cytokines, including TNFa. Analysing the functional properties of DICAM showed that LPS-induced TNFa-production in mononuclear phagocytes was effectively reduced by signalling through surface-bound DICAM. This discovery, together with the observation of a high prevalence of infiltrating DICAM + mononuclear phagocytes in individuals with no disease or in which disease was kept under control, suggests an immunomodulatory role of DICAM + mononuclear phagocytes. Also, DICAM can engage in homophilic interaction with DICAM on other cells, suggesting that the increased intrathecal sDICAM of natalizumab-treated patients may help regulate inflammation in a paracrine way. Overall, our data suggest that DICAM + mononuclear phagocytes play a role in controlling neuroinflammation. DICAM neuroinflammation mononuclear phagocytes monocytes macrophages multiple sclerosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Multiple sclerosis (MS) is a chronic, immune-mediated disease of the central nervous system (CNS) with increasing incidence, affecting 2.9 million people worldwide [ 1 ]. Approximately 90% are diagnosed with a relapsing-remitting form of MS (RRMS) characterized by episodic relapses of neurological symptoms. These attacks are mediated by activation of immune cells and their migration to the CNS where they participate in myelin sheath damage resulting in formation of inflammatory demyelinated lesions and neuroaxonal damage [ 2 , 3 ]. Microglia and macrophages are the main innate cells present in MS lesions where they directly or together with T and B cells cause neuroinflammatory tissue damage [ 4 ]. It has been proposed that CNS-resident microglia contribute to clearing of debris in lesion areas and that macrophages originating from blood monocytes are drivers of immunopathology [ 5 ]. In normal, steady-state conditions, blood monocytes also infiltrate the borders of the CNS as part of immune surveillance to monitor infection, malignancy, and tissue damage. Here, infiltrating monocytes differentiate into CNS border-patrolling macrophages which only cross the blood-brain-barrier (BBB) to enter the brain parenchyma in case of damage or pathogen-associated signals [ 5 ]. With this study we investigated cerebrospinal fluid (CSF) cells of monocyte origin expressing dual immunoglobulin domain-containing cell adhesion molecule (DICAM; with the alternative names MXRA8 and limitrin). DICAM is a membrane-bound receptor with proposed BBB-migratory abilities [ 6 ], likely through its homophilic interaction with DICAM and heterophilic interaction with aVb3 on endothelial cells [ 7 ]. Also, DICAM is likely implicated in intracellular signalling pathways affecting various functions of cells including proliferation [ 8 ], angiogenesis [ 9 ], apoptosis [ 9 ] and expression of inflammatory molecules, including IL-1b, TNFa, IL-6 and integrin b1 and b3 [ 10 ]. Studies have furthermore indicated an immunosuppressive role of DICAM [ 10 – 13 ] and implied that DICAM controls the activity of microglia [ 12 ]. The aim of this study was to investigate a possible role of DICAM-expressing cells of monocyte origin during steady state in control subjects without neurological disease and in neuroinflammation in patients with RRMS. As no surface markers to clearly distinguish between a CSF-infiltrating monocyte and a differentiated border-patrolling macrophage have been described, we generally refer to CSF cells of monocyte origin as mononuclear phagocytes. Results DICAM is primarily expressed on CD14 + mononuclear phagocytes in CSF To investigate a possible role of DICAM in neuroinflammation, we collected CSF samples from 14 symptomatic controls, 21 treatment-naïve patients with RRMS, and 12 patients treated with natalizumab, and used flow cytometry to determine the frequency of DICAM + leukocytes (Fig. 1 A-C). This showed that 11% (7.3; 21) of CSF leukocytes expressed DICAM in controls, 4.6% (2.9; 6.2) in untreated patients, and 25% (17; 30) in patients treated with natalizumab; median (IQR). Compared to both symptomatic controls (p = 0.002) and natalizumab-treated patients (p < 0.0001), untreated patients had a significantly lower frequency of DICAM + cells (Fig. 1 D). Furthermore, the majority of DICAM + leukocytes in both controls (87%) and natalizumab-treated patients (93%) were CD14 + mononuclear phagocytes (Fig. 1 D). Of the few DICAM + cells in CSF of treatment-naïve patients, 55% were CD14 + mononuclear phagocytes (Fig. 1 D). CD14 + mononuclear phagocytes are hereafter referred to as mononuclear phagocytes. Low prevalence of intrathecal DICAM mononuclear phagocytes in untreated patients Further analyzing mononuclear phagocytes in the CSF showed that the frequency of mononuclear phagocytes among leukocytes was greatly reduced in untreated patients compared to symptomatic controls (p = 0.001) and natalizumab-treated patients (p < 0.0001); Fig. 2 A. Most untreated patients had CSF pleocytosis (Fig. 2 B) and we observed that the absolute number of mononuclear phagocytes in untreated patients was similar to both symptomatic controls and natalizumab-treated patients (Fig. 2 C). Also, the frequency of mononuclear phagocytes expressing DICAM was lower in untreated patients compared to both symptomatic controls (p = 0.016) and natalizumab-treated patients (p = 0.016); Fig. 2 D. Despite an equal number of intrathecal mononuclear phagocytes in the three groups, the absolute number of DICAM + mononuclear phagocytes in untreated patients were lower compared to both symptomatic controls (p = 0.031) and natalizumab-treated patients (p = 0.028); Fig. 2 E. This difference was further emphasized by the observation that DICAM + mononuclear phagocytes constituted 24% of all leukocytes in CSF of natalizumab-treated patients and 10% in controls, compared to only 1.5% in untreated patients (Fig. 2 F). These observations were confirmed in a smaller reproduction cohort (Suppl. Figure 1). Altogether, this showed a decreased prevalence of DICAM + mononuclear phagocytes in CSF of untreated patients with RRMS. Surface phenotype of intrathecal DICAM mononuclear phagocytes To understand the role of intrathecal DICAM + mononuclear phagocytes in neuroinflammation, we used flow cytometry to characterize the surface expression of various proteins associated with the function of mononuclear phagocytes in untreated patients with RRMS. This showed that DICAM + mononuclear phagocytes in CSF predominantly were CD16 + and CCR2 − in contrast to DICAM − mononuclear phagocytes (p < 0.0001); Fig. 3 A-B. Additionally, DICAM + mononuclear phagocytes had an increased expression of CD40 (p < 0.0001), CD86 (p = 0.0001), and CD206 (p < 0.0001) compared to DICAM − mononuclear phagocytes, Fig. 3 C-E. Also, like DICAM − mononuclear phagocytes, 100% of DICAM + mononuclear phagocytes expressed CD49d, which is one of the two subunits of the adhesion molecule Very Late Activation Antigen-4 (VLA-4; data not shown). The same was found in symptomatic controls and natalizumab-treated patients (supplementary Fig. 1). Comparing the phenotype of DICAM + mononuclear phagocytes of untreated patients with symptomatic controls and natalizumab-treated patients showed a similar frequency of intrathecal DICAM + mononuclear phagocytes expressing CD16, CD40, CD86 and CD49d between the three groups (Fig. 3 F, and data not shown). In contrast, the frequency of DICAM + mononuclear phagocytes expressing CCR2 in untreated patients compared to controls and natalizumab-treated patients was increased (p = 0.004 and 0.006) and the expression of CD206 decreased (p = 0.005 and 0.046) (Fig. 3 G-H). Few circulating monocytes express DICAM In the blood, DICAM is expressed on less than 2% of circulating CD14 + monocytes, with no difference between healthy controls (n = 21), symptomatic controls (n = 21), untreated (n = 21) or natalizumab-treated (n = 13) patients with RRMS (Fig. 4 D-E). Subdividing circulating DICAM + monocytes into classical (CD16 − ), intermediate (CD16 + CCR2 + ) and non-classical (CD16 + CCR2 − ) monocytes (Fig. 4 A-C) showed a higher frequency of DICAM + intermediate monocytes in natalizumab-treated patients compared to both symptomatic controls (p = 0.029) and untreated patients (p = 0.035), Fig. 4 F. DICAM is strongly upregulated following monocyte differentiation to macrophages To investigate if the enrichment of DICAM + mononuclear phagocytes observed in the CSF was due to selective recruitment of DICAM + monocytes from the blood, differentiation of recruited monocytes to CNS border-patrolling macrophages or an effect of the local intrathecal microenvironment, monocytes from healthy controls were purified and cultured under macrophage differentiating conditions for 6 days. Thereafter, macrophages were grown in medium or CSF from either symptomatic control subjects (n = 14), untreated patients (n = 14), or natalizumab-treated patients (n = 14) for an additional 2 days and DICAM expression was measured (Fig. 4 G-H). Following macrophage differentiation, we observed a strong increase in DICAM + cells to a mean of 26% (dotted line in Fig. 4 I). This increase was marginally elevated when cells were cultured in CSF; however, not significantly and all CSF-samples affected the expression of DICAM equally (Fig. 4 I). The experiment was repeated, and the same observations obtained (data not shown). Soluble DICAM in CSF is increased in natalizumab-treated patients and correlate negatively with inflammation biomarkers In addition to a membrane bound form, DICAM exists in a soluble form (sDICAM). To investigate if sDICAM is present in CSF and whether there is a difference between patients and symptomatic controls, we measured sDICAM in CSF from 41 symptomatic controls, 43 treatment-naïve and 42 natalizumab-treated patients by ELISA. sDICAM could be detected in all CSF samples analyzed. The analysis showed higher sDICAM concentrations in natalizumab-treated patients compared to both untreated patients (p = 0.0008) and symptomatic controls (p = 0.015), Fig. 5 A. Besides mononuclear phagocytes, a small population of T cells [ 6 ], endothelial cells [ 9 ], and astrocytes [ 12 ] have been shown to express DICAM. The measured sDICAM in CSF is therefore likely of mixed origin; however, we found a positive correlation between sDICAM and the frequency of DICAM + mononuclear phagocytes in CSF of patients with RRMS (p = 0.011, r s =0.40; Fig. 5 B). Also, we found that mononuclear phagocytes stimulated in vitro with LPS for 14 h secreted sDICAM (Fig. 5 C); altogether suggesting that these cells are major contributors to CSF sDICAM. To investigate a clinical implication of DICAM in MS, we analyzed a possible association between sDICAM in CSF of 43 untreated patients with RRMS and the biomarker of neuroaxonal damage neurofilament light chain (NfL) [ 14 ] and the astrocyte activity biomarker glial fibrillary acidic protein (GFAP) [ 15 ]. This showed a weak association between sDICAM and an increased GFAP (p = 0.025; r s =0.35); Fig. 5 D. Further analyzing an association between sDICAM in CSF and various cytokines showed a negative correlation between sDICAM and TNFa (p = 0.014; r s =-0.39), IFNg (p < 0.0001; r s =-0.60), IL-12 (p = 0.0002; r s =-0.55), and IL-10 (p = 0.0004; r s =0.54); Fig. 5 D. To improve a link between these observations and the presence of DICAM + mononuclear phagocytes in CSF, we next analyzed correlations between the frequency of DICAM + mononuclear phagocytes in untreated patients with RRMS and the biomarkers that correlated with sDICAM. Despite a considerably lower sample size in this analysis (n = 18), we found a negative correlation between DICAM + mononuclear phagocytes and TNFa (p = 0.0026; r s =-0.66); Fig. 5 E. DICAM attenuates LPS-induced production of TNFa in mononuclear phagocytes Studies have indicated a role of DICAM in suppressing intracellular signaling pathways [ 9 ] and lipopolysaccharide (LPS)-induced production of proinflammatory cytokines including TNFa [ 10 ]. Based on our data suggesting a relation between DICAM and TNFa in CSF, we investigated DICAM’s direct ability to influence TNFa-production in mononuclear phagocytes. To study this functional property of DICAM, we sorted monocytes into CD14 + DICAM − and CD14 + DICAM + cells using antibodies targeting CD14 and DICAM, at a low temperature to ensure that no signals were induced through the DICAM-receptor by the anti-DICAM antibody during sorting. The two sorted populations were hereafter cultured in 10 ng/ml LPS for 6 h at 37°C allowing anti-DICAM induced signaling through surface expressed DICAM. The culture supernatants were collected, and TNFa-concentration measured. This showed a prompt LPS-induced production of TNFa without triggering of DICAM (Fig. 6 A, LPS); a production that was strongly reduced by anti-DICAM antibody triggering of DICAM (Fig. 6 A; LPS + aDICAM, p = 0.001). The experiment was repeated three times with a total of 7 donors. To validate this finding, the same cocktail of antibodies and LPS was added to freshly drawn CSF-cells from a patient with RRMS, and TNFa production was measured. Likewise, this showed that a signal through DICAM diminished TNFa production (Fig. 6 B). As a previous study described that DICAM accelerated apoptosis in endothelial cells [ 9 ] we measured cell death after 6h LPS-stimulation in the two sorted populations to ensure that the decrease in TNFa-production observed was not simply a result of cell death. This showed that cells in which DICAM was triggered were more sensitive to apoptosis, but not to a large degree (Fig. 6 C-D). Altogether, these data indicate that a functional property of DICAM is to attenuate LPS-induced TNFa production in mononuclear phagocytes. Figure 6 : Discussion With this study we investigated a possible role of DICAM in controlling neuroinflammation. Our findings showed that the primary CSF-resident immune cell to express DICAM was mononuclear phagocytes, including CSF-infiltrating monocytes and differentiated CNS-border patrolling macrophages. Investigating symptomatic controls as a representative of steady-state conditions showed a high prevalence of DICAM + mononuclear phagocytes, both in frequency and numbers, compared to treatment-naïve patients with MS. When patients were treated with natalizumab, an antibody blocking migration of VLA-4 + blood leukocytes to the CNS [ 16 ], we observed that DICAM + monocytes were still recruited to the CSF despite VLA-4 expression and that the frequency and numbers in CSF resembled those observed in the symptomatic controls. The observed decrease in DICAM + mononuclear phagocytes in untreated patients compared to both controls and natalizumab-treated individuals with no disease activity may indicate a regulatory role of this cell subset. The idea of DICAM as a mechanism to control inflammation is in line with previous studies showing that DICAM has a protective role in the mouse model of experimental autoimmune optic neuritis [ 13 ] and experimental colitis [ 11 ], that DICAM can protect against renal tubular injury through its anti-inflammatory properties [ 10 ], and that DICAM is involved in cancer-related signaling pathways promoting tumor malignancy and tumor immune dysfunction [ 17 ]. Furthermore, a study described how DICAM secreted in extracellular vesicles during neuroinflammation had the potential to control the activity of microglia, a mononuclear phagocyte of CNS origin, and overall neuroinflammation [ 12 ]. The mode of DICAM to control inflammation has not been thoroughly investigated; however, signals through surface-expressed DICAM likely suppress activation-induced intracellular signaling events of the AKT and p38 MAP kinase signaling pathways [ 9 ]. DICAM-triggering reduces production of both integrins and proinflammatory cytokines including TNFa, IL-1b, and IL-6 [ 10 ], and increases the level of occludin [ 10 ] and stabilizes barrier function [ 11 ]. In coherence, we found that triggering of surface expressed DICAM on mononuclear phagocytes during LPS-stimulation effectively reduced the production of TNFa. DICAM has been shown to interact homophilic with DICAM and heterophilic with aVb3 [ 7 ], but other ligands likely exist. Our observation of soluble DICAM in CSF and the ability of mononuclear phagocytes to secrete DICAM, along with a study documenting astrocyte secretion of DICAM in extracellular vesicles [ 12 ] may imply a regulatory role of DICAM distinct from the site where it was produced. In support of this notion, we observed an increased level of sDICAM in CSF of natalizumab-treated patients, possibly contributing to the attenuation of inflammation observed in these patients; and furthermore, we found a negative correlation between the concentration of sDICAM and TNFa, IFNg, IL-12, and IL-10 in CSF of patients with MS. This finding was substantiated by a negative correlation between DICAM + mononuclear phagocytes and TNFa in CSF. Also, we observed a tendency to a negative correlation with IFNg and IL-12; however, likely due to lack of power (n = 18 versus n = 43 in the sDICAM analysis) this was non-significant. In the blood, only few monocytes expressing DICAM were found (< 2% versus approximately 70% in CSF). We furthermore observed that DICAM was strongly upregulated in vitro following differentiation of monocytes to macrophages; suggesting DICAM to be a macrophage marker. It is therefore possible that circulating DICAM + mononuclear phagocytes represent a small population of macrophages rather than undifferentiated monocytes. DICAM has previously been suggested to be an adhesion molecule on a small subtype of inflammatory T cells, Th17 cells, involved in CNS-migration independent of the otherwise mandatory VLA-4 [ 6 ]. According to this and our observation that mononuclear phagocytes are still able to transmigrate to CNS despite natalizumab treatment, indicates that selective recruitment of the peripheral pool of DICAM-expressing mononuclear phagocytes to the CNS is possible, although the majority of intrathecal DICAM + mononuclear phagocytes likely are generated upon differentiation from infiltrated monocytes to CNS border-patrolling macrophages. Depending on the cytokine milieu in CNS, infiltrating monocytes are capable of adopting either a pro- or anti-inflammatory macrophage phenotype, a phenomenon of key relevance in MS where the pro-inflammatory state dominates [ 18 ]. As we observed a decreased prevalence of DICAM + mononuclear phagocytes in CSF of untreated patients with MS, we investigated the impact of the CSF-environment on DICAM expression. For this, we cultured in vitro differentiated macrophages in cell-free CSF from either symptomatic control subjects, untreated or natalizumab-treated patients and found no significant change in DICAM surface-expression following CSF incubation, and also not between the three groups. The local CNS-environment mirrored by cell-free CSF may therefore not be the key factor of DICAM upregulation in differentiating macrophages. Phenotypically, intrathecal DICAM + mononuclear phagocytes are predominantly CD16 + and CCR2 − . Additionally, a high frequency of DICAM + mononuclear phagocytes in CSF express CD40, CD86 and CD206. Where CD206 is considered representative of macrophage polarization towards an anti-inflammatory and homeostatic phenotype, CD40 and CD86 are associated with a pro-inflammatory state where TNFa, IFNg, and IL-12 are induced and inflammatory Th cells are primed [ 19 ]. However, we observe a negative correlation between these three cytokines and DICAM + mononuclear phagocytes; one could therefore speculate that DICAM + mononuclear phagocytes may use CD40 and CD86 to interact with inflammatory T cells to control their activity through DICAM-induced signals in the target cell. Altogether, our study indicates a protective immunomodulatory role of DICAM + mononuclear phagocytes in neuroinflammation in multiple sclerosis and proposes that the regulatory effect is directly delivered by DICAM. Soluble DICAM or vesicles containing DICAM may therefore represent a target to advance clinical intervention in MS and related diseases. Materials and methods Study population, protocol approvals and ethics In this observational case-control study, we included CSF cell samples from 14 symptomatic control subjects, 21 treatment-naïve patients with RRMS, 12 patients treated with natalizumab, and blood cell samples from 21 healthy controls, 21 symptomatic controls, 21 treatment-naïve patients with RRMS, and 13 patients treated with natalizumab. There was no significant difference in sex or age distribution between the groups. Furthermore, cell-free CSF samples from 41 symptomatic controls, 43 treatment-naïve patients with RRMS and 42 natalizumab-treated patients were included. There was no significant difference in sex or age distribution between the groups except a higher age of natalizumab-treated patients compared to untreated patients. Symptomatic control subjects were defined as patients with symptoms warranting a lumbar puncture but with no indication of inflammation or neurological disorder after diagnostic work-up [ 20 ]. All patients were diagnosed with RRMS fulfilled the 2017 McDonald criteria [ 21 ] and none had received methylprednisolone treatment within one month prior to sampling. All treatment-naïve patients with RRMS were diagnosed at the time of lumbar puncture and had a disease duration of 10 months (3; 25); median (IQR). Patients treated with natalizumab had been treated for 38 weeks (12; 97) at sampling time, and time since last treatment was 27 days (19; 44), median (IQR). Of the 42 natalizumab-treated patients, 4 switched to another treatment at sampling time due to disease activity. Healthy controls had no neurological, autoimmune, or chronic illness. CSF cell samples from a smaller reproduction cohort of 6 symptomatic controls, 8 untreated patients, and 7 natalizumab-treated patients with RRMS was further included. All participants gave informed, written consent to participation. The study was approved by the regional scientific ethics committee (protocol number H-17005703 and H-16047666). Blood and CSF samples Venous blood was collected, and peripheral blood mononuclear cells (PBMC) isolated by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway), washed twice in cold PBS/2 mM EDTA and applied directly to ex vivo flow cytometry or in vitro assays. In parallel with blood sampling, 10 ml of CSF was collected in a polypropylene tube on ice and immediately centrifuged for 10 min at 400 g to separate cells from fluid. Cell-free CSF was kept at -80°C until use and CSF cells applied to flow cytometry within one hour of sampling. PBMC were manually counted using a Neubauer chamber, and CSF cell counts were measured during routine assessment with counts below 3 cells/µl reported as 2 cells/µl. CSF cell count of natalizumab-treated patients below 3 cells/µl was reported as 1 cell/µl according to literature [ 22 ]. Flow cytometry analysis For ex vivo flow cytometry, freshly isolated PBMC and CSF cells were incubated in FcR-blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) to prevent nonspecific Ab binding, and thereafter stained in PBS/2% FBS/0.02% NaN 3 with a combination of fluorochrome-conjugated antibodies against (conjugate; clone): CD14 (BV605; M5E2), CD16 (PerCP; 3G8), CCR2 (BV421; K036C2), CD40 (APC-Cy7; 5C3), CD49d (APC; 9F10), CD86 (PE-Cy7; 2D10), CD206 (PE; 15 − 2) all from BioLegend (CA, USA). DICAM (unconjugated; 2H2G12A) from MBL International Corporation (USA, IL) was conjugated using the fast AF488-conjugation kit from Abcam (Cambridge, UK), according to manufacturer. Isotype matched controls were used to correct for nonspecific Ab binding and spectral overlap, where appropriate. For in vitro flow cytometry a live-dead stain (Thermo Fisher Scientific, MA, USA) was included in the staining procedure. TruCount staining of whole blood to measure absolute cell count was performed using BD Multitest 6-color TBNK Reagent including a CD14 antibody (BV605; M5E2), according to manufacturer (BD Biosciences, CA, USA). Data were acquired on a FACSymphony or FACS Canto II flow cytometer (BD Biosciences) and data analysis performed using the software FlowJo (TreeStar, OR, USA). In vitro macrophage differentiation PBMC were purified from healthy donors and monocytes negatively selected using the human monocyte enrichment kit without CD16 depletion from StemCell Technologies (Vancouver, Canada), according to manufacturer. The purity of monocytes was measured by flow cytometry of cells stained with anti-CD14 antibodies (APC-Cy7; M5E2) and live-dead stain (Thermo Fisher Scientific). Hereafter, cells were resuspended in monocyte attachment media (Sigma-Aldrich; MO, USA) at a concentration of 1×10 6 monocytes/ml and 200.000 monocytes plated in each well of a 96 wells flat bottom plate. After 1 h at 37°C, non-adherent cells were washed off, and medium changed to 100 ng/ml M-CSF (R&D Systems; MN, USA)/macrophage base medium DXF (Sigma-Aldrich), and cells incubated at 37°C, 5% CO 2 for 5 days. The growth medium was then replaced by fresh medium and cells incubated an additional 24 h. Hereafter, cell-free CSF was thawed and centrifuged at 3000g, 10 min to pellet any cell debris. The growth medium was removed from the cells and replaced by fresh growth medium with/without CSF from one of the 42 donors at a concentration of 37.5%. CSF was used from 14 symptomatic controls, 14 treatment-naïve patients with RRMS and 14 patients treated with natalizumab. After 48 h the cells were detached using accutase (Sigma-Aldrich) and analyzed by flow cytometry to measure expression of DICAM. For this, cells were stained with FcR-blocking reagent, DICAM-AF488 and live-dead stain as described in the flow cytometry analysis section above. In vitro monocyte secretion of sDICAM Monocytes were purified as described in the in vitro macrophage differentiation section and plated at a concentration of 1×10 6 monocytes/ml in RPMI with 1-1000 ng/ml LPS (InvivoGen, CA, USA). After 14 h at 37°C, 5% CO 2 the supernatant was applied to ELISA-measurement of sDICAM (human MXRA8-ELISA; MyBioSource, CA, USA), according to manufacturer. TNFa suppression assay Monocytes were purified from PBMC using the human monocyte enrichment kit without CD16 depletion (StemCell Technologies), and stained with antibodies targeting DICAM (AF488; 2H2G12A) and CD14 (APC-Cy7; M5E2) in PBS/2 mM EDTA/2% FBS and 9000 CD14 + DICAM − and 9000 CD14 + DICAM + cells sorted on a BD Melody FACS at 4°C. Cells were hereafter cultured with 10 ng/ml LPS (InvivoGen) for 6h at 37°C, 5% CO 2 and the supernatant applied to ELISA-measurement of TNFa (DuoSet from R&D Systems). The experiment was repeated using freshly drawn CSF-cells that was likewise incubated with DICAM and CD14 antibodies and 10 ng/ml LPS at 37°C, 5% CO 2 . Biomarker analysis Biomarker analysis was performed on cell-free CSF samples from 41 symptomatic controls, 43 untreated patients with RRMS and 42 natalizumab-treated patients. For measurement of IL-8, IL-10, IL-12, IL-15, TNFa, and IFNg electrochemiluminescence assays (Vplex kits) from Meso Scale Diagnostics (MD, USA) was used according to manufacturer. The maximum lower level of detection across plates measured for each analyte was used as a cut-off for detection. The limit of quantification was defined by the lowest standard with a CV below 25%, samples below this limit was assigned a value of 0. Samples were measured in duplicates and excluded if CV exceeded 20%, or 25% if the value was within the two lowest accepted standard. ELISA was used to measure CSF concentrations of sDICAM (MXRA8-ELISA from MyBioSource), NfL (Uman Diagnostics, Umeå, Sweden), and GFAP (SIMOA technology; Quanterix, MA, USA), according to manufacturer. NfL and GFAP were measured in duplicates. Statistical analysis For analysis of sex differences between groups a Chi-square test was performed, and for analysis of age differences between groups a Kruskal-Wallis test was applied. To compare cell frequencies or numbers between healthy controls, symptomatic controls, untreated and natalizumab-treated patients with RRMS, a Kruskal-Wallis and post-hoc Dunn’s test corrected for multiple comparisons was used. For comparison of DICAM + and DICAM − cell populations a Wilcoxon test was used. Correlations were assessed by Spearman rank correlation analysis. P < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 10.1. Abbreviations BBB = blood-brain-barrier, CFS = cerebrospinal fluid, CNS = central nervous system, DICAM = dual immunoglobulin domain-containing cell adhesion molecules, GFAP = glial fibrillary acidic protein, IFNg = interferon gamma, IL = interleukin, LPS = lipopolysaccharide, MS = multiple sclerosis, NfL = neurofilament light chain, RRMS = relapsing-remitting MS, sDICAM = soluble DICAM, TNFa = tumor necrosis factor alpha, VLA-4 = very late activation antigen-4 Declarations Ethics approval and consent to participate All participants gave informed, written consent to participation. The study was approved by the regional scientific ethics committee (protocol number H-17005703 and H-16047666). Consent to publication All authors consented to publication of the manuscript. Data availability Data are available in anonymized form and can be shared by request from any qualified investigator. Sharing requires approval of a data transfer agreement in accordance with GDPR and Danish data protection regulation. Competing interest M.E received speaker honoraria from Merck. M.M.H received non-financial support for conference participation from Merck and Sanofi. SEM has received speaker honoraria and non-financial support for conference participation from Merck. V.H.H. and M.B.H. declares no competing interest. S.B. received non-financial support for conference participation from Merck. F.S. has served on scientific advisory boards for, served as consultant for, received support for congress participation or received speaker honoraria from Alexion, Biogen, Bristol Myers Squibb, H. Lundbeck A/S, Merck, Novartis, Roche and Sanofi Genzyme. His laboratory has received research support from Biogen, Merck, Novartis, Roche and Sanofi Genzyme. Funding This work was supported by The Aase and Ejnar Danielsens Foundation, Johnsen and wife´s Foundation, and Simon Fougner Hartmanns family Foundation. Author’s contribution M.E. performed the experiments, analyzed data, conceptualized the research, and wrote the manuscript. M.M.H., S.M., V.H.H., M.B.H. and S.B. collected the patient material, performed the experiments, and analyzed data. F.S contributed to analyses of data and conceptualized the research. Acknowledgement We acknowledge Lisbeth Stolpe at the Danish Multiple Sclerosis Center for her excellent technical assistance. References Koch-Henriksen N, Magyari M. Apparent changes in the epidemiology and severity of multiple sclerosis. Nat Rev Neurol. 2021;17:676-688. Lublin FD, Reingold SC, Cohen JA, Cutter GR, Sorensen PS, Thompson AJ, et al. Defining the clinical course of multiple sclerosis: the 2013 revisions. Neurology 2014;83:278-286. Dendrou CA, Fugger L, Friese MA. Immunopathology of multiple sclerosis. 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DICAM inhibits angiogenesis via suppression of AKT and p38 MAP kinase signalling. Cardiovasc Res. 2013;98:73-82. Han J, Yook JM, Oh SH, Chung YK, Jung HY, Choi JY, et al. Dual Immunoglobulin Domain-Containing Cell Adhesion Molecule Increases Early in Renal Tubular Cell Injury and Plays Anti-Inflammatory Role. Curr Issues Mol Biol 2024;46:1757-1767. Han SW, Kim JM, Lho Y, Cho HJ, Jung YK, Kim JA, et al. DICAM Attenuates Experimental Colitis via Stabilizing Junctional Complex in Mucosal Barrier. Inflamm Bowel Dis 2019;25:853-861. Han J, Cho HJ, Park D, Han S. DICAM in the Extracellular Vesicles from Astrocytes Attenuates Microglia Activation and Neuroinflammation. Cells. 2022;11:2977. Chun BY, Kim JH, Jung YK, Choi YS, Kim G, Yonezawa T, et al. Protective Role of Limitrin in Experimental Autoimmune Optic Neuritis. Invest Ophthalmol Vis Sci 2021;62:8. Freedman MS, Gnanapavan S, Booth RA, Calabresi PA, Khalil M, Kuhle J, et al. Guidance for use of neurofilament light chain as a cerebrospinal fluid and blood biomarker in multiple sclerosis management. EBioMedicine 2024;101:104970. Park Y, Kc N, Paneque A, Cole PD. Tau, Glial Fibrillary Acidic Protein, and Neurofilament Light Chain as Brain Protein Biomarkers in Cerebrospinal Fluid and Blood for Diagnosis of Neurobiological Diseases. Int J Mol Sci 2024;25: Brandstadter R, Katz Sand I. The use of natalizumab for multiple sclerosis. Neuropsychiatr Dis Treat 2017;13:1691-1702. Tan L, Fu D, Liu F, Liu J, Zhang Y, Li X, et al. MXRA8 is an immune-relative prognostic biomarker associated with metastasis and CD8(+) T cell infiltration in colorectal cancer. Front Oncol 2022;12:1094612. Nally FK, De Santi C, McCoy CE. Nanomodulation of Macrophages in Multiple Sclerosis. Cells. 2019;8:543. Cutolo M, Campitiello R, Gotelli E, Soldano S. The Role of M1/M2 Macrophage Polarization in Rheumatoid Arthritis Synovitis. Front Immunol 2022;13:867260. Teunissen C, Menge T, Altintas A, Álvarez-Cermeño JC, Bertolotto A, Berven FS, et al. Consensus definitions and application guidelines for control groups in cerebrospinal fluid biomarker studies in multiple sclerosis. Mult Scler 2013;19:1802-1809. Thompson AJ, Banwell BL, Barkhof F, Carroll WM, Coetzee T, Comi G, et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria. Lancet Neurol. 2018;17:162-173. Stüve O, Marra CM, Jerome KR, Cook L, Cravens PD, Cepok S, et al. Immune surveillance in multiple sclerosis patients treated with natalizumab. Ann Neurol. 2006;59:743-747. Additional Declarations No competing interests reported. Supplementary Files Supplfigure1JNeuroinflamm.tif Suppl. Fig. 1 Surface phenotype of intrathecal DICAM + mononuclear phagocytes from symptomatic controls and natalizumab-treated patients with RRMS A-E. Frequency of CD16 + (A), CCR2 + (B), CD40 + (C), CD86 + (D), and CD206 + (E) DICAM + and DICAM - mononuclear phagocytes in CSF of 13 symptomatic controls (SC). F-J. Frequency of CD16 + (F), CCR2 + (G), CD40 + (H), CD86 + (I), and CD206 + (J) DICAM + and DICAM - mononuclear phagocytes in CSF of 12 natalizumab-treated patients with RRMS (NAT). Median value is shown for all groups analyzed. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5403219","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381031964,"identity":"9f58eef5-fdce-4203-a7ba-ac96573c3788","order_by":0,"name":"Marina Rode von Essen","email":"data:image/png;base64,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","orcid":"","institution":"Copenhagen University Hospital – Rigshospitalet","correspondingAuthor":true,"prefix":"","firstName":"Marina","middleName":"Rode","lastName":"von Essen","suffix":""},{"id":381031965,"identity":"7ce18683-b015-4258-b2a8-fc3ab28f4e3f","order_by":1,"name":"Marie Mathilde Hansen","email":"","orcid":"","institution":"Copenhagen University Hospital – Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"Mathilde","lastName":"Hansen","suffix":""},{"id":381031966,"identity":"b91f3afa-9580-49e7-9b87-6086bd67b10a","order_by":2,"name":"Sahla El Mahdaoui","email":"","orcid":"","institution":"Copenhagen University Hospital – Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Sahla","middleName":"El","lastName":"Mahdaoui","suffix":""},{"id":381031967,"identity":"79b2789c-da17-412a-b00d-bc4edadadb57","order_by":3,"name":"Victoria Hyslop Hvalkof","email":"","orcid":"","institution":"Copenhagen University Hospital – Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Victoria","middleName":"Hyslop","lastName":"Hvalkof","suffix":""},{"id":381031968,"identity":"d8873a10-0cb1-412c-83f6-7726b48748f6","order_by":4,"name":"Malene Bredahl Hansen","email":"","orcid":"","institution":"Copenhagen University Hospital – Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Malene","middleName":"Bredahl","lastName":"Hansen","suffix":""},{"id":381031969,"identity":"482536a3-ec38-49d7-8c6e-19f4e506a708","order_by":5,"name":"Sophie Buhelt","email":"","orcid":"","institution":"Copenhagen University Hospital – Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Sophie","middleName":"","lastName":"Buhelt","suffix":""},{"id":381031971,"identity":"0bfe32fa-baa6-4b9f-beb8-c28cfdee0b83","order_by":6,"name":"Finn Sellebjerg","email":"","orcid":"","institution":"Copenhagen University Hospital – Rigshospitalet","correspondingAuthor":false,"prefix":"","firstName":"Finn","middleName":"","lastName":"Sellebjerg","suffix":""}],"badges":[],"createdAt":"2024-11-06 13:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5403219/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5403219/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70960841,"identity":"5392d1b8-fbf4-4843-bd51-36bef27990de","added_by":"auto","created_at":"2024-12-09 15:20:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1106069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDICAM is primarily expressed on mononuclear phagocytes in CSF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-C.\u003c/strong\u003e Flow cytometry gating strategy. The CSF leukocyte population was defined (\u003cstrong\u003eA\u003c/strong\u003e), doublet cells excluded, DICAM\u003csup\u003e+\u003c/sup\u003e leukocytes gated (\u003cstrong\u003eB\u003c/strong\u003e), and CD14\u003csup\u003e+\u003c/sup\u003e DICAM\u003csup\u003e+\u003c/sup\u003e cells found (\u003cstrong\u003eC\u003c/strong\u003e). \u003cstrong\u003eD\u003c/strong\u003e. Frequency of DICAM\u003csup\u003e+\u003c/sup\u003e leukocytes in CSF of 14 symptomatic controls (SC), 21 untreated patients with RRMS (UNT) and 12 natalizumab-treated patients with RRMS (NAT). Black bars represent percent CD14\u003csup\u003e+\u003c/sup\u003e DICAM\u003csup\u003e+\u003c/sup\u003e leukocytes, grey bars CD14\u003csup\u003e-\u003c/sup\u003e DICAM\u003csup\u003e+ \u003c/sup\u003eleukocytes.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1JNeuroinflamm.png","url":"https://assets-eu.researchsquare.com/files/rs-5403219/v1/ff8d55e3934fff36d72940ef.png"},{"id":70960839,"identity":"e6d620c6-2f3c-4b26-a856-5be9dc82902d","added_by":"auto","created_at":"2024-12-09 15:20:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1792036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLow prevalence of intrathecal DICAM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mononuclear phagocytes in untreated patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Frequency of mononuclear phagocytes in CSF of 14 symptomatic controls (SC), 21 untreated patients with RRMS (UNT) and 12 natalizumab-treated patients with RRMS (NAT). \u003cstrong\u003eB.\u003c/strong\u003e White blood cell count in CSF. \u003cstrong\u003eC.\u003c/strong\u003e Cell count of mononuclear phagocytes in CSF. \u003cstrong\u003eD-F.\u003c/strong\u003e Frequency and absolute number of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF. Median value is shown for all groups analyzed.\u003c/p\u003e","description":"","filename":"Figure2JNeuroinflamm.png","url":"https://assets-eu.researchsquare.com/files/rs-5403219/v1/71f878271952632ff4338621.png"},{"id":70960840,"identity":"72190499-0434-4365-808b-200131a5a3a8","added_by":"auto","created_at":"2024-12-09 15:20:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1731231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurface phenotype of intrathecal DICAM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mononuclear phagocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-E.\u003c/strong\u003e Frequency of CD16\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eA\u003c/strong\u003e), CCR2\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eB\u003c/strong\u003e), CD40\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eC\u003c/strong\u003e), CD86\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eD\u003c/strong\u003e), and CD206\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eE\u003c/strong\u003e) DICAM\u003csup\u003e+\u003c/sup\u003e and DICAM\u003csup\u003e- \u003c/sup\u003emononuclear phagocytes in CSF of 21 untreated patients with RRMS. \u003cstrong\u003eF-H.\u003c/strong\u003e Frequency of CD16\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eF\u003c/strong\u003e), CCR2\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eG\u003c/strong\u003e), and CD206\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eH\u003c/strong\u003e) DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes of 14 symptomatic controls (Control), 21 untreated patients with RRMS (UNT) and 12 natalizumab-treated patients with RRMS (NAT). Median value is shown for all groups analyzed.\u003c/p\u003e","description":"","filename":"Figure3JNeuroinflamm.png","url":"https://assets-eu.researchsquare.com/files/rs-5403219/v1/ba877d9feee2ee840f9f5b1f.png"},{"id":70960843,"identity":"fb732c4a-06ec-4026-87d3-dfec2c6f85cd","added_by":"auto","created_at":"2024-12-09 15:20:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3760023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDICAM is upregulated following monocyte differentiation to macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-D.\u003c/strong\u003e Flow cytometry gating strategy. The blood leukocyte population was defined (\u003cstrong\u003eA\u003c/strong\u003e), doublet cells excluded, CD14\u003csup\u003e+\u003c/sup\u003eCD16\u003csup\u003e-\u003c/sup\u003e and CD14\u003csup\u003e+\u003c/sup\u003eCD16\u003csup\u003e+\u003c/sup\u003e monocyte populations gated (\u003cstrong\u003eB\u003c/strong\u003e), and classical CD16\u003csup\u003e- \u003c/sup\u003eCCR2\u003csup\u003e+\u003c/sup\u003e, CD16\u003csup\u003e+\u003c/sup\u003e CCR2\u003csup\u003e+\u003c/sup\u003e intermediate, and CD16\u003csup\u003e+\u003c/sup\u003e CCR2\u003csup\u003e-\u003c/sup\u003e non-classical monocyte subpopulations defined (\u003cstrong\u003eC\u003c/strong\u003e), and percent DICAM\u003csup\u003e+\u003c/sup\u003e monocytes found (\u003cstrong\u003eD\u003c/strong\u003e). \u003cstrong\u003eE.\u003c/strong\u003e Frequency of DICAM\u003csup\u003e+\u003c/sup\u003e leukocytes in blood of 21 healthy controls (HC), 21 symptomatic controls (SC), 21 untreated patients with RRMS (UNT) and 13 natalizumab-treated patients with RRMS (NAT).\u003cstrong\u003e F.\u003c/strong\u003e Distribution of circulating DICAM\u003csup\u003e+\u003c/sup\u003e monocyte subsets of untreated and natalizumab-treated patients. \u003cstrong\u003eG.\u003c/strong\u003e Flow cytometry dot plot example showing \u003cem\u003ein vitro\u003c/em\u003e differentiated macrophages. \u003cstrong\u003eH.\u003c/strong\u003e DICAM\u003csup\u003e+\u003c/sup\u003e macrophages after doublet cell and dead cell exclusion, left panel shows macrophages cultured 48 h in medium and right panel macrophages cultured in CSF from natalizumab-treated patients with RRMS. \u003cstrong\u003eI.\u003c/strong\u003e Frequency of DICAM\u003csup\u003e+\u003c/sup\u003e macrophages after 48 h culture in CSF from 14 symptomatic controls (SC), 14 untreated patients with RRMS (UNT) and 14 natalizumab-treated patients with RRMS (NAT). Dotted line shows media frequency of macrophages grown for 48 h in medium with no CSF. Median value is shown for all groups analyzed.\u003c/p\u003e","description":"","filename":"Figure4JNeuroinflamm.png","url":"https://assets-eu.researchsquare.com/files/rs-5403219/v1/739465bfc70a8bc5ed487a7c.png"},{"id":70960838,"identity":"4bcd76e2-001a-4ea7-a577-81f9edd49387","added_by":"auto","created_at":"2024-12-09 15:20:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1985141,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003esDICAM, DICAM+ mononuclear phagocytes and biomarkers in CSF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e sDICAM concentration in CSF of 41 symptomatic controls (SC), 43 untreated patients with RRMS (UNT) and 42 natalizumab-treated patients with RRMS (NAT). Median value is shown for all groups analyzed. \u003cstrong\u003eB.\u003c/strong\u003e Correlation analysis between sDICAM and percent DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF of patients with RRMS (blue circles represents untreated patients, green represents natalizumab treated patients; r\u003csub\u003es\u003c/sub\u003e is the spearman correlation coefficient). \u003cstrong\u003eC. \u003c/strong\u003esDICAM secreted by mononuclear phagocytes of three healthy donors stimulated for 14 h with the indicated concentrations of LPS. \u003cstrong\u003eD.\u003c/strong\u003e Correlation matrix of sDICAM and biomarkers in CSF of 43 untreated patients with RRMS.\u003cstrong\u003e E.\u003c/strong\u003e Correlation matrix of percent DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes and biomarkers in CSF of 18 untreated patients with RRMS. Only biomarkers with a significant correlation to sDICAM was included in the analysis of percent DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes. Correlation coefficients are depicted by colors. * p\u0026gt;0.05; **p\u0026lt;0.005; ***p\u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Figure5JNeuroinflamm.png","url":"https://assets-eu.researchsquare.com/files/rs-5403219/v1/9e2e99883bc3f2c22ae42ab4.png"},{"id":70960844,"identity":"12729f80-6e63-4a98-a4ff-e9a60fca65dc","added_by":"auto","created_at":"2024-12-09 15:20:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1110997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDICAM attenuates production of TNFa\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e TNFa production by monocytes from 7 healthy donors after 6 h of stimulation with 10 ng/ml LPS with or without DICAM triggering by anti-DICAM antibodies. Monocytes stimulated by LPS are referred to as LPS and monocytes stimulated by LPS and anti-DICAM antibody are referred to as LPS+aDICAM on the graph. \u003cstrong\u003eB.\u003c/strong\u003e TNFa production by mononuclear phagocytes in CSF after 6 h of stimulation with 10 ng/ml LPS with or without anti-DICAM antibodies. \u003cstrong\u003eC.\u003c/strong\u003e Flow cytometry dot plot example of cell death analysis of LPS-stimulated monocytes from the healthy donors.\u003c/p\u003e","description":"","filename":"Figure6JNeuroinflamm.png","url":"https://assets-eu.researchsquare.com/files/rs-5403219/v1/5320b2d3eb0c9388c40fad84.png"},{"id":77646539,"identity":"c7557397-7f11-46e9-8971-e1d3e38f5484","added_by":"auto","created_at":"2025-03-04 00:01:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13715198,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5403219/v1/f74ff049-ed9a-4944-b48d-2cb7e2f1e5df.pdf"},{"id":70961273,"identity":"abcbb1b0-d3c2-4fe0-9dc0-29811df4d7b7","added_by":"auto","created_at":"2024-12-09 15:28:45","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":343116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppl. Fig. 1 Surface phenotype of intrathecal DICAM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mononuclear phagocytes from symptomatic controls and natalizumab-treated patients with RRMS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-E.\u003c/strong\u003e Frequency of CD16\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eA\u003c/strong\u003e), CCR2\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eB\u003c/strong\u003e), CD40\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eC\u003c/strong\u003e), CD86\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eD\u003c/strong\u003e), and CD206\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eE\u003c/strong\u003e) DICAM\u003csup\u003e+\u003c/sup\u003e and DICAM\u003csup\u003e- \u003c/sup\u003emononuclear phagocytes in CSF of 13 symptomatic controls (SC). \u003cstrong\u003eF-J.\u003c/strong\u003e Frequency of CD16\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eF\u003c/strong\u003e), CCR2\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eG\u003c/strong\u003e), CD40\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eH\u003c/strong\u003e), CD86\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eI\u003c/strong\u003e), and CD206\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eJ\u003c/strong\u003e) DICAM\u003csup\u003e+\u003c/sup\u003e and DICAM\u003csup\u003e- \u003c/sup\u003emononuclear phagocytes in CSF of 12 natalizumab-treated patients with RRMS (NAT). Median value is shown for all groups analyzed.\u003c/p\u003e","description":"","filename":"Supplfigure1JNeuroinflamm.tif","url":"https://assets-eu.researchsquare.com/files/rs-5403219/v1/b14f2697b8c6660d41babd6c.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"A role for DICAM+ mononuclear phagocytes in controlling neuroinflammation in multiple sclerosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultiple sclerosis (MS) is a chronic, immune-mediated disease of the central nervous system (CNS) with increasing incidence, affecting 2.9\u0026nbsp;million people worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Approximately 90% are diagnosed with a relapsing-remitting form of MS (RRMS) characterized by episodic relapses of neurological symptoms. These attacks are mediated by activation of immune cells and their migration to the CNS where they participate in myelin sheath damage resulting in formation of inflammatory demyelinated lesions and neuroaxonal damage [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Microglia and macrophages are the main innate cells present in MS lesions where they directly or together with T and B cells cause neuroinflammatory tissue damage [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It has been proposed that CNS-resident microglia contribute to clearing of debris in lesion areas and that macrophages originating from blood monocytes are drivers of immunopathology [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn normal, steady-state conditions, blood monocytes also infiltrate the borders of the CNS as part of immune surveillance to monitor infection, malignancy, and tissue damage. Here, infiltrating monocytes differentiate into CNS border-patrolling macrophages which only cross the blood-brain-barrier (BBB) to enter the brain parenchyma in case of damage or pathogen-associated signals [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith this study we investigated cerebrospinal fluid (CSF) cells of monocyte origin expressing dual immunoglobulin domain-containing cell adhesion molecule (DICAM; with the alternative names MXRA8 and limitrin). DICAM is a membrane-bound receptor with proposed BBB-migratory abilities [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], likely through its homophilic interaction with DICAM and heterophilic interaction with aVb3 on endothelial cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Also, DICAM is likely implicated in intracellular signalling pathways affecting various functions of cells including proliferation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], angiogenesis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], apoptosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and expression of inflammatory molecules, including IL-1b, TNFa, IL-6 and integrin b1 and b3 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Studies have furthermore indicated an immunosuppressive role of DICAM [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and implied that DICAM controls the activity of microglia [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The aim of this study was to investigate a possible role of DICAM-expressing cells of monocyte origin during steady state in control subjects without neurological disease and in neuroinflammation in patients with RRMS. As no surface markers to clearly distinguish between a CSF-infiltrating monocyte and a differentiated border-patrolling macrophage have been described, we generally refer to CSF cells of monocyte origin as mononuclear phagocytes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDICAM is primarily expressed on CD14\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF\u003c/h2\u003e \u003cp\u003eTo investigate a possible role of DICAM in neuroinflammation, we collected CSF samples from 14 symptomatic controls, 21 treatment-na\u0026iuml;ve patients with RRMS, and 12 patients treated with natalizumab, and used flow cytometry to determine the frequency of DICAM\u003csup\u003e+\u003c/sup\u003e leukocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). This showed that 11% (7.3; 21) of CSF leukocytes expressed DICAM in controls, 4.6% (2.9; 6.2) in untreated patients, and 25% (17; 30) in patients treated with natalizumab; median (IQR). Compared to both symptomatic controls (p\u0026thinsp;=\u0026thinsp;0.002) and natalizumab-treated patients (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), untreated patients had a significantly lower frequency of DICAM\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Furthermore, the majority of DICAM\u003csup\u003e+\u003c/sup\u003e leukocytes in both controls (87%) and natalizumab-treated patients (93%) were CD14\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Of the few DICAM\u003csup\u003e+\u003c/sup\u003e cells in CSF of treatment-na\u0026iuml;ve patients, 55% were CD14\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). CD14\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes are hereafter referred to as mononuclear phagocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLow prevalence of intrathecal DICAM mononuclear phagocytes in untreated patients\u003c/h3\u003e\n\u003cp\u003eFurther analyzing mononuclear phagocytes in the CSF showed that the frequency of mononuclear phagocytes among leukocytes was greatly reduced in untreated patients compared to symptomatic controls (p\u0026thinsp;=\u0026thinsp;0.001) and natalizumab-treated patients (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001); Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Most untreated patients had CSF pleocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and we observed that the absolute number of mononuclear phagocytes in untreated patients was similar to both symptomatic controls and natalizumab-treated patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlso, the frequency of mononuclear phagocytes expressing DICAM was lower in untreated patients compared to both symptomatic controls (p\u0026thinsp;=\u0026thinsp;0.016) and natalizumab-treated patients (p\u0026thinsp;=\u0026thinsp;0.016); Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD. Despite an equal number of intrathecal mononuclear phagocytes in the three groups, the absolute number of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in untreated patients were lower compared to both symptomatic controls (p\u0026thinsp;=\u0026thinsp;0.031) and natalizumab-treated patients (p\u0026thinsp;=\u0026thinsp;0.028); Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE. This difference was further emphasized by the observation that DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes constituted 24% of all leukocytes in CSF of natalizumab-treated patients and 10% in controls, compared to only 1.5% in untreated patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These observations were confirmed in a smaller reproduction cohort (Suppl. Figure\u0026nbsp;1). Altogether, this showed a decreased prevalence of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF of untreated patients with RRMS.\u003c/p\u003e\n\u003ch3\u003eSurface phenotype of intrathecal DICAM mononuclear phagocytes\u003c/h3\u003e\n\u003cp\u003eTo understand the role of intrathecal DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in neuroinflammation, we used flow cytometry to characterize the surface expression of various proteins associated with the function of mononuclear phagocytes in untreated patients with RRMS. This showed that DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF predominantly were CD16\u003csup\u003e+\u003c/sup\u003e and CCR2\u003csup\u003e\u0026minus;\u003c/sup\u003e in contrast to DICAM\u003csup\u003e\u0026minus;\u003c/sup\u003e mononuclear phagocytes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001); Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B. Additionally, DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes had an increased expression of CD40 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), CD86 (p\u0026thinsp;=\u0026thinsp;0.0001), and CD206 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to DICAM\u003csup\u003e\u0026minus;\u003c/sup\u003e mononuclear phagocytes, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E. Also, like DICAM\u003csup\u003e\u0026minus;\u003c/sup\u003e mononuclear phagocytes, 100% of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes expressed CD49d, which is one of the two subunits of the adhesion molecule Very Late Activation Antigen-4 (VLA-4; data not shown). The same was found in symptomatic controls and natalizumab-treated patients (supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eComparing the phenotype of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes of untreated patients with symptomatic controls and natalizumab-treated patients showed a similar frequency of intrathecal DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes expressing CD16, CD40, CD86 and CD49d between the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, and data not shown). In contrast, the frequency of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes expressing CCR2 in untreated patients compared to controls and natalizumab-treated patients was increased (p\u0026thinsp;=\u0026thinsp;0.004 and 0.006) and the expression of CD206 decreased (p\u0026thinsp;=\u0026thinsp;0.005 and 0.046) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eFew circulating monocytes express DICAM\u003c/h3\u003e\n\u003cp\u003eIn the blood, DICAM is expressed on less than 2% of circulating CD14\u003csup\u003e+\u003c/sup\u003e monocytes, with no difference between healthy controls (n\u0026thinsp;=\u0026thinsp;21), symptomatic controls (n\u0026thinsp;=\u0026thinsp;21), untreated (n\u0026thinsp;=\u0026thinsp;21) or natalizumab-treated (n\u0026thinsp;=\u0026thinsp;13) patients with RRMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E). Subdividing circulating DICAM\u003csup\u003e+\u003c/sup\u003e monocytes into classical (CD16\u003csup\u003e\u0026minus;\u003c/sup\u003e), intermediate (CD16\u003csup\u003e+\u003c/sup\u003eCCR2\u003csup\u003e+\u003c/sup\u003e) and non-classical (CD16\u003csup\u003e+\u003c/sup\u003eCCR2\u003csup\u003e\u0026minus;\u003c/sup\u003e) monocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C) showed a higher frequency of DICAM\u003csup\u003e+\u003c/sup\u003e intermediate monocytes in natalizumab-treated patients compared to both symptomatic controls (p\u0026thinsp;=\u0026thinsp;0.029) and untreated patients (p\u0026thinsp;=\u0026thinsp;0.035), Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDICAM is strongly upregulated following monocyte differentiation to macrophages\u003c/h3\u003e\n\u003cp\u003eTo investigate if the enrichment of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes observed in the CSF was due to selective recruitment of DICAM\u003csup\u003e+\u003c/sup\u003e monocytes from the blood, differentiation of recruited monocytes to CNS border-patrolling macrophages or an effect of the local intrathecal microenvironment, monocytes from healthy controls were purified and cultured under macrophage differentiating conditions for 6 days. Thereafter, macrophages were grown in medium or CSF from either symptomatic control subjects (n\u0026thinsp;=\u0026thinsp;14), untreated patients (n\u0026thinsp;=\u0026thinsp;14), or natalizumab-treated patients (n\u0026thinsp;=\u0026thinsp;14) for an additional 2 days and DICAM expression was measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H). Following macrophage differentiation, we observed a strong increase in DICAM\u003csup\u003e+\u003c/sup\u003e cells to a mean of 26% (dotted line in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). This increase was marginally elevated when cells were cultured in CSF; however, not significantly and all CSF-samples affected the expression of DICAM equally (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). The experiment was repeated, and the same observations obtained (data not shown).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSoluble DICAM in CSF is increased in natalizumab-treated patients and correlate negatively with inflammation biomarkers\u003c/h2\u003e \u003cp\u003eIn addition to a membrane bound form, DICAM exists in a soluble form (sDICAM). To investigate if sDICAM is present in CSF and whether there is a difference between patients and symptomatic controls, we measured sDICAM in CSF from 41 symptomatic controls, 43 treatment-na\u0026iuml;ve and 42 natalizumab-treated patients by ELISA. sDICAM could be detected in all CSF samples analyzed. The analysis showed higher sDICAM concentrations in natalizumab-treated patients compared to both untreated patients (p\u0026thinsp;=\u0026thinsp;0.0008) and symptomatic controls (p\u0026thinsp;=\u0026thinsp;0.015), Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003eBesides mononuclear phagocytes, a small population of T cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], endothelial cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and astrocytes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] have been shown to express DICAM. The measured sDICAM in CSF is therefore likely of mixed origin; however, we found a positive correlation between sDICAM and the frequency of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF of patients with RRMS (p\u0026thinsp;=\u0026thinsp;0.011, r\u003csub\u003es\u003c/sub\u003e=0.40; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Also, we found that mononuclear phagocytes stimulated \u003cem\u003ein vitro\u003c/em\u003e with LPS for 14 h secreted sDICAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC); altogether suggesting that these cells are major contributors to CSF sDICAM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate a clinical implication of DICAM in MS, we analyzed a possible association between sDICAM in CSF of 43 untreated patients with RRMS and the biomarker of neuroaxonal damage neurofilament light chain (NfL) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and the astrocyte activity biomarker glial fibrillary acidic protein (GFAP) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This showed a weak association between sDICAM and an increased GFAP (p\u0026thinsp;=\u0026thinsp;0.025; r\u003csub\u003es\u003c/sub\u003e=0.35); Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD. Further analyzing an association between sDICAM in CSF and various cytokines showed a negative correlation between sDICAM and TNFa (p\u0026thinsp;=\u0026thinsp;0.014; r\u003csub\u003es\u003c/sub\u003e=-0.39), IFNg (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; r\u003csub\u003es\u003c/sub\u003e=-0.60), IL-12 (p\u0026thinsp;=\u0026thinsp;0.0002; r\u003csub\u003es\u003c/sub\u003e=-0.55), and IL-10 (p\u0026thinsp;=\u0026thinsp;0.0004; r\u003csub\u003es\u003c/sub\u003e=0.54); Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD. To improve a link between these observations and the presence of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF, we next analyzed correlations between the frequency of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in untreated patients with RRMS and the biomarkers that correlated with sDICAM. Despite a considerably lower sample size in this analysis (n\u0026thinsp;=\u0026thinsp;18), we found a negative correlation between DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes and TNFa (p\u0026thinsp;=\u0026thinsp;0.0026; r\u003csub\u003es\u003c/sub\u003e=-0.66); Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDICAM attenuates LPS-induced production of TNFa in mononuclear phagocytes\u003c/h3\u003e\n\u003cp\u003eStudies have indicated a role of DICAM in suppressing intracellular signaling pathways [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and lipopolysaccharide (LPS)-induced production of proinflammatory cytokines including TNFa [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Based on our data suggesting a relation between DICAM and TNFa in CSF, we investigated DICAM\u0026rsquo;s direct ability to influence TNFa-production in mononuclear phagocytes. To study this functional property of DICAM, we sorted monocytes into CD14\u003csup\u003e+\u003c/sup\u003eDICAM\u003csup\u003e\u0026minus;\u003c/sup\u003e and CD14\u003csup\u003e+\u003c/sup\u003eDICAM\u003csup\u003e+\u003c/sup\u003e cells using antibodies targeting CD14 and DICAM, at a low temperature to ensure that no signals were induced through the DICAM-receptor by the anti-DICAM antibody during sorting. The two sorted populations were hereafter cultured in 10 ng/ml LPS for 6 h at 37\u0026deg;C allowing anti-DICAM induced signaling through surface expressed DICAM. The culture supernatants were collected, and TNFa-concentration measured. This showed a prompt LPS-induced production of TNFa without triggering of DICAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, LPS); a production that was strongly reduced by anti-DICAM antibody triggering of DICAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA; LPS\u0026thinsp;+\u0026thinsp;aDICAM, p\u0026thinsp;=\u0026thinsp;0.001). The experiment was repeated three times with a total of 7 donors. To validate this finding, the same cocktail of antibodies and LPS was added to freshly drawn CSF-cells from a patient with RRMS, and TNFa production was measured. Likewise, this showed that a signal through DICAM diminished TNFa production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). As a previous study described that DICAM accelerated apoptosis in endothelial cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] we measured cell death after 6h LPS-stimulation in the two sorted populations to ensure that the decrease in TNFa-production observed was not simply a result of cell death. This showed that cells in which DICAM was triggered were more sensitive to apoptosis, but not to a large degree (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). Altogether, these data indicate that a functional property of DICAM is to attenuate LPS-induced TNFa production in mononuclear phagocytes. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e:\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWith this study we investigated a possible role of DICAM in controlling neuroinflammation. Our findings showed that the primary CSF-resident immune cell to express DICAM was mononuclear phagocytes, including CSF-infiltrating monocytes and differentiated CNS-border patrolling macrophages. Investigating symptomatic controls as a representative of steady-state conditions showed a high prevalence of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes, both in frequency and numbers, compared to treatment-na\u0026iuml;ve patients with MS. When patients were treated with natalizumab, an antibody blocking migration of VLA-4\u003csup\u003e+\u003c/sup\u003e blood leukocytes to the CNS [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], we observed that DICAM\u003csup\u003e+\u003c/sup\u003e monocytes were still recruited to the CSF despite VLA-4 expression and that the frequency and numbers in CSF resembled those observed in the symptomatic controls. The observed decrease in DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in untreated patients compared to both controls and natalizumab-treated individuals with no disease activity may indicate a regulatory role of this cell subset. The idea of DICAM as a mechanism to control inflammation is in line with previous studies showing that DICAM has a protective role in the mouse model of experimental autoimmune optic neuritis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and experimental colitis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], that DICAM can protect against renal tubular injury through its anti-inflammatory properties [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and that DICAM is involved in cancer-related signaling pathways promoting tumor malignancy and tumor immune dysfunction [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, a study described how DICAM secreted in extracellular vesicles during neuroinflammation had the potential to control the activity of microglia, a mononuclear phagocyte of CNS origin, and overall neuroinflammation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mode of DICAM to control inflammation has not been thoroughly investigated; however, signals through surface-expressed DICAM likely suppress activation-induced intracellular signaling events of the AKT and p38 MAP kinase signaling pathways [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. DICAM-triggering reduces production of both integrins and proinflammatory cytokines including TNFa, IL-1b, and IL-6 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and increases the level of occludin [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and stabilizes barrier function [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In coherence, we found that triggering of surface expressed DICAM on mononuclear phagocytes during LPS-stimulation effectively reduced the production of TNFa.\u003c/p\u003e \u003cp\u003eDICAM has been shown to interact homophilic with DICAM and heterophilic with aVb3 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], but other ligands likely exist. Our observation of soluble DICAM in CSF and the ability of mononuclear phagocytes to secrete DICAM, along with a study documenting astrocyte secretion of DICAM in extracellular vesicles [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] may imply a regulatory role of DICAM distinct from the site where it was produced. In support of this notion, we observed an increased level of sDICAM in CSF of natalizumab-treated patients, possibly contributing to the attenuation of inflammation observed in these patients; and furthermore, we found a negative correlation between the concentration of sDICAM and TNFa, IFNg, IL-12, and IL-10 in CSF of patients with MS. This finding was substantiated by a negative correlation between DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes and TNFa in CSF. Also, we observed a tendency to a negative correlation with IFNg and IL-12; however, likely due to lack of power (n\u0026thinsp;=\u0026thinsp;18 versus n\u0026thinsp;=\u0026thinsp;43 in the sDICAM analysis) this was non-significant.\u003c/p\u003e \u003cp\u003eIn the blood, only few monocytes expressing DICAM were found (\u0026lt;\u0026thinsp;2% versus approximately 70% in CSF). We furthermore observed that DICAM was strongly upregulated \u003cem\u003ein vitro\u003c/em\u003e following differentiation of monocytes to macrophages; suggesting DICAM to be a macrophage marker. It is therefore possible that circulating DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes represent a small population of macrophages rather than undifferentiated monocytes. DICAM has previously been suggested to be an adhesion molecule on a small subtype of inflammatory T cells, Th17 cells, involved in CNS-migration independent of the otherwise mandatory VLA-4 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. According to this and our observation that mononuclear phagocytes are still able to transmigrate to CNS despite natalizumab treatment, indicates that selective recruitment of the peripheral pool of DICAM-expressing mononuclear phagocytes to the CNS is possible, although the majority of intrathecal DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes likely are generated upon differentiation from infiltrated monocytes to CNS border-patrolling macrophages.\u003c/p\u003e \u003cp\u003eDepending on the cytokine milieu in CNS, infiltrating monocytes are capable of adopting either a pro- or anti-inflammatory macrophage phenotype, a phenomenon of key relevance in MS where the pro-inflammatory state dominates [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As we observed a decreased prevalence of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF of untreated patients with MS, we investigated the impact of the CSF-environment on DICAM expression. For this, we cultured \u003cem\u003ein vitro\u003c/em\u003e differentiated macrophages in cell-free CSF from either symptomatic control subjects, untreated or natalizumab-treated patients and found no significant change in DICAM surface-expression following CSF incubation, and also not between the three groups. The local CNS-environment mirrored by cell-free CSF may therefore not be the key factor of DICAM upregulation in differentiating macrophages.\u003c/p\u003e \u003cp\u003ePhenotypically, intrathecal DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes are predominantly CD16\u003csup\u003e+\u003c/sup\u003e and CCR2\u003csup\u003e\u0026minus;\u003c/sup\u003e. Additionally, a high frequency of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF express CD40, CD86 and CD206. Where CD206 is considered representative of macrophage polarization towards an anti-inflammatory and homeostatic phenotype, CD40 and CD86 are associated with a pro-inflammatory state where TNFa, IFNg, and IL-12 are induced and inflammatory Th cells are primed [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, we observe a negative correlation between these three cytokines and DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes; one could therefore speculate that DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes may use CD40 and CD86 to interact with inflammatory T cells to control their activity through DICAM-induced signals in the target cell.\u003c/p\u003e \u003cp\u003eAltogether, our study indicates a protective immunomodulatory role of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in neuroinflammation in multiple sclerosis and proposes that the regulatory effect is directly delivered by DICAM. Soluble DICAM or vesicles containing DICAM may therefore represent a target to advance clinical intervention in MS and related diseases.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStudy population, protocol approvals and ethics\u003c/h2\u003e \u003cp\u003eIn this observational case-control study, we included CSF cell samples from 14 symptomatic control subjects, 21 treatment-na\u0026iuml;ve patients with RRMS, 12 patients treated with natalizumab, and blood cell samples from 21 healthy controls, 21 symptomatic controls, 21 treatment-na\u0026iuml;ve patients with RRMS, and 13 patients treated with natalizumab. There was no significant difference in sex or age distribution between the groups. Furthermore, cell-free CSF samples from 41 symptomatic controls, 43 treatment-na\u0026iuml;ve patients with RRMS and 42 natalizumab-treated patients were included. There was no significant difference in sex or age distribution between the groups except a higher age of natalizumab-treated patients compared to untreated patients. Symptomatic control subjects were defined as patients with symptoms warranting a lumbar puncture but with no indication of inflammation or neurological disorder after diagnostic work-up [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All patients were diagnosed with RRMS fulfilled the 2017 McDonald criteria [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and none had received methylprednisolone treatment within one month prior to sampling. All treatment-na\u0026iuml;ve patients with RRMS were diagnosed at the time of lumbar puncture and had a disease duration of 10 months (3; 25); median (IQR). Patients treated with natalizumab had been treated for 38 weeks (12; 97) at sampling time, and time since last treatment was 27 days (19; 44), median (IQR). Of the 42 natalizumab-treated patients, 4 switched to another treatment at sampling time due to disease activity. Healthy controls had no neurological, autoimmune, or chronic illness.\u003c/p\u003e \u003cp\u003eCSF cell samples from a smaller reproduction cohort of 6 symptomatic controls, 8 untreated patients, and 7 natalizumab-treated patients with RRMS was further included.\u003c/p\u003e \u003cp\u003e All participants gave informed, written consent to participation. The study was approved by the regional scientific ethics committee (protocol number H-17005703 and H-16047666).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBlood and CSF samples\u003c/h2\u003e \u003cp\u003eVenous blood was collected, and peripheral blood mononuclear cells (PBMC) isolated by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway), washed twice in cold PBS/2 mM EDTA and applied directly to \u003cem\u003eex vivo\u003c/em\u003e flow cytometry or \u003cem\u003ein vitro\u003c/em\u003e assays. In parallel with blood sampling, 10 ml of CSF was collected in a polypropylene tube on ice and immediately centrifuged for 10 min at 400 g to separate cells from fluid. Cell-free CSF was kept at -80\u0026deg;C until use and CSF cells applied to flow cytometry within one hour of sampling. PBMC were manually counted using a Neubauer chamber, and CSF cell counts were measured during routine assessment with counts below 3 cells/\u0026micro;l reported as 2 cells/\u0026micro;l. CSF cell count of natalizumab-treated patients below 3 cells/\u0026micro;l was reported as 1 cell/\u0026micro;l according to literature [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eFor \u003cem\u003eex vivo\u003c/em\u003e flow cytometry, freshly isolated PBMC and CSF cells were incubated in FcR-blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) to prevent nonspecific Ab binding, and thereafter stained in PBS/2% FBS/0.02% NaN\u003csub\u003e3\u003c/sub\u003e with a combination of fluorochrome-conjugated antibodies against (conjugate; clone): CD14 (BV605; M5E2), CD16 (PerCP; 3G8), CCR2 (BV421; K036C2), CD40 (APC-Cy7; 5C3), CD49d (APC; 9F10), CD86 (PE-Cy7; 2D10), CD206 (PE; 15\u0026thinsp;\u0026minus;\u0026thinsp;2) all from BioLegend (CA, USA). DICAM (unconjugated; 2H2G12A) from MBL International Corporation (USA, IL) was conjugated using the fast AF488-conjugation kit from Abcam (Cambridge, UK), according to manufacturer. Isotype matched controls were used to correct for nonspecific Ab binding and spectral overlap, where appropriate. For \u003cem\u003ein vitro\u003c/em\u003e flow cytometry a live-dead stain (Thermo Fisher Scientific, MA, USA) was included in the staining procedure. TruCount staining of whole blood to measure absolute cell count was performed using BD Multitest 6-color TBNK Reagent including a CD14 antibody (BV605; M5E2), according to manufacturer (BD Biosciences, CA, USA). Data were acquired on a FACSymphony or FACS Canto II flow cytometer (BD Biosciences) and data analysis performed using the software FlowJo (TreeStar, OR, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003emacrophage differentiation\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePBMC were purified from healthy donors and monocytes negatively selected using the human monocyte enrichment kit without CD16 depletion from StemCell Technologies (Vancouver, Canada), according to manufacturer. The purity of monocytes was measured by flow cytometry of cells stained with anti-CD14 antibodies (APC-Cy7; M5E2) and live-dead stain (Thermo Fisher Scientific). Hereafter, cells were resuspended in monocyte attachment media (Sigma-Aldrich; MO, USA) at a concentration of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e monocytes/ml and 200.000 monocytes plated in each well of a 96 wells flat bottom plate. After 1 h at 37\u0026deg;C, non-adherent cells were washed off, and medium changed to 100 ng/ml M-CSF (R\u0026amp;D Systems; MN, USA)/macrophage base medium DXF (Sigma-Aldrich), and cells incubated at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e for 5 days. The growth medium was then replaced by fresh medium and cells incubated an additional 24 h. Hereafter, cell-free CSF was thawed and centrifuged at 3000g, 10 min to pellet any cell debris. The growth medium was removed from the cells and replaced by fresh growth medium with/without CSF from one of the 42 donors at a concentration of 37.5%. CSF was used from 14 symptomatic controls, 14 treatment-na\u0026iuml;ve patients with RRMS and 14 patients treated with natalizumab. After 48 h the cells were detached using accutase (Sigma-Aldrich) and analyzed by flow cytometry to measure expression of DICAM. For this, cells were stained with FcR-blocking reagent, DICAM-AF488 and live-dead stain as described in the \u003cspan refid=\"Sec14\" class=\"InternalRef\"\u003e\u003cem\u003eflow cytometry analysis\u003c/em\u003e\u003c/span\u003e section above.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003emonocyte secretion of sDICAM\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMonocytes were purified as described in the \u003cem\u003ein vitro macrophage differentiation\u003c/em\u003e section and plated at a concentration of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e monocytes/ml in RPMI with 1-1000 ng/ml LPS (InvivoGen, CA, USA). After 14 h at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e the supernatant was applied to ELISA-measurement of sDICAM (human MXRA8-ELISA; MyBioSource, CA, USA), according to manufacturer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTNFa suppression assay\u003c/h2\u003e \u003cp\u003eMonocytes were purified from PBMC using the human monocyte enrichment kit without CD16 depletion (StemCell Technologies), and stained with antibodies targeting DICAM (AF488; 2H2G12A) and CD14 (APC-Cy7; M5E2) in PBS/2 mM EDTA/2% FBS and 9000 CD14\u003csup\u003e+\u003c/sup\u003eDICAM\u003csup\u003e\u0026minus;\u003c/sup\u003e and 9000 CD14\u003csup\u003e+\u003c/sup\u003eDICAM\u003csup\u003e+\u003c/sup\u003e cells sorted on a BD Melody FACS at 4\u0026deg;C. Cells were hereafter cultured with 10 ng/ml LPS (InvivoGen) for 6h at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e and the supernatant applied to ELISA-measurement of TNFa (DuoSet from R\u0026amp;D Systems). The experiment was repeated using freshly drawn CSF-cells that was likewise incubated with DICAM and CD14 antibodies and 10 ng/ml LPS at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBiomarker analysis\u003c/h2\u003e \u003cp\u003eBiomarker analysis was performed on cell-free CSF samples from 41 symptomatic controls, 43 untreated patients with RRMS and 42 natalizumab-treated patients. For measurement of IL-8, IL-10, IL-12, IL-15, TNFa, and IFNg electrochemiluminescence assays (Vplex kits) from Meso Scale Diagnostics (MD, USA) was used according to manufacturer. The maximum lower level of detection across plates measured for each analyte was used as a cut-off for detection. The limit of quantification was defined by the lowest standard with a CV below 25%, samples below this limit was assigned a value of 0. Samples were measured in duplicates and excluded if CV exceeded 20%, or 25% if the value was within the two lowest accepted standard. ELISA was used to measure CSF concentrations of sDICAM (MXRA8-ELISA from MyBioSource), NfL (Uman Diagnostics, Ume\u0026aring;, Sweden), and GFAP (SIMOA technology; Quanterix, MA, USA), according to manufacturer. NfL and GFAP were measured in duplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor analysis of sex differences between groups a Chi-square test was performed, and for analysis of age differences between groups a Kruskal-Wallis test was applied. To compare cell frequencies or numbers between healthy controls, symptomatic controls, untreated and natalizumab-treated patients with RRMS, a Kruskal-Wallis and post-hoc Dunn\u0026rsquo;s test corrected for multiple comparisons was used. For comparison of DICAM\u003csup\u003e+\u003c/sup\u003e and DICAM\u003csup\u003e\u0026minus;\u003c/sup\u003e cell populations a Wilcoxon test was used. Correlations were assessed by Spearman rank correlation analysis. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 10.1.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBBB = blood-brain-barrier, CFS = cerebrospinal fluid, CNS = central nervous system, DICAM =\u0026nbsp;dual immunoglobulin domain-containing cell adhesion molecules, GFAP =\u0026nbsp;glial fibrillary acidic protein,\u0026nbsp;IFNg\u0026nbsp;= interferon gamma, IL = interleukin, LPS =\u0026nbsp;lipopolysaccharide, MS = multiple sclerosis, NfL = neurofilament light chain, RRMS = relapsing-remitting MS, sDICAM = soluble DICAM, TNFa\u0026nbsp;= tumor necrosis factor alpha, VLA-4 = very late activation antigen-4\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants gave informed, written consent to participation. The study was approved by the regional scientific ethics committee (protocol number\u0026nbsp;H-17005703 and H-16047666).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent to publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consented to publication of the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available in anonymized form and can be shared by request from any qualified investigator. Sharing requires approval of a data transfer agreement in accordance with GDPR and Danish data protection regulation.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.E received speaker honoraria from Merck. M.M.H received non-financial support for conference participation from Merck and Sanofi. SEM has received speaker honoraria and non-financial support for conference participation from Merck. V.H.H. and M.B.H. declares no competing interest. S.B. received non-financial support for conference participation from Merck. F.S. has served on scientific advisory boards for, served as consultant for, received support\u0026nbsp;for congress participation or received speaker honoraria from Alexion, Biogen, Bristol Myers Squibb, H. Lundbeck A/S, Merck, Novartis, Roche and Sanofi Genzyme. His laboratory has received research support from Biogen, Merck, Novartis, Roche and Sanofi Genzyme.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;The Aase and Ejnar Danielsens Foundation, Johnsen and wife\u0026acute;s Foundation, and\u0026nbsp;Simon Fougner Hartmanns family Foundation.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.E. performed the experiments, analyzed data, conceptualized the research, and wrote the manuscript. M.M.H., S.M., V.H.H., M.B.H. and S.B. collected the patient material,\u0026nbsp;performed the experiments, and analyzed data. F.S contributed to analyses of data and conceptualized the research.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Lisbeth Stolpe at the Danish Multiple Sclerosis Center for her excellent technical assistance.\u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKoch-Henriksen N, Magyari M. Apparent changes in the epidemiology and severity of multiple sclerosis. Nat Rev Neurol. 2021;17:676-688.\u003c/li\u003e\n\u003cli\u003eLublin FD, Reingold SC, Cohen JA, Cutter GR, Sorensen PS, Thompson AJ, et al. Defining the clinical course of multiple sclerosis: the 2013 revisions. Neurology 2014;83:278-286.\u003c/li\u003e\n\u003cli\u003eDendrou CA, Fugger L, Friese MA. Immunopathology of multiple sclerosis. Nat Rev Immunol 2015;15:545-558.\u003c/li\u003e\n\u003cli\u003eFischer MT, Sharma R, Lim JL, Haider L, Frischer JM, Drexhage J, et al. NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury. Brain 2012;135:886-899.\u003c/li\u003e\n\u003cli\u003eMundt S, Greter M, Becher B. The CNS mononuclear phagocyte system in health and disease. Neuron 2022;110:3497-3512.\u003c/li\u003e\n\u003cli\u003eCharabati M, Grasmuck C, Ghannam S, Bourbonni\u0026egrave;re L, Fournier AP, L\u0026eacute;cuyer MA, et al. DICAM promotes T(H)17 lymphocyte trafficking across the blood-brain barrier during autoimmune neuroinflammation. Sci Transl Med. 2022;14:eabj0473.\u003c/li\u003e\n\u003cli\u003eGadani SP, Kornberg MD. DICAM, a molecular passport for T(H)17 cell entry into the brain. Sci Transl Med 2022;14:eabm7204.\u003c/li\u003e\n\u003cli\u003eHan S, Park HR, Lee EJ, Jang JA, Han MS, Kim GW, et al. Dicam promotes proliferation and maturation of chondrocyte through Indian hedgehog signaling in primary cilia. Osteoarthritis Cartilage 2018;26:945-953.\u003c/li\u003e\n\u003cli\u003eHan SW, Jung YK, Lee EJ, Park HR, Kim GW, Jeong JH, et al. DICAM inhibits angiogenesis via suppression of AKT and p38 MAP kinase signalling. Cardiovasc Res. 2013;98:73-82.\u003c/li\u003e\n\u003cli\u003eHan J, Yook JM, Oh SH, Chung YK, Jung HY, Choi JY, et al. Dual Immunoglobulin Domain-Containing Cell Adhesion Molecule Increases Early in Renal Tubular Cell Injury and Plays Anti-Inflammatory Role. Curr Issues Mol Biol 2024;46:1757-1767.\u003c/li\u003e\n\u003cli\u003eHan SW, Kim JM, Lho Y, Cho HJ, Jung YK, Kim JA, et al. DICAM Attenuates Experimental Colitis via Stabilizing Junctional Complex in Mucosal Barrier. Inflamm Bowel Dis 2019;25:853-861.\u003c/li\u003e\n\u003cli\u003eHan J, Cho HJ, Park D, Han S. DICAM in the Extracellular Vesicles from Astrocytes Attenuates Microglia Activation and Neuroinflammation. Cells. 2022;11:2977.\u003c/li\u003e\n\u003cli\u003eChun BY, Kim JH, Jung YK, Choi YS, Kim G, Yonezawa T, et al. Protective Role of Limitrin in Experimental Autoimmune Optic Neuritis. Invest Ophthalmol Vis Sci 2021;62:8.\u003c/li\u003e\n\u003cli\u003eFreedman MS, Gnanapavan S, Booth RA, Calabresi PA, Khalil M, Kuhle J, et al. Guidance for use of neurofilament light chain as a cerebrospinal fluid and blood biomarker in multiple sclerosis management. EBioMedicine 2024;101:104970.\u003c/li\u003e\n\u003cli\u003ePark Y, Kc N, Paneque A, Cole PD. Tau, Glial Fibrillary Acidic Protein, and Neurofilament Light Chain as Brain Protein Biomarkers in Cerebrospinal Fluid and Blood for Diagnosis of Neurobiological Diseases. Int J Mol Sci 2024;25:\u003c/li\u003e\n\u003cli\u003eBrandstadter R, Katz Sand I. The use of natalizumab for multiple sclerosis. Neuropsychiatr Dis Treat 2017;13:1691-1702.\u003c/li\u003e\n\u003cli\u003eTan L, Fu D, Liu F, Liu J, Zhang Y, Li X, et al. MXRA8 is an immune-relative prognostic biomarker associated with metastasis and CD8(+) T cell infiltration in colorectal cancer. Front Oncol 2022;12:1094612.\u003c/li\u003e\n\u003cli\u003eNally FK, De Santi C, McCoy CE. Nanomodulation of Macrophages in Multiple Sclerosis. Cells. 2019;8:543.\u003c/li\u003e\n\u003cli\u003eCutolo M, Campitiello R, Gotelli E, Soldano S. The Role of M1/M2 Macrophage Polarization in Rheumatoid Arthritis Synovitis. Front Immunol 2022;13:867260.\u003c/li\u003e\n\u003cli\u003eTeunissen C, Menge T, Altintas A, \u0026Aacute;lvarez-Cerme\u0026ntilde;o JC, Bertolotto A, Berven FS, et al. Consensus definitions and application guidelines for control groups in cerebrospinal fluid biomarker studies in multiple sclerosis. Mult Scler 2013;19:1802-1809.\u003c/li\u003e\n\u003cli\u003eThompson AJ, Banwell BL, Barkhof F, Carroll WM, Coetzee T, Comi G, et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria. Lancet Neurol. 2018;17:162-173. \u003c/li\u003e\n\u003cli\u003eSt\u0026uuml;ve O, Marra CM, Jerome KR, Cook L, Cravens PD, Cepok S, et al. Immune surveillance in multiple sclerosis patients treated with natalizumab. Ann Neurol. 2006;59:743-747.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"DICAM, neuroinflammation, mononuclear phagocytes, monocytes, macrophages, multiple sclerosis","lastPublishedDoi":"10.21203/rs.3.rs-5403219/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5403219/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). In MS, CNS-infiltrating monocytes differentiate to tissue resident macrophages which are found in large numbers within the injured areas of the brain where they play a central role in driving disease progression through demyelination and tissue destruction. However, infiltrating monocytes and their derivative macrophages can also serve protective functions. In this study we investigated a possible role of intrathecal mononuclear phagocytes (infiltrating monocytes and macrophages) expressing dual immunoglobulin domain-containing cell adhesion molecule (DICAM) in neuroinflammation. Compared to symptomatic controls, treatment-na\u0026iuml;ve patients with relapsing-remitting MS had a reduced prevalence of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF. When patients were treated with natalizumab, an antibody blocking migration of blood leukocytes to the CNS, we observed that DICAM\u003csup\u003e+\u003c/sup\u003e monocytes were still recruited to the CSF and that the level of soluble DICAM (sDICAM) in CSF was significantly increased compared to untreated patients. sDICAM and the prevalence of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in CSF furthermore correlated negatively with concentrations of various cytokines, including TNFa. Analysing the functional properties of DICAM showed that LPS-induced TNFa-production in mononuclear phagocytes was effectively reduced by signalling through surface-bound DICAM. This discovery, together with the observation of a high prevalence of infiltrating DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes in individuals with no disease or in which disease was kept under control, suggests an immunomodulatory role of DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes. Also, DICAM can engage in homophilic interaction with DICAM on other cells, suggesting that the increased intrathecal sDICAM of natalizumab-treated patients may help regulate inflammation in a paracrine way. Overall, our data suggest that DICAM\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes play a role in controlling neuroinflammation.\u003c/p\u003e","manuscriptTitle":"A role for DICAM+ mononuclear phagocytes in controlling neuroinflammation in multiple sclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-09 15:20:40","doi":"10.21203/rs.3.rs-5403219/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4ff9b08c-769e-448a-a436-077bebde83a8","owner":[],"postedDate":"December 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-03T23:53:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-09 15:20:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5403219","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5403219","identity":"rs-5403219","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}