MS is an inflammatory demyelinating disease characterized by massive infiltration of monocytederived macrophages into the central nervous system (CNS). Infiltrated macrophages, as well as brain-resident activated microglia, produce a variety of cytotoxic factors and cytokines and thereby contribute to CNS damage and associated neurodegeneration. Macrophage depletion in an experimental MS animal model significantly reduces clinical symptoms underscoring the pathogenic role of these cells (1). However, macrophages and microglia also exert neuroprotective properties. Notably, intravenous administration of anti-inflammatory macrophages or microglia reduced clinical signs in the MS animal model experimental autoimmune encephalomyelitis (EAE) (2, 3). A well-known mechanism by which phagocytes promote regeneration is via clearance of myelin debris. Removal of damaged myelin components and apoptotic cells is a requisite for remyelination (4, 5). Although the cellular mechanisms involved in clearance of myelin debris by phagocytes are poorly understood, evidence is emerging that lipoprotein-lipase (LPL), an enzyme involved in lipid-processing, plays an important role during initiation of remyelination (4, 6). Activity of this enzyme is significantly increased in brain tissue of the EAE model at the point

**86**

when clinical symptoms start to decrease and shows to be involved in lipid and lipoprotein uptake in microglia (6). This suggests that LPL-expressing phagocytes might contribute to repair and support remyelination through the clearance and reuptake of lipid debris. Regulation of phagocyte function and activity is under control of astrocytes (7) and recently, our group described the presence of, angiopoietin-like 4 (ANGPTL4), a potent inhibitor of LPL, in astrocytes (8). We here provide evidence for decreased astrocytic expression of ANGPTL4 within active inflammatory MS lesions. We show that the cellular communication between astrocytes and macrophages is necessary for the downregulation of astrocytic ANGPTL4 expression. Furthermore, we show that ANGPTL4-mediated inhibition of LPL activity reduced myelin-lipid uptake by phagocytes, without affecting phagocytosis. Taken together, our findings suggest that the strong reduction in astrocytic ANGPTL4 expression in active demyelinating MS lesions enables LPL-immunopositive phagocytes to adequately clear myelin debris, paving the way for remyelination.

# MATERIALS AND METHODS

#### Immunohistochemistry

Blocks of formalin-fixed paraffin-embedded post-mortem brains samples were obtained from the VUmc MS Centrum Amsterdam and the Netherlands Brain Bank from 12 MS patients and 3 non-neurological controls. Detailed clinical data are summarized in **Table 1**. Sections (5µm) from each block were cut with a microtome. Sections were deparaffinized in xylene and rehydrated through graded alcohol into distilled water. Antigen retrieval was performed using citrate buffer pH 6.0 (0.01M) at 100◦C for 10 min. Sections were incubated overnight with appropriate antibodies (see **Table 2**) in phosphate buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA) and subsequently stained with the EnVision horseradish peroxidase kit (Dako, K4061, Belgium) for 30 min at room temperature and followed by 3,3′diaminobenzidine-tetrachloridedihydrate (DAB). Between incubation steps, the sections were thoroughly washed with PBS. After a short rinse in tap water the sections were incubated with haematoxylin for 1 min and intensely washed with tap water for 10 min. Finally, the sections were dehydrated with ethanol followed by xylene and mounted with Entellan (Merck, #107960, Germany).

For cellular localization sections were incubated overnight with antibodies applied simultaneously at 4◦C. After washing with PBS, secondary antibodies consisting of donkey-anti-mouse Alexa Fluor 488 (1:200, Abcam, ab150105, UK), rabbit-antigoat Alexa Fluor 647 (1:200, Abcam, ab150143, UK), or goatanti-rabbit Alexa Fluor 647 (1:200, Abcam, ab150079, UK) were applied for 1 h at room temperature. Fluorescent preparations were embedded and analysis was performed with a Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems, Heidelberg, Germany). GFAP and ANGPTL4 fluorescence intensity were measured by a blinded observer using ImageJ software (freely available from: U.S. National Institutes of Health, Bethesda, MD, USA). Per slide, 3 independent locations were analyzed. Relative ANGPTL4 intensity to GFAP was calculated by dividing ANGPTL4 intensity over GFAP intensity. Next, intensity was normalized to the relative intensity of ANGPTL4 over GFAP in NAWM slides.

# Human Astrocyte Culture

Human astrocytoma cells (U373) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Life Technologies, #11320033, USA) containing 10% fetal bovine serum (FBS; Biowest, S1810-500, USA), and penicillin/streptomycin (50 mg/ml; Life Technologies, #15140122, USA) in 5% CO2 at 37◦C.

## RNA Isolation and Real-Time Quantitative PCR

RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) according to manufacturer's protocol. mRNA concentration and quality (OD 280/260 ratios of 1.8 or higher) was measured using Nanodrop (Nanodrop Technologies, USA). cDNA syntheses was performed using the Reverse Transcription System kit (Applied Biosystems, #4368814, USA) following manufacture's guidelines. Expression was assessed by quantitative RT-PCR using SYBR Green Power mix (Applied Biosystems, #4367659, USA). All primer sequences are listed in **Table 3**. qPCR reaction was performed using the Step-one (Applied Bioscience, USA) Real-Time PCR System with the following program: 2 min at 50◦C, 10 min at 95◦C and then 40 cycles of 15 s at 95 ◦C and 1 min at 60◦C. mRNA expression levels were quantified using the 2−11CT method as described in (9).

# Myelin Isolation

Myelin was isolated and labeled as described previously (10). In short, human CNS white matter tissue was obtained from three healthy controls. Myelin was isolated by homogenization and sucrose gradient centrifugation after which protein concentration was assessed by the bicinchoninic acid method. Isolated myelin was subsequently labeled with NHS-activated Atto633.

# Human Macrophages Cultures

Human blood monocytes were isolated from buffy coats of healthy donors (Sanquin Blood Bank, The Netherlands) using Ficoll (LymphoprepTM, Axis-Shield, #1114544 Norway) and subsequent Percoll density gradient centrifugation. Monocytes were differentiated into macrophages in culture medium (IMDM, Gibco) containing 10% FBS, penicillin (100IU/ml), streptomycin (50 mg/ml) and 50 ng/ml MCSF, for 6 days at 37◦C, 5% CO2. The alternatively activated macrophages phenotype was induced by culturing M-CSF differentiated macrophages in the presence of 10 ng/ml human IL-4 (ImmunoTools, #13340043, Germany) for 48 h as described before by (11).

# Macrophage/Astrocyte Coculture

Human monocytes were cultured on top of a transwell filter (Corning, #3421, USA) with a pore size of 0.3µm and differentiated into macrophages as described above. Human astrocytoma cells (U373) were plated in a culture plate. After cell adhesion, the transwell filter containing macrophages was transferred to the culture plate containing astrocytes. After 24 h at 37◦C in 5% CO2, the transwell filter containing macrophages

*MS, multiple sclerosis; SP, secondary progressive MS; RR, relapse remitting MS; PP, primary progressive MS; ND, non-determined; NA, non-applicable; M, male; F, female.*

TABLE 2 | Antibody details.

*HLA-DR, human leukocyte antigen-DR; LPL, Lipoprotein-lipase; ANGPTL4, Angiopoietinlike 4; GFAP, glial fibrillary acidic protein; PLP, proteolipid protein; Iba1, ionized calcium binding adaptor molecule 1.*

was discarded and mRNA from astrocytes was isolated as described above.

#### Oil-Red-O Staining

Alternatively activated macrophages were cultured on glass coverslips and fixed with 4%PFA for 10 min. Oil-Red-O stock solution was prepared by dissolving in 0.25 g Oil-Red-O (Sigma-Aldrich, #O0265, USA) in 50 ml isopropanol. Oil-Red-O working solution was prepared by mixing 3 parts Oil-Red-OO stock solution with 2 parts dH2O, followed by filtration. Coverslips were immersed in Oil-Red-O working solution for 10 min, followed by two rinses with 60% isopropanol and one rinse with dH2O. Nuclear staining was performed using Haematoxylin for 5 min followed by several rinses with dH2O. Quantification of Oil-Red-O staining was performed by extracting Oil-Red-O using 100% isopropanol for 5 min. Absorbance was subsequently measuring at 492 nm. 100% isopropanol was used as background control.

#### Myelin Phagocytosis Assay

For phagocytosis experiments, alternatively activated macrophages were exposed to 5µg/ml atto633-labeled myelin for 4 h at 37◦C in 5% CO2, with or without addition of 1µg/ml ANGPTL4 (R&D systems, 4487-AN, USA) or 10µM Cytochalasin D (Sigma-Aldrich, C8273, USA). Subsequently, cells were thoroughly washed with PBS to remove extracellular myelin. Phagocytosis of fluorescent myelin particles was quantified using intracellular FACS analysis (Calibur flow cytometer, Becton & Dickinson, USA).

#### Lipoprotein Lipase Activity (LPL) Assay

Lipoprotein lipase activity on alternatively activated monocyte derived macrophages was measured using a fluorometric assay kit (Abcam, ab204721, UK) according to the manufacturer's instructions.

#### Statistics

All data reflect mean ± SEM and all comparisons were statistically tested in GraphPad Prism 5.0. For comparing two experimental groups with normal distribution, unpaired twotailed Student's t-tests was used. For comparing two experimental groups without normal distribution Mann–Whitney U-test was used. One-way analysis of variance (ANOVA) was used to compare more than two groups.

#### RESULTS

### Decreased Astrocytic ANGPTL4 Expression in Multiple Sclerosis Lesions

First, we investigated the cellular expression of ANGPTL4 in well-characterized MS lesions. Active white matter lesions were identified by the absence of myelin (proteolipid protein) and the presence of MHC class II+ cells with some MHC

#### TABLE 3 | Primer sequences.

FIGURE 1 | ANGPTL4 expression is strikingly reduced in active MS lesions.  Inactive white matter lesions display a loss of PLP immunoreactivity and absence of HLA-DR positive cells. Active white matter lesions display a marked decreased expression of ANGPTL4 compared to normal appearing white matter and inactive lesions. ANGPTL4 is predominantly localized to astrocytes (insert) (scale bar = 500µm). (B) Double immunofluorescence labeling shows co-localization of ANGPTL4 (green) with GFAP-immunoreactive astrocytes (red) (scale bar = 5µm). (C) Quantification of fluorescence intensity of ANGPTL4 in NAWM, active lesions and inactive lesions shows downregulation of ANGPTL4 in active MS lesions (One-way ANOVA, *N* = 6 for NAWM, 5 for active lesion and 4 for inactive lesion). \**p* < 0.05.

class II cells containing myelin proteins. Inactive lesions are characterized with a demyelinated core with little evidence of ongoing inflammation. We observed no clear differences in cellular expression nor intensity of ANGPTL4 immunoreactivity comparing control white matter with normal appearing white matter (NAWM). ANGPTL4 was found to be expressed in normal appearing white matter by cells with an astrocytic morphology. Interestingly we observed a marked decreased expression of ANGPTL4 in active MS lesions compared to NAWM, while in inactive lesions the expression of ANGPTL4 displayed similar immunoreactivity as observed in NAWM (**Figure 1A**). To confirm the cellular source of ANGPTL4, we performed co-localization studies which confirmed that ANGPTL4 is predominantly expressed by glial fibrillary acidic protein (GFAP)-positive astrocytes (**Figure 1B**). Fluorescence intensity of ANGPTL4 was measured in NAWM, active and inactive lesions. These quantitative analyses showed that ANGPTL4 immunoreactivity is consistently reduced in active MS lesions compared to surrounding NAWM and to inactive lesions (**Figure 1C**).

## Lipoprotein-Lipase Is Expressed by Iba-1 Positive Cells in MS Lesions

Since ANGPTL4 is a known inhibitor of lipoprotein-lipase (LPL), we next analyzed the cellular distribution of LPL in MS brain specimens. LPL was weakly expressed in NAWM and abundantly expressed in active lesions, localized to cells with the morphological appearance of macrophages (**Figure 2A**). Immunofluorescent double staining with Allograft inflammatory factor 1 (iba1, macrophage/microglia marker) confirmed the cellular localization of LPL in macrophages/microglia (**Figure 2B**). Taken together, ANGPTL4 expression is virtually absent in astrocytes in active lesions, while LPL, the target of ANGPTL4, is expressed by Iba1 positive phagocytes in active lesions.

#### Crosstalk Between Phagocytes and Astrocyte Underlies Downregulation of ANGPTL4

To determine what underlies the observed decrease in astrocytic ANGPTL4 expression, human astrocytes were exposed to myelin

FIGURE 3 | Astrocytic ANGPTL4 expression is not influenced by myelin, but is by crosstalk with macrophages. (A) Astrocytes express ANGTPL4 at similar levels under normal conditions compared to 24 h treatment with myelin (Mann Whitney, N=8 for control and 11 for myelin treatment). Expression of ANGPTL4 is significantly reduced in astrocytes cultures in the presence of alternatively activated macrophages (Student's *t*-test, *N* = 7) (B). \*\**p* < 0.01.

for 24 h. Exposure to myelin did not affect ANGPTL4 expression in astrocytes (**Figure 3A**). Based on the co-occurrence of LPLpositive phagocytes and ANGPTL4-deficient astrocytes in active lesions, we hypothesized that macrophages might be responsible for the observed loss of astrocytic ANGPTL4. Astrocytes cultured in the presence of activated macrophages showed a significant decreased expression of ANGPTL4 compared to astrocytes cultured in the absence of macrophages (**Figure 3**).

## ANGPTL4 Inhibits (Myelin) Lipid Uptake via Modulation of LPL Activity

Recent reports have highlighted the pivotal role of microglial LPL in remyelination (4, 6, 12), and it is thought that microglial LPL is able to process myelin after which the myelin derived lipids can be taken up via scavenger receptors expressed on microglia (13). Here we investigated whether LPL expression on macrophages is indeed involved in myelin uptake and tested the hypothesis that ANGPTL4 inhibits this uptake by decreasing LPL activity. Macrophages were exposed to myelin, in the absence or presence of ANGPTL4. We first analyzed whether myelin treatment induced lipid accumulation in macrophages. As shown by Oil-Red-O staining, we observed increased lipid accumulation after myelin treatment (**Figures 4A,B**). Macrophages that were treated with ANGPTL4 peptide during exposure to myelin displayed decreased Oil-Red-O staining, demonstrating that ANGPTL4 inhibits (myelin) lipid uptake. To see if a well-known LPL inhibitor also causes a reduction in Oil-Red-O staining, we treated the cells with the LPL inhibitor Orlistat. Treatment with Orlistat resulted in similar reduction as ANGPTL4. Using an LPL activity assay, we confirmed that ANGPTL4 causes a significant decrease in LPL activity in macrophages compared to non ANGPTL4 treated macrophages (**Figure 4C**). To confirm that ANGPTL4 inhibits myelin uptake by blocking LPL activity and not by affecting phagocytosis, we exposed macrophages to atto633 labeled myelin, and quantified the amount of atto633-labeled myelin inside the cells by FACS (**Supplementary Figure 1**). Phagocytosis of atto633-labeled myelin was not inhibited by ANGPTL4, while treatment with Cytochalasin D, which blocks phagocytosis, completely prevented the myelin phagocytosis by macrophages (**Figures 4D,E**). Our findings suggest that macrophages utilize LPL in the process of myelin clearance. In addition, these findings show that astrocyte derived ANGPTL4 inhibits LPL activity, leading to decreased myelin derived lipid uptake by macrophages by mechanisms other than phagocytosis.

# DISCUSSION

We previously demonstrated that angiopoietin-like 4 (ANGPTL4) is expressed by astrocytes in white matter and in gray matter of patients suffering from capillary cerebral amyloid angiopathy (8). Here, we show that the astrocytic expression

of ANGPTL4 is markedly reduced in active demyelinating MS lesions compared to surrounding normal appearing white matter (NAWM). ANGPTL4 is a potent inhibitor of lipoprotein lipase (LPL), an enzyme involved in lipid uptake. We provide evidence that ANGPTL4 inhibits uptake of myelin-derived lipids by LPL-immunoreactive phagocytes. This finding suggests that loss of astrocytic ANGPTL4 expression in active demyelinating MS lesions might be a protective mechanism enabling phagocytes to effectively remove myelin debris paving the way for repair.

Phagocytes, i.e., microglia and macrophages, play a critical role in pathogenesis of MS. In MS, phagocytes promote the clearance of cellular debris after myelin damage, which is a prerequisite for remyelination (14, 15). Our work suggests a novel concept how astrocytes, via decreased production of ANGPTL4, regulate clearance of myelin debris. Reduced astrocytic expression of ANGPTL4 in active lesions might enhance LPL activity on phagocytes, thereby enabling the processing and subsequent clearance of myelin debris. However, full global knockout of ANGPTL4 in mice did not show any significant differences in LPL activity in the brain (16). This might be explained by the lack of LPL protein expression in control white matter brain tissue (17). LPL mRNA expression was almost exclusively detected in microglia/macrophage and specifically in alternatively activated microglia/macrophages (18, 19). Although microglia and macrophage activation states are complex, in particular in MS where they obtain an intermediate activation status (11), it appears that lipid-laden microglia/macrophages are immunosuppressive (20) which corresponds to an alternative activation status. We therefore opted to use alternatively activated macrophages in this study.

We next investigated which factors might be responsible for the decreased expression of ANGPTL4 on astrocytes in active MS lesions. We first cultured astrocytes in the presence of myelin debris, which has been shown to induce a reactive astrocyte profile (21) However, the expression of ANGPTL4 was not affected by myelin uptake. Astrocytes cultured in the presence of alternatively stimulated macrophages showed a significant reduction in the expression in astrocytic ANGPTL4. It thus appears that crosstalk between alternatively activated macrophages and astrocytes is involved in the downregulation of astrocytic ANGPTL4. Future studies are needed to gain more insight in the mechanisms how macrophages regulate astrocytic ANGPTL4 production.

Recent reports have highlighted the pivotal role of microglial LPL in remyelination. LPL hydrolyses triglycerides into glycerol and free fatty acids (FFA). The resulting FFAs can in turn be taken up via scavenger receptors, such as CD36 and CD68 by microglia (22). These scavenger receptors have been suggested to involved in myelin phagocytosis and have been shown to be upregulated in and around demyelinating areas in MS tissue (13).

Upon exposure to myelin fragments, the lipid storage in macrophages was dramatically increased as determined by Oil-Red-O staining. Inhibition of LPL in macrophages by addition of recombinant ANGPTL4 or the LPL inhibitor Orlistat significantly reduced the amount of Oil-Red-O positive lipid droplets. Interestingly, we did not observe differences in their capacity to phagocytose myelin. These findings suggest that ANGPTL4 inhibits the breakdown and subsequent uptake of myelin derived lipids by macrophages, but not myelin uptake via phagocytosis. In line with these observations, ANGPTL4 mediated inhibition of LPL has been shown to limit the formation of foam cells in the context of atherosclerosis (23, 24).

In conclusion, we provide evidence that ANGPTL4 expression is strongly reduced in reactive astrocytes in active MS lesions, which might be a protective mechanism enabling phagocytes to effectively remove myelin debris setting the stage for repair.

# ETHICS STATEMENT

VUmc MS Centrum Amsterdam and the Netherlands Brain Bank received permission to perform autopsies for the use of tissue and for access to medical records for research purposes from the Ethical Committee of the VU University Medical Center, Amsterdam, The Netherlands. All patients and controls, or their next of kin, had given informed consent for autopsy and use of brain tissue for research purposes. Buffy coats were obtained from volunteer donors at Sanquin after written informed consent was obtained.

# AUTHOR CONTRIBUTIONS

AK with the help of JvH and HdV conceived the study. AK, JvH, MR, and HdV designed the experiments. AK with the help of AC performed most of the experiments. SvdP performed FACS experiments. AK with the help of JvH, MR, and HdV wrote the manuscript, which was reviewed by all authors.

# FUNDING

This work was supported by the MS research foundation (MS-14- 358e, AK), which had no role in study design, data collection and analyses, decision to publish, or preparation of the manuscript.

# ACKNOWLEDGMENTS

Expert technical support by the AO|2M facility (Advanced Optical Microscopy facility in O|2, VU Medical Center, Amsterdam) was highly appreciated.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00950/full#supplementary-material

Supplementary Figure 1 | Gating strategy. Non-myelin treated monocyte derived macrophages were gated on forward scatter/side scatter (FCS/SSC) dot plot. These events were next visualized using an FSC/myelin-633 dot plot and a gate was placed for atto6330-labeled myelin negative cells. Then atto633-labeled myelin treated monocyte derived macrophages were analyzed to confirm that treated macrophages lay inside the gate.

# REFERENCES

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

Copyright © 2019 Kamermans, Rijnsburger, Chakraborty, van der Pol, de Vries and van Horssen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Oncostatin M, an Underestimated Player in the Central Nervous System

Evelien Houben, Niels Hellings and Bieke Broux\*

*Department of Immunology, Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium*

For a long time, the central nervous system (CNS) was believed to be an immune privileged organ. In the last decades, it became apparent that the immune system interacts with the CNS not only in pathological, but also in homeostatic situations. It is now clear that immune cells infiltrate the healthy CNS as part of immune surveillance and that immune cells communicate through cytokines with CNS resident cells. In pathological conditions, an enhanced infiltration of immune cells takes place to fight the pathogen. A well-known family of cytokines is the interleukin (IL)-6 cytokine family. All members are important in cell communication and cell signaling in the immune system. One of these members is oncostatin M (OSM), for which the receptor is expressed on several cells of the CNS. However, the biological function of OSM in the CNS is not studied in detail. Here, we briefly describe the general aspects related to OSM biology, including signaling and receptor binding. Thereafter, the current understanding of OSM during CNS homeostasis and pathology is summarized.

#### Edited by:

*Fabienne Brilot, University of Sydney, Australia*

#### Reviewed by:

*Carl D. Richards, McMaster University, Canada Yoshiro Ohara, Kanazawa Medical University, Japan*

> \*Correspondence: *Bieke Broux<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology*

Received: *21 February 2019* Accepted: *08 May 2019* Published: *29 May 2019*

#### Citation:

*Houben E, Hellings N and Broux B (2019) Oncostatin M, an Underestimated Player in the Central Nervous System. Front. Immunol. 10:1165. doi: 10.3389/fimmu.2019.01165* Keywords: Oncostatin M (OSM), central nervous system, cell biology, homeostasis, pathology

# INTRODUCTION

A well-orchestrated transmission of signals is crucial to maintain and restore homeostasis in humans. A class of messenger molecules that play an important role in interaction and communication of cells in the immune system, are cytokines. They are produced by immune cells but also by other resident cells of the human body as a response to changes in their microenvironment. The central nervous system (CNS) was long believed to be immune privileged. However, in the last decades it has become clear that communication between the CNS and the immune system is very important, even during homeostasis, and that it needs to be strictly regulated.

Many cytokines are thoroughly characterized in the CNS, yet others remain less studied, including oncostatin M (OSM). In 1991, OSM was categorized as a member of the interleukin (IL)-6 cytokine family (1, 2). Next to OSM and IL-6, this family of cytokines includes IL-11, IL-27, leukemia inhibitory factor (LIF) ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), neuropoietin (NP) and IL-31 (3, 4). These cytokines all signal through a multi-unit receptor complex, containing the common glycoprotein 130 (gp130) subunit, except for IL-31 which binds to the OSM receptor beta (OSMRβ)/IL-31 receptor alpha (IL-31Rα) complex (5). Because of the related receptor complexes, signaling properties are shared by the members of the IL-6 family. For more insights see (4, 6–9). Here, we briefly describe the biological effects of OSM and summarize the current understanding of OSM activity in the CNS.

# OSM BIOLOGY

OSM binds to the heterodimeric OSM receptor (OSMR), consisting of the gp130/OSMRβ complex, also referred to as OSMR type II in humans. Moreover, in humans and rats, OSM signaling is also possible through the LIF receptor (LIFR), consisting of the heterodimer gp130 and LIF receptor beta (LIFRβ), also known as OSMR type I in humans (4, 10). However, in mice, murine OSM (mOSM) does not transmit signals through the LIFR (11, 12). Only extremely high concentrations of mOSM may lead to weak LIFR signaling (11). While there are reports on mOSM signaling via the LIFR in mouse osteocytes (13, 14), no literature hints to LIFR activation by mOSM in neural cells. Cross-species activities of OSM in mice, rats and humans are also possible (summarized in **Table 1**) (10, 15). Understanding of the cross-species activities is crucial for proper interpretation of results obtained in experimental studies. For example, experiments in which OSM only signals via the LIFR show receptor and cell signaling of LIF rather than that of OSM.

OSM contains BC loops which form a steric hindrance for OSMRβ and LIFRβ. Therefore, OSM first binds with gp130 to subsequently recruit OSMRβ or LIFRβ (16). In both cases, formation of the heterodimeric complex leads to activation of different signaling cascades [extensively reviewed in (4, 6, 7, 17)]. First, receptor binding can activate Janus kinase (JAK)s, JAK1, JAK2 and Tyk2, which recruit signal transducer and activation transcription (STAT)s, STAT1, STAT3, STAT5, and STAT6. The activated STATs translocate to the nucleus to induce transcription of target genes. Second, activation of the LIFR and OSMR can also induce the mitogen activated protein kinases (MAPK) cascade. The different MAPKs involved are extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 and c-jun N-terminal kinases (JNK) (18). Finally, activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway and the protein kinase C delta (PKCδ) have been described after OSMR activation (19, 20). These different signaling pathways lead to the diverse nature of OSM in various cell types and environmental conditions.

# OSM IN THE CNS

The role of OSM has already been specified in joint, skin, lung, and vascular homeostasis and disease [reviewed in (4, 7, 19)]. Also in cancer, depending on the cancer type, various actions of OSM are reported (4, 7, 19). Yet, less is known about the role of OSM in the CNS. Here, we summarize the reports for which OSM is described in the healthy and pathological CNS (**Figure 1**).

# Source of OSM and OSMRβ Expression in the CNS

Many cells of the immune system, i.e. dendritic cells, neutrophils, monocytes/macrophages, and T-cells, have been identified as a source of OSM (21–23). Hematopoietic cells of the bone marrow also produce OSM, regardless of inflammation (24). In the CNS, OSM is expressed by different cell types, namely neurons, astrocytes and microglia (25–27). In pathological conditions, such as multiple sclerosis (MS), OSM expression in the CNS is increased, in part by OSM production via infiltrating leukocytes (25, 28). With regard to expression of OSMRβ in the CNS, the first reports described expression of OSMRβ RNA in most regions of the murine CNS (forebrain, cortex, midbrain, hindbrain and spinal cord) (29, 30). Later reports investigated the cellular specificity of OSMRβ expression, and indicated that the OSMRβ protein is expressed on neurons (31), astrocytes (31–34), endothelial cells (33), and oligodendrocytes (31). For microglia conflicting reports are found and discussed later. In addition, OSMRβ transcripts are also present in epithelial cells of the pia mater and the choroid plexus (29, 35). The extensive expression of the OSMRβ in the brain implies important CNS related effects of OSM in different cell types both in health and disease.

# Effects of OSM on Neural Cells

In physiological conditions, OSM has been implied in the homeostasis of neural precursor cells (NPCs). NPCs are a pool of cells for the continuous production of new neural cells, located in the subventricular zone (SVZ) (36), hippocampus (37), and olfactory bulb (38) in the adult mammalian brain. In mice, the OSMR is expressed on a subpopulation of NPC in the SVZ and in the subgranular zone of the dentate gyrus within the hippocampus (39). Functionally, OSM induces in vitro repression of neurosphere formation, indicating inhibition of NPC proliferation isolated from the SVZ and olfactory bulb, (39) while NPCs isolated from SVZ, olfactory bulb and hippocampus of OSMRβ knock-out animals lead to enhanced formation of neurospheres (39).

In pathological conditions, the vast majority of papers report neuroprotective effects of OSM. To start, OSM inhibits N-methyl-D-aspartate (NMDA)-induced excitotoxicity in a dose-dependent way. This effect is even more pronounced after pre-treatment with OSM (40). Neuroprotective effects against excitotoxicity are among others mediated by inhibitory adenosine A1 receptors (A1Rs), suppressing excitatory transmission (41). Inhibition of glutamate-induced excitotoxicity by OSM is completely abolished after A1Rs blockage and knockout, indicating the requirement of adenosine A1R function for neuroprotection (42). Also, a protective effect of OSM is observed after amyloid beta-peptide (Aβ) induced neurotoxicity (43), known to cause mitochondrial dysfunction in Alzheimer's disease (44). Furthermore, OSM protects against 3-nitropropionic acid induced mitochondrial dysfunction in rat cortical neurons through induction of myeloid cell leukemia-1 (Mcl-1). Mcl-1 enhances mitochondrial respiration and ATP production (43) and is described as an anti-apoptotic protein with neuroprotective functions (45, 46). Since rat neuronal cells and rOSM are used in these experiments, both the involvement of LIFR and OSMR signaling needs to be considered. Moreover, we reported that OSM enhances neuronal cell viability after withdrawal of B27, a vital supplement for growth and differentiation of primary neurons and enhances neurite outgrowth in vitro (47). Only one study reported a potential neurotoxic effect of OSM. In this study, neuronal cell growth was inhibited when cultured in the presence of the secretome of peripheral blood mononuclear cells (PBMC) from HIV-1-infected patients. Analysis of the secretome, identified OSM as the key molecule involved in inhibition of neuronal

TABLE 1 | Cross-species signaling of OSM via the LIFR and the OSMR in mice, rats and humans.

\**OSMR type I;* \*\**OSMR type II.*

FIGURE 1 | The role of OSM in CNS pathology, an overview. This figure depicts all reported activities of OSM on different CNS resident cells as investigated in *in vitro* and *in vivo* studies. For further details see accompanying text.

proliferation and viability (48). Another study reported an indirect neurotoxic activity of OSM by inducing TNF-α secretion by microglia (49). Altogether, we can conclude that OSM has been widely reported to have a direct neuroprotective activity. However, indirect neurotoxic effects are possible and need to be kept under consideration.

Astrocytes usually prevent neuronal excitotoxicity via sequestration of extracellular glutamate through the glutamate aspartate transporter (GLAST/EAAT1) and glutamate transporter-1 (GLT-1/EAAT2) (50). OSM downregulates the expression of these receptors on astrocytes, leading to reduced glutamate uptake and consequently, excitotoxic injury (26). Astrocytes also secrete different molecules in response to OSM. Plasminogen activator inhibitor-1 (PAI-1) and α1-antichymotrypsin (ACT) (51) expression is induced by OSM in astrocytes. Co-treatment of OSM and IL-1, leads to matrix metalloproteinase (MMP)-1 and MMP-3 production by astrocytes (52). Moreover, OSM works synergistically with the pro-inflammatory cytokines, IL-1β and TNF-α, to induce IL-6 (53) and prostaglandin E2 (PGE2) (54) production in human astrocytes. These OSM-induced astrocytic molecules are linked to pro-inflammatory and tissue remodeling processes. However, OSM also induces astrocytic secretion of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) (55) and SOCS3 (32), which quench inflammation. Therefore, the net outcome of OSM signaling in astrocytes depends on the microenvironment and other cytokines present herein.

For microglia/macrophages, contradictory reports are present about OSMRβ expression and therefore the effect of OSM on these cells. Different research groups do not observe OSMRβ expression in primary mouse microglia (34, 42), nor in the C8-B4 microglia cell line (34). Moreover, no phosphorylation of STAT1 or STAT3 is observed in microglia after OSM treatment (34). In contrast, tumor necrosis factor-α (TNF-α) and nitric oxide (NO) production is reported after NF-κB pathway stimulation via OSM treatment of primary microglia and the BV2 microglia cell line (49). Yet, others did not see OSM-induced activation of the NF-κB pathway nor OSM-induced NO production in microglia (34). Moreover, our group found OSMRβ expression on Iba-1+ cells, a marker for both microglia and macrophages, in naive and cuprizone treated animals (31). In naive animals, no infiltration of macrophages is expected, yet perivascular, meningeal and choroid plexus macrophages are present (56). In the cuprizone challenged mice, macrophages infiltrate the brain (57). Therefore, it is possible that the Iba-1+ cells are macrophages. However, Hsu and colleagues did not detect OSMRβ in bone marrow-derived macrophages and the RAW 264.7 macrophage cell line (34). Yet, in other tissues, OSMRβ expression is seen on macrophages, i.e. in adipose tissue and atherosclerotic lesions (58, 59). In conclusion, more research is needed to address whether OSMR signaling is active in microglia and macrophages.

The blood brain-barrier (BBB) is very important to protect the brain from unwanted intruders. On human cerebral endothelial cells (HCECs), expression of OSMRβ, but not LIFRβ, is seen, despite low RNA levels of LIFRβ (25). The latter implies that OSM only signals through the OSMR type II in HCECs. OSM treatment increases the percentage of HCECs expressing intracellular adhesion molecule-1 (ICAM-1), yet no effect on vascular cell adhesion molecule-1 (VCAM-1) expression is detectable (25). Next to adhesion molecules, OSM augments the secretion of IL-6 and monocyte chemotactic protein-1 (MCP-1) in HCECs (25). This effect is further enhanced after co-treatment of HCECs with OSM and TNF-α (25). Also, a decreased BBB permeability is attributed to persistently high activation of the JAK/STAT3 signaling pathway (60). Here, rat brain capillary endothelial cells (RBECs) were treated with mOSM, implying OSMR and not LIFR signaling as indicated in **Table 1** (10). Both increased permeability for sodium fluorescein and decreased transendothelial electrical resistance (TEER) are seen in mOSM treated RBECs (60, 61). Moreover, delocalization of the tight junction molecules, claudin-5 and zonula occludens-1 (ZO-1) is apparent after OSM treatment (60). Together, these studies imply a pro-inflammatory state of BBB-ECs when treated with OSM.

Finally, for oligodendrocytes and myelination, protective effects of LIF have been described [reviewed in (62, 63)] and tested preclinically via therapeutic delivery through nanoparticles or lentiviral vectors (64, 65). However, only a few in vivo studies reported on the role of OSM on oligodendrocytes and its repair mechanisms after myelin damage. These results are described in the next section. Overall, both protective and detrimental effects on cells of the CNS are described for OSM. Even though, the described in vitro experiments investigated the effect of OSM on distinctive cell types, the interplay between different cells is more complex and needs to be studied using in vivo models.

#### OSM in Murine Models of CNS Pathology

Murine neurological disease models are used to investigate the biological role of OSM in a more complex in vivo setting and to further allow development of OSM based treatment strategies based on these insights. Inducing disease in these models can influence the level of OSM and OSMRβ expression. Indeed, upregulation of OSM and OSMRβ in whole spinal cord is observed in the mouse spinal cord hemisection model (47). During cuprizone-induced demyelination increased OSMRβ expression is seen mainly on astrocytes and Iba-1+ cells (microglia/macrophages) (31). In contrast, middle cerebral artery occlusion (MCAO)/reperfusion reduces expression of OSMRβ on neurons in brain areas of disturbed perfusion, i.e. ipsilateral cortex and striatum (66).

To test the effect of OSM signaling, both OSM treatment and transgenic mice [OSMRβ overexpressing animals or OSMRβ knock-out (KO) mice] can be used. While OSM KO and OSMRβ KO mice are healthy and fertile, phenotypical changes observed are a disturbed hematopoiesis in both KO strains (67, 68) and severe obesity upon a high-fat diet in OSMRβ KO animals (69). It is unknown whether there is a phenotype in the CNS, since no neurological deficits are reported to date. For the OSMRβ KO mice, the IL-31 receptor consisting of OSMRβ/IL-31Rα is also affected. However, to date there are no reports of IL-31 signaling in the CNS, only the presence of the IL-31 receptor is described in dorsal root ganglia (70–73).Therefore, it is difficult to evaluate the effect of disturbed IL-31 signaling in CNS studies using OSMRβ deficient mice.

OSM was studied in the experimental autoimmune encephalomyelitis (EAE) mouse model, a MOG autoreactive T-cell mediated model for MS. Mice receiving intraperitoneal injections with OSM did not develop any sign of paralysis, the clinical outcome of EAE induction (74). The absence of symptoms was in agreement with limited immune cell infiltration in the brain of these animals (74). However, it needs to be mentioned that human OSM was used to treat the animals. Therefore, LIFR signaling and not OSMR signaling is studied here as indicated in **Table 1**. Indeed, systemic LIF treatment of EAE mice reduces the disease symptoms (75). We have shown that local overexpression of OSM through lentiviral vectors reduces cell-death of oligodendrocytes in the cuprizone model and therefore limits subsequent demyelination. The latter is linked to a reduced microglial response, increased IL-4 expression and M2 polarization (31). OSM-induced M2 polarization is also seen in other organ/tissue models, i.e. lung, adipose tissue (58, 76–78) and cancer models (79, 80). In line with the cuprizone model, OSM treatment in the ethidium bromide (EtBr) toxin-induced demyelination model counteracts a reduction in mRNA expression of oligodendrocyte precursor marker (Pdgfra), oligodendrocyte lineage transcription factors (Olig1 and Olig2) and myelin genes (Plp and Mog) indicative for oligodendrocyte and myelin sparing (33). Moreover, neuroprotective effects are reported in vivo. Local OSM treatment in a spinal cord injury (SCI) mouse model improves functional recovery, and histological analysis reveals a reduced lesion size with less astrogliosis, less CD4+ T cell infiltration and an increased nerve fiber sprouting (47). Also, stereotaxic injection of NMDA together with OSM reduces the NMDAinduced lesion volume in a model of neurotoxic injury. This could be attributed to a reduced expression of the NR2C subunit of the NMDA-receptor and attenuated increase of intracellular calcium, preceding NMDA-induced cell death (40). Correspondingly, OSMRβ overexpression in neurons improves stroke outcome following ischemia/reperfusion (I/R) induced cerebral injury (66). The latter is due to a protective role of OSMRβ against neuronal apoptosis via JAK2/STAT3 signal activation, leading to transcription of genes involved in neuronal survival (66). When mice lack OSMRβ, opposite effects on neurons and oligodendrocytes are described. OSMRβ deficiency leads to an increased infarct volume and more severe neurological deficits after I/R damage (66) and an aggravation

#### REFERENCES

of demyelination in the cuprizone model (31). Taken together, these studies indicate protective effects of OSM signaling in different mouse models for neurodegenerative diseases, with both oligodendrocytes and neurons being directly or indirectly protected by OSM. The most important intermediate players in these processes are astrocytes and microglia/macrophages.

# CONCLUSION

Within the CNS, the major cellular sources of OSM are astrocytes, neurons, microglia and infiltrating immune cells. OSM can signal though both the LIFR (OSMR type I in humans) and OSMR (OSMR type II in humans) in humans and rats, while in mice, only signaling via the OSMR is possible. Since OSM can signal via two receptors, caution should be taken when interpreting research findings, because OSM-mediated effects can be attributed to LIFR and/or OSMR signaling, depending on the model and species used. With regard to the effect of OSM on neurons, the majority of papers report protective effects of OSM. For oligodendrocytes, astrocytes, and the BBB a limited amount of studies is available. OSM has protective effects at the level of myelination, which is very important for good signal transduction and protection of axons. For astrocytes, there is a role in excitotoxicity and secretion of inflammatory molecules. Finally, at the level of the BBB a pro-inflammatory readout is observed. To conclude, OSM can exhibit different functions depending on the variety of cell types that express the receptor and the cellular and molecular microenvironment. While in vivo models demonstrated that OSM has beneficial effects in the diseased CNS, more research is warranted to reveal the true role of OSM in the CNS.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### ACKNOWLEDGMENTS

This work was financially supported by the Research Foundation of Flanders (FWO Vlaanderen, 1106817N). **Figure 1** was adapted from smart servier medical art (https://smart.servier.com/).

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

Copyright © 2019 Houben, Hellings and Broux. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Role of BDNF in the Neuroimmune Axis Regulation of Mood Disorders

#### Yang Jin1 , Li Hua Sun1 , Wei Yang1 , Ran Ji Cui 1 and Song Bai Xu2 \*

*1 Jilin Provincial Key Laboratory on Molecular and Chemical Genetic, Second Hospital of Jilin University, Changchun, China, 2 Department of Neurosurgery, First Hospital of Jilin University, Changchun, China*

The neuroimmune system plays a crucial role in the regulation of mood disorders. Moreover, recent studies show that brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is a key regulator in the neuroimmune axis. However, the potential mechanism of BDNF action in the neuroimmune axis' regulation of mood disorders remains unclear. Therefore, in this review, we focus on the recent progress of BDNF in influencing mood disorders, by participating in alterations of the neuroimmune axis. This may provide evidence for future studies in this field.

Keywords: BDNF, neuroimmune axis, mood disorders, depression, inflammation, cytokines

#### Edited by:

*Bieke Broux, University of Hasselt, Belgium*

#### Reviewed by:

*Christina Pabelick, Mayo Clinic, United States Bonaventura Casanova, Hospital Universitari i Politècnic La Fe, Spain*

> \*Correspondence: *Song Bai Xu<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Neurology*

Received: *14 February 2019* Accepted: *30 April 2019* Published: *04 June 2019*

#### Citation:

*Jin Y, Sun LH, Yang W, Cui RJ and Xu SB (2019) The Role of BDNF in the Neuroimmune Axis Regulation of Mood Disorders. Front.  Epidemiological studies have found that there are approximately 350 million people affected by depression in the world, and the number is increasing year on year (1, 2). According to the results of the global burden of disease study, years lost to disability of depression ranks first among the top 10 disabling diseases in the world (3). The main clinical features are marked by consistent emotional upsurge or depression, often accompanied by corresponding changes in thinking and behavior (4). The performance of mood disorders is highly variable. Lighter ones may respond to certain negative life events, while heavier ones may become a seriously recurrent or even chronic disabling disorder. Clinically, mood disorders can be divided into four types: depressive episode, manic episode, bipolar disorder, and a persistent mood disorder. It not only brings severe mental pain to patients but also leads to other diseases, such as heart disease and cerebrovascular diseases. However, the pathogenesis of mood disorders is still unclear, so it is difficult for patients to be cured. Although mood disorders can currently be treated with drugs, psychotherapy or a combination thereof, the efficacy is limited and side effects may also occur (5, 6). It is therefore necessary to explore the etiology and mechanism of mood disorders to treat and prevent mood disorders.

There are many theories about the nosogenesis of depression disorder, such as monoamine neurotransmitter hypothesis, hypothalamus-pituitary-adrenal (HPA) axis dysfunction, neurotrophic hypothesis and cytokine hypothesis (7, 8). In recent years, more and more studies have focused on the relationship between mood disorders and neuroimmune regulation (9). Many neurotrophins are associated with the pathogenesis of mood disorders, such as the nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) (10, 11). Several studies have shown that BDNF may be indispensable to the neuroimmune regulation of mood disorders. However, the potential mechanism for BDNF to affect mood disorders, by participating in changes in the neural-immune axis, has not been elucidated. In this review, we summarize the latest progress in the role of BDNF in the neuroimmune axis regulation of mood disorders. It may provide new ideas for the research and treatment of mood disorders in the future.

### BDNF AND MOOD DISORDERS

BDNF, an important member of the neurotrophic factor family, is a protein synthesized in the brain and widely distributed in the central nervous system (CNS), as well as the peripheral nervous system (12). It can promote the survival, growth, differentiation, and development of neurons and plays a crucial role in the neural structure and functional plasticity (13, 14). A large number of human and animal studies have implicated the close links between BDNF and the occurrence and treatment of various diseases, including schizophrenia (15), Alzheimer's disease (16, 17), mood disorders (18), and Parkinson's disease (19).

#### Studies About BDNF in Depression

The neurotrophic hypothesis suggests that pathological changes in brain areas associated with depression, are closely related to BDNF expression and functional down-regulation (20). Animal models of depression suggest the vital function of BDNF in the pathophysiological mechanism of depression. In animal experiments, chronic stress and depression conditions decreased BDNF expression, increased apoptosis and decreased regeneration of neurons in the hippocampus, and also decreased BDNF expression in other parts of the brain (21, 22). However, whether these speculations apply to humans still remain to be tested in clinical studies. A large number of clinical studies have found that various kinds of stress can reduce the activity of the BDNF pathway in the hippocampus and prefrontal cortex (23–25). The postmortem studies of Karege et al. (26) reported that the analysis of brain tissue samples from patients with depression after self-killing found that BDNF and TrkB expression in the hippocampus decreased. Moreover, in the hippocampus of patients who have received antidepressant treatment before their death, BDNF and TrkB expression increased (26). The vast majority of studies have found abnormally lower serum BDNF levels in patients with depression than that of people without depression (25, 27, 28). Ristevsk-Dimitrovska et al. found that the serum BDNF level of depressed patients was lower than that of the control group (29), while significantly higher BDNF levels were found after antidepressant treatment (24). A meta-analysis showed that the serum or plasma BDNF increased during treatment in severe mental illness inpatients, but was not restored (18). Treatment with an antidepressant, Agomelatine, could increase the hippocampal BDNF level and BDNF positive neurons in CUMS rats (30). Kreinin et al. found that the serum BDNF level was positively correlated with depression in women with severe MDD, which further supported the role of BDNF in the pathogenesis and treatment of MDD (31). Moreover, increased BDNF levels suggests that BDNF may serve as a marker for a therapeutic response to ECT in MDD patients (32). Thus, it has been speculated that BDNF may be a biomarker of depression. But to understand the role of BDNF in depression, it is also necessary to further clarify the regulatory factors affecting BDNF expression, namely the upstream and downstream signaling pathways of BDNF in the nervous system.

### BDNF and Neurotransmitters in Depression

The monoaminergic hypothesis is also one of the most important hypotheses to study in the pathogenesis of depression. It points out that depression may be caused by low levels of monoamine neurotransmitters in the brain (33). Meanwhile, the function of BDNF is closely related to the plasticity of 5-HT, choline lipids, DA neurons and the survival of central neurons. For example, BDNF could promote the regeneration of 5-HT neurons in the CNS, so the large consumption of 5-HT in the CNS reduces the BDNF level, which leads to the atrophy and death of nerve cells, affects neural plasticity, and thus aggravates depressive symptoms (34). In addition, the regulatory effect of BDNF on the 5-HT2A receptor level is also an important mechanism of BDNF's role in affective disorder (35). A study suggested that BDNF was implicated in the neuroprotective effects of the selective 5-HT1A receptor agonist, 8-OH-DPAT, against CA1 neurons apoptotic death after transient global cerebral ischemia (36). BDNF also plays an essential role in the mesolimbic DA pathway. Studies have shown that the blockade of BDNF activity in the ventral tegmental area-nucleus accumbens pathway exerts an antidepressant-like activity in rodent models of stress (37–39). BDNF controls the expression of the D3 receptor in part of the brain, and induction of BDNF by antidepressant treatments is associated with its behavioral activity (40).

#### Signaling Pathway of BDNF in Depression

Tyrosine receptor kinase B (TrkB), a member of the tyrosine kinase family, can specifically bind to BDNF with a high affinity (41). Comprehensive research has indicated that BDNF is involved in the regulation of CNS, mainly by binding to TrkB (**Figure 1**). BDNF activates intracellular tyrosine kinase activity by binding to TrkB, causing the autophosphorylation of TrkB, thereby activating the mitogenactivated protein kinase (MAPK) pathway, the phospholipase C-gamma (PLC-γ) pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, and other signaling pathways (42). Finally, CREB is activated at the Ser133 site of the cAMP response element binding protein (CREB). CREB promotes the survival of nerve cells and increases synaptic plasticity and neurogenesis by boosting the expression of the BDNF and BCL-2 genes (43). BDNF-TrkB not only affects the survival, development, and functions of neurons but also promotes the formation of the dendritic spine, provides a structural basis for synapse formation and improves the transmission efficiency of synapses. BDNF/TrkB signaling has a major impact on the production of antidepressant effects (44). Knock-out of the BDNF gene or the reduction of the levels of BDNF in the forebrain blocks the behavioral effects of antidepressants (45). In recent years, more and more studies have found that antidepressants might play an anti-depressant role by up-regulating brain BDNF levels or activating TrkB receptors. Song et al. found that silibinin mitigated the depression-like symptoms of Aβ1- 42-treated rats by decreasing the BDNF/TrkB expression, suggesting the role of the BDNF/TrkB signaling pathway in the activity of antidepressants (46). Likewise, sesquiterpenoids from

ginseng root treatment, ameliorate depression-like behaviors induced by LPS by upregulating the BDNF/TrkB Pathway (47). The BDNF-TrkB pathway in the nucleus accumbens of α7 nACHR knockout mice was demonstrated to be up-regulated, which was considered to be involved in their depressionlike behavior (48). The antidepressant role of fisetin was confirmed by Wang et al. which was achieved by activating TrkB rather than regulating its overall level (49). All of these studies suggest that BDNF and the mediated TrkB signaling pathway may provide new approaches for the treatment of depression.

The N-methyl-D-aspartic acid receptor (NMDA receptor) is associated with depression. NMDA receptor antagonism has a significant antidepressant effect. On the one hand, it exerts its antidepressant effect by inhibiting NMDA receptors, which not only promote the establishment of new synaptic connections but also restore the synaptic connections caused by stress damage. On the other hand, antagonizing the NMDA receptor also activates the AMPA receptor, which provides a fast antidepressant effect through its signaling pathway. This provides a new method in the treatment of traditional depression (50). BDNF may play a role through the NMDA receptor. BDNF enhances the AMPA-dependent synaptic signaling in the hippocampus through downstream pathways mediated by NMDA receptors (51). Wang et al. enhanced the BDNF/TrkB signaling pathway by means of transcranial magnetic stimulation (TMS). At the same time, the activity of the NMDA receptor in the cerebral cortex was strongly correlated with the degree of TrkB activation (52). In cultured hippocampal neurons and rat neocortical cells, the activation of TrkB or chronic administration of BDNF can enhance the expression of the NMDA receptor NR1 and NR2A/2B through transcriptional activation. BDNF can also promote the release of glutamate through the presynaptic receptor signal transduction pathway, and enhance the AMPA receptor and NMDA through the postsynaptic receptor pathway and then participate in and promote the formation of LTP (53). Duncan et al. through a study of 30 depressive patients treated with ketamine, found that the antidepressant effect of ketamine might be due to the enhancement of inter-synaptic communication by BDNF (54).

#### Effects of Antidepressants on BDNF

In recent years, the major clinical antidepressants are monoamine oxidase inhibitors, tricyclic inhibitors, and tetracyclic inhibitors (55, 56). Some clinical studies suggest that antidepressant therapy for a period of time could reverse the decrease of peripheral BDNF levels of depressed patients. Serum BDNF levels of depressed patients taking SSRIs were markedly higher than that of the control group and depressed patients not taking SSRIs (57). Treatment with venlafaxine or paroxetine also increased BDNF in patients with depression (58). However, whether all antidepressants affect BDNF levels, remains controversial. A meta-analysis showed that the level of peripheral BDNF increased during antidepressant treatment of SSRIs and SNRIs, among which sertraline could improve the BDNF level after short-term treatment (59). Treatment with fluoxetine (SSRI) was found to alter BDNF levels in patients with depression, whereas venlafaxine did not (60). Freire et al. found that neither the combined group, nor the pharmacological group resulted in the increase of the serum BDNF level in patients with depression, although both significantly improved the depressive symptoms of patients (61). These studies indicate that different antidepressants may have different effects on the peripheral BDNF during treatment.

#### NEURO-IMMUNE AXIS AND MOOD DISORDERS

#### Neuro-Immune Axis

Nowadays, plenty of research has been conducted on the interaction between the central nervous system and immune system. It is neurogenic inflammation that determines whether the immune response is caused by a local threat, through the connection of nerve fibers to immune cells. In recent research, a CNS with complicated innate immune responses is demonstrated to have high immunocompetence (62). Microglia, resident immune cells within the brain parenchyma, can secrete some soluble factors, such as chemokines, cytokines, and neurotrophic factor, to adjust the CNS immune response and tissue repair (63). In addition, astrocytes also play an essential role in central immunity. They respond to an inflammatory environment not only in an immunological way by changing their cell phenotype, but also modulate the immune response of lymphocyte in the brain by releasing associated protein molecules, like chemokines and cytokines (64).

Some immune cells, such as non-specific leucocytes and lymphocytes, produce neurotransmitters and neuropeptides. Opioids may serve as an example. It is suggested that opioids are secreted in inflammatory tissues and act to alleviate clinical pain under stress by activating peripheral opioid receptors (65). There are also some neurotrophins produced by activated lymphocytes, such as BNDF and NGF (66, 67). In turn, non-specific leucocytes and lymphocytes can also express classic neuronal receptors. For example, the activated non-neuron A7 nicotinic cholinergic receptor has anti-inflammatory and immunomodulatory effects on multiple cell types, T cells, B cells, dendritic cells, and mononuclear phagocytes included (68).

Cytokines, chemokines and their receptors were reported to express on the central and peripheral nervous systems. For instance, IFN-α not only affected CNS directly but also had an indirect action through inflammatory cytokines of the central and peripheral nervous systems (69). Previous studies found that interleukin and chemokine receptors, which participated in neuronal inflammation and CNS diseases, were expressed by neurons (70). Cytokines like interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) could influence the behavior, by directly functioning in the nervous system. Cytokines are conducive to the growth and function of the brain and regulate neural activity and neurotransmitter systems, which result in behavioral changes. Chronic exposure to high levels of inflammatory cytokines and constant alterations of central neurotransmitters may contribute to psychiatric disorders like schizophrenia and mood disorders (71, 72). Cytokines induce behavioral effects by activating inflammatory signaling pathways in the brain, leading to the reduction of growth factors such as BDNF for instance (72).

#### Cytokines and Depression

In recent years, studies have found that immune dysfunction was closely related to depression, and pro-inflammatory cytokines produced by innate immune activation were especially closely related to the occurrence and development of depression. Therefore, the hypothesis of cytokine is gradually proposed. The cytokine hypothesis suggests that depression is an inflammatory disease caused by neuroimmune regulation disorders, emphasizing that the body's immune system plays an important role in depression. Cytokines are intercellular information transfer molecules, which mainly have immunomodulatory and multiple effector functions. Different cytokines play different roles in inflammation. Some have proinflammatory effects, while others have anti-inflammatory effects. For instance, IL-1β, IL-6, and tumor necrosis factor α (TNF-α) are relatively advanced proinflammatory cytokines, while IL-4 and IL-10 is the main area of research in anti-inflammatory cytokines. Clinical studies have shown that patients with depression were often associated with varying degrees of inflammatory activation or increased inflammatory molecules, suggesting that the occurrence of depression might be closely related to cytokines (73, 74).

#### Studies About Cytokines in Depression

This hypothesis has been supported by a large number of clinical cases in recent years: patients with autoimmune diseases and chronic viral infections often showed depressive symptoms (75, 76). Autopsy studies have found that cytokines were significantly increased, as well as the synthesis of carbon monoxide synthase in macrophages, microglia, and astrocyte (77). A number of studies have indicated that various cytokines, such as IL-1β, IL-2, IL-6, TNF-α, and IFN-γ in serum or plasma of patients with depression were significantly increased (78). The results of the meta-analysis showed that the levels of inflammatory factors in patients with depression, including IL-1, IL-6, and TNF-α, were significantly higher than those of healthy people and in positive relevance to the serious extent of depressive symptoms (79–81), and antidepressants could lower these cytokines in people with depression (82). According to the study of the cytokine overview, Zou et al. found that the expression of IL-1, IL-10 and TNF in MDD patients increased significantly, while the expression of IL-8 decreased significantly. Such aberrant changes in the levels of inflammatory cytokines demonstrated that it is depression that activates the inflammatory process (83). Animal studies have also shown that the levels of IL-1β, IL-6, and TNF-α in the brain increased significantly after lipopolysaccharide treatment, as well as depressive behaviors, such as sleep disorders, loss of pleasure, and insufficiency of power (84). Depressive behaviors of animals were blocked after injecting IL-1 receptor antagonist IL-1rA into animals before stress (85). The results suggested that inflammatory pathways might be involved in the development of depression.

#### Actions of Cytokines in Depression

People realize that there is a two-way effect between immunity and nerves. Both physiological and psychological stress can activate the immune system and make the cytokines secreted, and then influence the central nervous system, such as neurogenesis, neurotransmitter level, neuroendocrine function, neuroplasticity, and behavior related neural pathways (86–88). It leads to changes in the neurochemistry and endocrinology associated with depression and influences the development of depression. Cytokines can promote oxidative stress and damage glial cells in emotionally related brain regions, such as the prefrontal cortex and amygdala (89). In addition, dysfunctions of glutamate-induced by cytokine can reduce the generation of neurotrophic factors (88). Under stress, the increase of proinflammatory cytokines in the human body activates the indolamine 2,3- dioxygenase (IDO), an enzyme that can directly act on the metabolism of tryptophan (TRP). IDO can increase the level of kynurenine produced by TRP metabolism, thereby reducing the level of 5-HT and promoting the occurrence of depression (90). Cytokines are also momentous to the dysfunction of the HPA axis. They induce the hyperactivity of the HPA axis to increase the glucocorticoid for a long time, and the abnormal glucocorticoid signal can affect the production, maintenance, and development of depressive behaviors (91). At the same time, these cytokines and excess glucocorticoids also inhibit nerve regeneration in the brain. Cytokine signaling pathways, for example NF-kB, can disrupt the function and expression of glucocorticoid receptors, leading to an unrestricted inflammatory response, further exacerbating depressive symptoms (92). Additionally, cytokines can also contribute to depression by influencing neural plasticity. Ben et al. confirmed that IL-1 could inhibit neuronal regeneration, and inflammatory cytokines—IL-6, as an example, could disrupt neuronal function (93). Currently, though the causal relationship between cytokines and depression is still considered controversial, it is undeniable that the negative regulation of neuroplasticity in the brain has a significant impact on the developmental progress of depression. Furthermore, the study also shows that abnormally changed levels of cytokines are associated with an increased risk of delirium and suicide (94–96).

#### Immune Regulation and Antidepressant Effects

Some anti-inflammatory drugs have antidepressant effects or enhance antidepressant effects. Studies have shown that the anti-inflammatory drug COX-2 inhibitors could directly or indirectly affect the 5-HT system through the CNS and play an antidepressant effect. Giving rats a dose of rofecoxib can increase the level of 5-HT in the prefrontal and parietal cortex (97). Celecoxib in depressed rats was found to decrease cytokine levels and improve behavior in the hypothalamus (98). Through the drug combinations of celecoxib with antidepressants, such as reboxetine, fluoxetine, and sertraline, it was found that the combined group was better than an antidepressant alone in patients with depression (99–101). Etanercept, a kind of tumor necrosis factor (TNF- α) antagonist, also has a strong antidepressant effect, which can improve depression symptoms and patient fatigue (102). Many other clinical cases show that antidepressants can reduce proinflammatory cytokines and other inflammatory markers in patients (103). Tricyclic antidepressants, SSRI, SNRI, and other antidepressants have been shown to increase anti-inflammatory immunomodulatory cytokine levels by inhibiting inflammatory cytokines and th1-like cytokines (such as IFN-γ). Réus et al. revealed that imipramine, an antidepressant, could reduce the levels of TNF-α and IL-1β in cerebrospinal fluid of maternally deprived adult rats (104). Studies have shown that some non-pharmacological treatments can also improve depressive symptoms by regulating immune inflammatory pathways. Kim et al. believe that acupuncture can reduce the levels of peripheral and central proinflammatory factors (IL-1, IL-6, TNF- α) and proinflammatory neuropeptides, and the results are better in the treatment of depression (105). In addition, exercise can play a synergistic role by inhibiting the immune and inflammatory pathways (106).

#### BDNF, NEUROIMMUNE AXIS, AND MOOD DISORDERS

BDNF is a relatively mature neurotrophic factor, which can promote the proliferation of neurons and glial cells in the inflammation of the nervous system through various molecular mechanisms (107, 108). Glial cells are innate immune cells in the center. They not only synthesize and release multiple inflammatory mediators but also express many inflammatory mediator receptors on the cell surface. Microglia, the first line of defense for the central immune response, exerts essential influence on the inflammatory response in the brain (109). Although there is no direct evidence that microglia are correlated with the nosogenesis of depression, many studies have examined if there is a significant increase in the amount of microglia in the brain in patients with suicidal depression. A previous study observed microgliosis in the dorsolateral prefrontal cortex, anterior cingulate cortex, mediodorsal thalamus, and hippocampus of suicidal patients (110). Torres-Platas et al. also observed a relative increase of primed microglia in depressed suicides (77). Microglia can regulate the release of BDNF. Microglia may take effect on pathogenesis by reducing BDNF expression as well as its high-affinity receptor TrkB. Studies demonstrated that microglia was of extensive and diverse importance for the formation of appropriate synaptic connections during development and maturation, which were frequently mediated by BDNF (111). In addition, high levels of IL-6, IL-1β, and BDNF in LPS-stimulated normal human astrocytes (NHAs) was observed, using an LPS-induced in vitro injury model of astroglial cultures. Vice versa, BDNF can promote the growth of astrocytes and regulate the viability and proliferation of LPS-induced NHA through the PI3K/AKT pathway (112, 113).

Reports repeatedly demonstrated that inflammatory cytokines affect neuronal development as well as apoptosis (114, 115). As a matter of fact, stress and its associated activation of inflammatory cytokines might have a negative effect on neurogenesis and neuroplasticity (84, 116, 117). Considerable research efforts have been devoted to the effect of inflammation on the BDNF expression in the brain. The significant reduction in BDNF was caused by the administration of pro-inflammatory cytokines or lipopolysaccharide (LPS), an inducer for cytokines, serve as an example. LPS injections could significantly reduce mature BDNF levels in the hippocampus and cerebral cortex (118), as well as IFN-α administration, which decreased systemic BDNF levels

(119). Furthermore, other neurotrophic factors also decreased to varying degrees: NGF and neurotrophic factor−3 (NT-3), for instance (120).

It was demonstrated in a number of research studies that inflammation inhibits BDNF/TrkB expression. Inflammatory cytokines influence the phosphorylation of the BDNF receptor (TrkB), thereby further interfering with BDNF signaling (121). Gibney et al. found that poly-I:C administration upregulated the expression of the inflammatory cytokines, which caused the occurrence of an inflammatory reaction. At the same time, BDNF and TrkB expression in the hippocampus and cortex were downregulated, which might lead to behavioral defects of depression and anxiety (122). In addition, it is under integrated BDNF signaling that antidepressants are able to reverse LPS-induced apoptosis, which agrees well with the abovementioned studies.

The anti-inflammatory mechanism of antidepressive agents has not been elucidated yet. Imipramine has been shown to suppress proinflammatory cytokines in rat neural stem cells, stimulating the expression of BDNF (123). Studies have shown that the production of inflammatory cytokines was regulated by complex signaling pathways, especially the nuclear factor-κb (NF-κB) inflammatory response signal pathway (BDNF-TrkB-MEK-ERK-NF-κB pathway) whose activation plays a central regulatory role in the inflammatory response. Investigation of the effect and potential mechanism of salidroside on depression showed that salidroside could down-regulate the expression of BDNF, TrkB, and the NF-κB protein (124). Ge et al. thought that the antidepressant effect of resveratrol is mainly to reduce the expression of inflammatory cytokines and improve NFκB activation (125). Chrysophanol could inhibit the NF-κB signaling pathway (126), and the high dose of fisetin could regulate the expression of NF-κB in the hippocampus to antagonize the expression of iNOS mRNA (127). Similarly, the antidepressant effect of aesculetin may be achieved by inhibiting the NF-κB pathway as well as activating BDNF/TrkB signaling (128). Furthermore, as an inflammatory intracellular signaling molecule, p38 mitogen-activated protein kinase is now a target for clinical studies of chronic inflammatory diseases due to the potential antidepressant effects of its inhibitors (129). All these studies provide a basis for the development of new clinical antidepressants and the continued development of antidepressant treatments.

# CONCLUSION

Based on many clinical and basic research studies, a variety of theories were proposed to expound the nosogenesis of mood disorders, especially depression. In this review, the neuroimmune axis has been related to mood disorders (**Figure 2**). BDNF is thought to be involved in the neuroimmune axis regulation. On the one hand, the expression of BDNF is affected by immune cells and the immune factors they secrete. On the other hand, the immunomodulatory process also requires the regulation of BDNF-mediated signaling pathways. Unfortunately, the specific mechanism of how BDNF participates in the regulation of the neuroimmune axis in mood disorders is still unclear and it is therefore necessary to conduct more in-depth research. The treatment of mood disorders in the past often only focus on a certain aspect of research. The characteristics of the varied symptoms of depression determine that these treatments are not effective. Exploring a treatment strategy for depression based on neuroimmune axis regulation may be more helpful to further guide the development of anti-mood disorders drugs.

# AUTHOR CONTRIBUTIONS

YJ and RJC contributed conception and design of the review. WY organized the documents. YJ wrote the first draft of the manuscript. SBX and LHS wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

#### FUNDING

This work was supported by grants from Jilin Science and Technology Agency funding<PHONE_NUMBER>3JH) and the National Key Research and Development Program of China (2018YFC1311603).

#### REFERENCES

elevated plasma interleukin-6 are inflammatory signatures of post-stroke delirium. J Neuroinflammation. (2018) 15:111. doi: 10.1186/s12974-018- 1156-y

density in schizophrenia and depression is associated with suicide. J Psychiatr Res. (2008) 42:151–7. doi: 10.1016/j.jpsychires.2006.10.013

by depression and anxiety-like behaviours, kynurenine pathway activation and reduced BDNF expression. Brain Behav Immun. (2013) 28:170–81. doi: 10.1016/j.bbi.2012.11.010

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

Copyright © 2019 Jin, Sun, Yang, Cui and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Emerging Role of Triggering Receptor Expressed on Myeloid Cells 2 as a Target for Immunomodulation in Ischemic Stroke

#### Pascal Gervois\* and Ivo Lambrichts

*Department of Morphology, Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium*

Stroke is the second most common cause of death and permanent disability. It is characterized by loss of neural tissue in which inflammation plays a crucial role in both the acute contribution to ischemic damage as in the late-stage impact on post-ischemic tissue regeneration. Microglia play a key role in the inflammatory stroke microenvironment as they can adapt a disease-promoting pro-inflammatory- or pro-regenerative phenotype thereby contributing to the exacerbation or alleviation of ischemic damage, respectively. Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor which in the central nervous system is mainly expressed on microglia. This receptor has been shown to play an essential role in microglial phagocytosis and function but its contribution in stroke pathobiology remains unclear. TREM2 was shown to be activated by nucleotides and lipid mediators, key factors that are secreted in the extracellular stroke environment by apoptotic neurons and cell/myelin debris. These factors in turn stimulate TREM2 signaling which mediates microglial migration toward- and phagocytosis of myelin debris and apoptotic cells. Moreover, microglial TREM2 appears to counteract the toll-like receptor response, thereby decreasing the production of pro-inflammatory cytokines. Finally, TREM2 is involved in microglial migration, survival, and is suggested to directly stimulate pro-regenerative phenotype shift. Therefore, this receptor is an attractive target for microglial modulation in the treatment of ischemic stroke and it provides additional information on microglial effector functions. This short review aims to elaborate on these TREM2-mediated microglial functions in the pathobiology and resolving of ischemic stroke.

Keywords: ischemic stroke, microglia, TREM2, immunomodulation, phagocytosis

# INTRODUCTION

Worldwide, stroke, of which ischemic stroke is the most prominent, is the second most common cause of death and is a major cause of permanent disability (1).  Microglia, the resident phagocytes of the central nervous system (CNS), continuously monitor the status of the brain parenchyma through extension and retraction of their processes (2). Furthermore, they are the first cells to become activated after stroke which migrate toward

#### Edited by:

*Jack Van Horssen, VU University Medical Center, Netherlands*

#### Reviewed by:

*Robert Adam Harris, Karolinska Institutet (KI), Sweden Long-Jun Wu, Mayo Clinic, United States*

> \*Correspondence: *Pascal Gervois<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology*

Received: *09 October 2018* Accepted: *03 July 2019* Published: *17 July 2019*

#### Citation:

*Gervois P and Lambrichts I (2019) The Emerging Role of Triggering Receptor Expressed on Myeloid Cells 2 as a Target for Immunomodulation in Ischemic Stroke. Front. Immunol. 10:1668. doi: 10.3389/fimmu.2019.01668* the site of injury to act as a first line of immune defense. Stroke outcome is determined by a fine balance of tissue-damaging pro-inflammatory and recovery-promoting pro-regenerative responses. Microglia play an important role in this inflammatory balance by obtaining a broadly defined disease promoting proinflammatory- or pro-regenerative phenotype (3–7). It should be noted that this classification will be used in the context of this review of ischemic stroke. There is an ongoing debate on phagocyte classification based on their activation state, activation stimuli, polarization, cellular origin, and phenotypical shift. However, this discussion will be left out of consideration but is thoroughly discussed by Ma et al. (7), Guilliams et al. (8), and Murray et al. (9).

Pro-inflammatory microglia dominate during the acute phase after stroke and are characterized by increased tumor necrosis factor alfa (TNF-α), interleukin (IL)-1β, IL-12, and inducible nitric oxide synthase (iNOS) expression amongst others. Pro-regenerative microglia (characterized by i.e., CD206 and Arginase-1 expression) play a protective role by stimulating tissue and vascular remodeling after stroke during the subacute and chronic phase. Pro-regenerative microglia secrete antiinflammatory cytokines such as IL-4, IL-10, and transforming growth factor β (TGF-β) and remodeling factors including vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) (7, 10, 11). Unfortunately, these microglia numbers decline following an initial peak 12– 24 h after stroke, whereas the number of pro-inflammatory microglia continue to rise, contributing to tissue damage and neuroinflammation (3, 10). Nonetheless, microglial polarization and the secretion of pro- and anti-inflammatory soluble factors remains a topic of interest in stroke research (10).

The clearance of apoptotic neurons and/or neuronal debris is an aspect of stroke repair that is often overlooked. Phagocytic clearance is indispensable in enabling the reconstruction and reorganization of neural networks and triggering tissue repair (12, 13). Moreover, in contrast to neuronal debris, phagocytosis of apoptotic cells is a controlled process that does not induce inflammation, thereby circumventing self-catalyzing secondary tissue damage that is characterized by free radical and nitric oxide production, glutamate excitotoxicity and the release of pro-inflammatory factors (14, 15). Recently, triggering receptor expressed on myeloid cells 2 (TREM2) emerged as a novel microglial target in stroke that is involved in phagocytosis and microglial function. TREM2 is expressed on myeloid cells including microglia in the CNS where its expression is over 400-fold higher than on other glial cells and neurons (16). The importance of this receptor in myeloid and microglial function is stressed by Nasu-Hakola disease, also known as polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy. This disease develops in patients with a deleterious or loss of function mutation in the TREM2 gene, or in its signaling adapter protein DNAX activating protein of 12 kDa (DAP12) (17, 18). These patients develop bone cysts due to dysfunctional osteoclasts (19) but also present with progressive dementia, motor dysfunction, seizures and shortened life span (18, 20). Moreover, the R47H TREM2 mutation increases the risk for Alzheimer's disease (AD) 3–4 fold (21, 22) and mutations in the TREM2 gene are thought to be linked with the impaired uptake of Aβ-lipoprotein complexes by microglia (23).

The significance of TREM2 in ischemic stroke was shown by Kawabori et al. who showed that TREM2 deficiency was detrimental for ischemic injury and microglial phagocytosis (24). Moreover, TREM2 overexpression suppressed the proinflammatory reaction of microglia in vitro (25, 26). More strikingly, a recent study by Kurisu et al. demonstrated that specifically microglial TREM2 expression is fundamental in stroke outcome and not TREM2 expression on circulating macrophages (27). In addition to microglia, perivascular and perimeningeal macrophages can be seen as a distinct brainresident macrophage with several indications for CNS diseases [reviewed in Faraco et al. (28)] which role has also recently emerged in stroke pathogenesis regulating vascular permeability and leukocyte chemotaxis in acute stroke (29). However, no reports are available describing the role of TREM2 in these cells and will therefore be left out of consideration. This review aims to provide an overview on the regulation and potential role of TREM2 in stroke pathobiology and resolution with a focus on microglial function as its role in these processes remains to be elucidated.

### STROKE PHAGOCYTOSIS AND INFLAMMATION

The accurate clearance of apoptotic neurons and neuronal debris is crucial for the resolution of CNS damage. Following (ischemic) injury, microglia are activated, migrate toward the lesion site, secrete pro- and anti-inflammatory factors and clear cellular debris and apoptotic cells (13, 30). In general, proper clearance of apoptotic cells is mediated through four essential processes. The first step is initiated by apoptotic cells that release "find-me" signals to attract phagocytes to the site of neuronal injury. These signals include the nucleotides (2, 31) adenosine triphosphate (ATP) and uridine triphosphate (UTP), lysophosphatidylcholine (LPC) (32), sphingosine-1-phosphate (S1P) (33), and fractalkine/CX3CL1 (34). The second step involves recognition of the dying cells by "eat me" signals of which phosphatidylserine is the most prominent and well-studied (35). After recognition, the target cell becomes internalized and the engulfed target becomes degraded in the maturating phagosome (36). Interestingly, in the fourth phase, the phagocyte releases anti-inflammatory mediators such as TGF-β1, IGF-1, and IL-10 (37–39) and suppresses the production of pro-inflammatory factors (40). For in-depth information on phagocytosis and the associated cellular and molecular mechanisms, see reviews by Medina and Ravichandran (41), Park and Kim (42).

These phagocytosis mechanisms hold when injury is limited and these "find me" signals are present at relatively low concentrations. However, in ischemic stroke, extracellular ATP levels increase dramatically due to neuronal and glial depolarization waves and membrane leakage (14). These high levels of ATP then not only serve as a chemoattractant for microglia, but can activate P2 × 7 receptors which leads to the production of pro-inflammatory factors (43). In the subsequent phase of ischemic cell death, the dying neuronal cells leak intracellular signal molecules that together with extracellular molecules can be seen as danger signals that activate the immune system (44). These so-called danger associated molecular pattern molecules (DAMPs) include high concentrations of extracellular ATP (45), extracellular matrix breakdown proteins (46), heatshock proteins (Hsp) (47), and the high mobility group box 1 protein (48). These DAMPs activate purine receptors and scavenger- or pattern recognition receptors on microglia, leading to the production of pro-inflammatory mediators by resident brain cells and infiltrating leukocytes (49). In this context, one of the most studied pattern recognition receptors is the membrane– bound Toll-like receptor (TLR) family in which the TLR2 and TLR4 subtypes are known to play an important role in ischemic stroke. TLR2 is most prominently expressed on microglia and TLR2 signaling increases the expression of pro-inflammatory and pro-apoptotic genes in the transient middle cerebral artery occlusion (tMCAO) mouse stroke model. Moreover, TLR2 deficient mice have a smaller infarct volume compared to controls (50). Similarly, TLR4-deficient mice had lower infarct volumes in a mouse model with permanent distal middle cerebral artery occlusion (dMCAO). Additionally, TLR4-deficient mice had a better score on a neurological and behavioral test. The lack of TLR4 decreased the expression of iNOS, cyclooxygenase-2 and matrix metalloprotease-9 amongst others, demonstrating an attenuation of the pro-inflammatory milieu that is created post-stroke (51).

Ultimately, acute stroke-associated inflammation is a selflimiting process that is coordinated by lipid mediators (52) that contribute to the effective removal of dead cells and debris, the generation of pro-regenerative milieu and the production of growth factors such as microglial IGF-1, VEGF, and BDNF that favor tissue regeneration. As will be discussed in the next section, TREM2 can interfere in the stroke immune response at multiple levels to determine stroke outcome.

#### THE NEUROIMMUNOLOGICAL FUNCTION OF TREM2—IMPLICATIONS FOR STROKE PATHOBIOLOGY

Microglia are key regulators of innate immunity in the CNS, protecting the parenchymal cells from injury. In addition to detecting extrinsic insults such as bacterial lipopolysaccharide (LPS), microglia are equipped with intrinsic DAMP-recognizing receptors like TLRs, as discussed previously. Similar to TLRs, TREM2 was shown to be capable of binding LPS and intrinsic ligands that are released during neuronal degeneration including lipids (53, 54), nucleic acids (24), and Hsps (55). However, the identification of the entire spectrum of endogenous TREM2 ligands and clarification of the ligandspecific physiological TREM2-mediated response in health and disease remains challenging [reviewed by Kober and Brett (56)]. Interestingly, fractalkine/CX3CL1-expressing damaged neurons stimulate TREM2 expression and attenuate neuronal excitotoxicity by enhancing the clearance of these injured neuronal cells by CX3CR1-expressing microglia (57, 58).

TREM2 is a single-pass transmembrane receptor that belongs to the immunoglobulin-like superfamily of receptors. The immunoglobulin-like ligand-binding domain is situated in the extracellular domain whereas the transmembrane domain facilitates the interaction with DAP12. This leads to the phosphorylation of the immunoreceptor tyrosine activation motif (ITAM) in DAP12 by Src kinases which subsequently activate Syk kinase to trigger intracellular signaling cascades involving the downstream signaling molecules phosphatidylinositol 3-kinase (PI3K), extracellular signalregulated protein kinase (ERK), phospholipase Cγ (PLCγ), and Vav that influence microglial activation, function, survival, and phagocytosis (53, 54, 59–61). Interestingly, TREM2 can be shed from cell membranes by proteolytic cleavage by for example the sheddase ADAM17 (a disintegrin and metalloproteinase domain containing protein 17), forming soluble TREM2 (sTREM2) that is able to cross to the cerebrospinal fluid (62, 63). The current knowledge on the role of sTREM2 in CNS pathology and microglial function is mainly based on AD research. A recent paper by Zhong et al. demonstrated that the stereotactic injection of sTREM2 or adeno-associated virus mediated activation of sTREM2 reduced the amyloid plaque load and reduced functional memory deficits. Moreover, sTREM2 stimulated microglial proliferation and homing toward amyloid plaques where amyloid-β uptake and degradation was increased. Interestingly, these effects were specifically mediated by microglia as they were absent upon microglial depletion (64). Remarkably, sTREM2 levels in the cerebrospinal fluid were recently proposed as a biomarker for AD and the associated inflammatory response (63, 65, 66). Unfortunately, the role of sTREM2 in ischemic stroke is to date unexplored, and will therefore be left out of consideration for stroke specific microglial TREM2 function in this context.

Based on these TREM2 effects on microglial function and the role of microglia in ischemic stroke, several mechanisms are proposed to be mediated by microglial TREM2 activity (**Figure 1**). The first and potentially the most thorough investigated role of TREM2 in stroke is its function in phagocytosis. A pioneer study by Takahashi et al. (59) demonstrated that microglial TREM2 is involved in the phagocytosis of apoptotic neurons. In this study, knockdown of TREM2 with short hairpin TREM2 RNA (shRNA) in primary mouse microglia decreased the phagocytic clearance of apoptotic neurons which coincided with the increased expression of proinflammatory TNF-α and nitric oxide synthase-2. Lentiviral overexpression and activation of TREM2 increased phagocytosis and attenuated the pro-inflammatory response. In addition to its role in phagocytosis, Takahashi et al. showed that the migratory capacity of microglia with activated TREM2 toward CCL21 is increased significantly. CCL21 is used by ischemic injured neurons as a "find me" signal to attract microglia, suggesting a role of TREM2 in the early phase after ischemic injury (67). Kawabori et al. demonstrated that TREM2 deficiency exacerbates ischemic injury in a dMCAO mouse stroke model in which TREM2 knockout (KO) and wildtype (WT) mice were compared

FIGURE 1 | Microglial TREM2 in stroke pathogenesis. Ischemic stroke leads to the massive loss of neuronal tissue by triggering apoptosis and/or necrosis. In turn, the dying tissue releases damage-associated molecular pattern (DAMP) molecules including nucleotides and ECM breakdown products in addition to lipid mediators being present in myelin and neuronal debris and on the surface of apoptotic cells. These events trigger both TLR and TREM2 activation on microglia. While classically the TLR response leads to a pro-inflammatory microglial phenotype characterized by the release of pro-inflammatory cytokines, TREM2 activation appears to counteract the TLR response, leading to a decrease in the production of pro-inflammatory cytokines. Moreover, microglial TREM2 stimulates migration of microglia toward the lesion site and promotes the phagocytic clearance of apoptotic cells and debris. Finally, TREM2 was shown to be associated with a pro-regenerative microglial phenotype and the production of pro-regenerative cytokines and is suspected to play a role in microglial survival. Based on these functions of TREM2, this receptor is an attractive target for microglial modulation in the treatment of ischemic stroke. This Figure was created using Servier Medical Art licensed under a Creative Common Attribution 3.0 Generic License, available online at http://smart.servier.com/.

(24). In vitro, knockdown of TREM2 in the BV-2 mouse microglial cell line using small interfering RNA (siRNA) directed against TREM2 inhibited phagocytosis of Neuro-2A cells that were rendered apoptotic by oxygen- and glucose deprivation (OGD), an in vitro model that mimics ischemic damage. This role of TREM2 in phagocytosis was also demonstrated in vivo. TREM2 deficient mice showed a reduced tissue resorption and therefore increased infarct size following ischemic damage compared to WT controls. Confocal microscopy confirmed these results as a significantly reduced direct contact between activated macrophages and TUNEL-positive dying cells at the stroke site in TREM2 KO mice was observed (<4% compared to almost 40% in WT animals). The reduced phagocytic clearance of apoptotic cells and debris in TREM KO mice was also reflected by the virtual absence of Oil Red O-stained foamy macrophages which stained positive due to the uptake of lipid-containing cellular debris and dead cells. In line with these histological findings, the Bederson score and ladder test, which both measure neurological deficits, demonstrated a worsened functional outcome in the TREM2 KO mice. Moreover, WT mice showed a gradual improvement in the ladder test to nearly baseline levels while TREM2 KO mice did not reach these values over 2 weeks (24). This stresses the importance of clearing neuronal debris to enable synaptic and axonal repair, as stated previously (13). More recently, Kurisu et al. demonstrated that it is specifically microglial TREM2 that is essential in stroke outcome by using bone marrow chimeric mice in which TREM2 KO mice were used as donor and WT mice as recipient. The results of this study indicated improved neurological outcome in the elevated body swing and adhesive removal test and smaller infarct volume in mice with intact TREM2. In addition, mice with no intact microglial TREM2 demonstrated a lower number of phagocytes near the stroke lesion that also showed reduced phagocytic activity (27). Similar observations have been made in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (68) and in the cuprizone-induced non-autoimmune demyelination model (54, 69). In EAE, TREM2 overexpressing bone-marrow derived macrophages demonstrated enhanced phagocytosis of apoptotic neurons and after intravenous injection, these cells cleared myelin debris and ameliorated the disease outcome (68). Supporting the role of TREM2 in EAE, Piccio et al. showed that inhibiting TREM2 activation is detrimental for EAE outcome (70). In the cuprizone model, the corpus callosum in TREM2 KO mice showed extensive demyelination, axonal damage and decreased clearance of myelin debris compared to WT controls (54, 69). Furthermore, Poliani et al. showed that myelin-associated lipids triggered microglial TREM2 signaling, stressing the need of microglial TREM2 for effective clearance of myelin debris (54). More support for the importance of microglial TREM2 in the phagocytic clearance of apoptotic cells, comes from AD research. Atagi et al. demonstrated that apoptotic neuronal cells have the ability to bind Apolipoprotein E (APOE), the major cholesterol carrier protein in the brain. APOE was demonstrated to be a TREM2 ligand that enhanced the microglial phagocytosis of apoptotic neurons and the phagocytic capacity of microglia was diminished in the ADassociated TREM2-R47H mutant (71). While ongoing research is focusing on the link in AD between TREM2 mutants and the APOEε4 isoform, which is the major risk factor for AD, the association of TREM2 and APOE in ischemic stroke and their putative intertwined signaling remains unclear. However, given the ability of apoptotic neurons to bind APOE and use it as an opsonisation mechanism to enhance TREM2-mediated microglial phagocytosis demonstrates that this ligand-receptor interaction plays in important role in the clearance of damaged neurons (72).

Second, TREM2 can drive the pro-inflammatory stroke microenvironment toward a pro-regenerative environment and can therefore exert a neuroprotective effect on the ischemic brain. In a study by Wu et al., TREM2 expression is upregulated in microglia following OGD in vitro, and in the ischemic penumbra after tMCAO in mice (25). In vivo, TREM2 silencing exacerbated stroke outcome as demonstrated by an increased lesion volume, the number of apoptotic neurons and a decrease in neurological function measured with the Modified Garcia Score. This correlated with an increase in TNF-α, IL-1β, and iNOS mRNA levels in vitro in microglia exposed to OGD. Conversely, TREM2 overexpression reduced TNF-α, IL-1β, and iNOS mRNA levels and decreased neuronal apoptosis in vitro. Moreover, this study demonstrated the role of the ERK and NF-κB in TREM2 signaling. TREM2 silencing increased phosphorylation of ERK and NF-κB in primary microglia exposed to OGD. Taken together, a tempering role of TREM2 signaling in proinflammatory pathways and a critical protective role for TREM2 in ischemia-induced neuronal injury is suggested (25). These findings are in line with Zhai et al. (26) that demonstrated that intraperitoneal administration of hsp60, a TREM2 agonist (55), or intraventricular injection of a TREM2 lentivirus had a neuroprotective effect in vivo. This study reported a decreased number of apoptotic cells and a smaller infarct volume. Interestingly, TREM2 expression is increased by an IL-4/IL-13 treatment, cytokines that induce a pro-regenerative phenotype in microglia whereas its expression is decreased after exposure to the pro-inflammatory polarizing factors LPS and interferonγ. Therefore, the authors suggest TREM2 as a novel proregenerative microglial marker. Moreover, pro-inflammatory iNOS, TNF-α, and IL-6 expression was increased in OGDexposed primary microglia and treated with TREM2 shRNA while Arginase-1 and BDNF levels were increased when these cells were treated with a TREM2 vector, indicating a proregenerative phenotype. In contrast to these studies and the research of Kawabori et al. that suggest a role of TREM2 in alleviating the pro-inflammatory response and its beneficial role in stroke outcome, Sieber et al. showed that in the subacute phase after stroke, the transcription of the pro-inflammatory cytokines IL-1α, IL-1β, and TNF-α was reduced in TREM2 KO mice (73). However, a difference in lesion size in WT and TREM2 KO mice was not observed. These opposing results may be explained from differences between the timing of the analysis, although with the exception of Zhai et al., peak TREM2 levels were observed 7 days post-ischemia. Given this 7 days period for TREM2 to reach peak levels and the abovementioned experiments, it can be postulated that a gradual increase in TREM2 expression is associated with a dampening of the pro-inflammatory microglial response elicited in the acute and sub-acute stroke phase. Another possibility for these opposing results is the stroke model used, although only Kawabori et al. used the dMCAO model compared to the filament model used by Zhai et al., Wu et al., and Sieber et al. Despite these different results, the majority of these stroke studies suggest a beneficial role of TREM2 in ischemic stroke (24–26). It should be noted that to date, no stroke stage-specific reports are available that go into detail in TREM2 expression dynamics and pathways that lead to TREM2 upregulation and effector mechanisms. This is of specific importance as TREM2 is a microglia-specific receptor involved in stroke resolution and microglial function varies between the acute, sub-acute and chronic stroke phase balancing between a pro-inflammatory and pro-regenerative phenotype (6, 7).

A third indication of the role of TREM2 in stroke is that TREM2 can inhibit the TLR-mediated pro-inflammatory response. This was first demonstrated in myeloid cells by Turnbull et al. who showed that in macrophages isolated from WT and TREM2 KO mice, the production of pro-inflammatory mediators was elevated in TREM2 KO macrophages after LPS stimulation (74). Similarly, Hamerman et al. showed an increased TLR-LPS response in DAP12 deficient mice and demonstrated that TLR responses are negatively regulated by TREM2 and DAP12 (75, 76). Although these studies show a connection between the TLR response and TREM2 signaling, mechanistic insights in the TLR-TREM2 interaction come from a study by Peng et al. using bone-marrow derived macrophages (77). The ERK pathway can be activated by TLR4 binding of LPS leading to the production of pro-inflammatory cytokines (78, 79). In the study by Peng et al., it was shown that the adapter protein Dok3 is phosphorylated by DAP12-ITAM which subsequently inhibits the RAS-ERK pathway, thereby reducing the production of pro-inflammatory cytokines such as IL-6 and TNF-α (77). However, the role of the TREM2-TLR interaction in ischemic stroke remains to studied into more detail. A recent study by Rosciszewski et al. (80) investigated this interaction on astrocytes, although it should be noted that TREM2 expression in microglia is almost 400 times higher than in astrocytes (16). This study showed that DAP12 overexpression suppressed LPS-induced astrocyte NF-κB activation in vitro, diminishing the pro-inflammatory response. Moreover, TREM2 expression in astrocytes was observed up to 14 days post-lesion in the ischemic penumbra. These results suggest that TREM2 expression in astrocytes negatively regulates the TLR4-mediated pro-inflammatory response by reducing NF-κB activation (80).

Finally, TREM2 signaling is associated with microglial survival. Sieber et al., Kurisu et al., and Kawabori et al. observed a decreased number of (activated) microglia in TREM2 KO models compared to WT controls (24, 27, 73). However, Kurisu et al. demonstrated that the number of microglia in the nonischemic brain is similar between TREM2 KO and WT mice (27). Therefore, the diminished number of microglia associated with the stroke lesion in TREM2 KO mice is due to the reduced chemotactic capacity in KO mice, which is supported by a previous study that reports a reduced microglial recruitment to sites of laser-induced neuronal injury in TREM2 KO mice (81). These results were in line with the cuprizone model used by Poliani et al. and Cantoni et al. who also showed decreased microglia numbers in TREM2 KO mice (54, 69). However, no plausible mechanism is identified. Nonetheless, the role of TREM2 in microglial survival and proliferation cannot be excluded, as Zheng et al. reported a reduction in microglial proliferation rate after TREM2 downregulation (82). However, no analysis was performed to unravel the TREM2 targets involved with the decreased proliferation rate. Other reports on the involvement of TREM2 in microglial proliferation come from AD research. A possible mechanism of TREM2-sustained microglial survival come from Wang et al. who demonstrated in an AD model using TREM2 KO 5XFAD mice that microglial survival is diminished compared to 5XFAD control mice. They determined that TREM2 signaling is essential for microglial survival at low concentrations of colony-stimulating factor 1 (CSF-1). The same group showed that TREM2 synergizes with CSF-1-CSF-1 receptor signaling, contributing to microglial survival. Moreover, this study suggests that the lipid-sensing function of TREM2 provides a survival function in addition to triggering phagocytosis of apoptotic cells and myelin debris (53).

#### CONCLUDING REMARKS

Microglia play an essential role in both the pro-inflammatory and pro-regenerative response that is triggered following ischemic injury and can therefore be seen as a double-edged sword in stroke pathobiology. TREM2 signaling mediates microglial phagocytosis of myelin debris and apoptotic cells, microglial migration and survival, and can drive the microglial activation status toward an anti-inflammatory phenotype. Therefore, targeting TREM2 signaling has become a therapeutic target as it was shown that the systemic administration of a TREM2 agonist or TREM2 overexpression had a neuroprotective effect in

#### REFERENCES

ischemic injury. However, it should be noted that the temporal dynamics of TREM2 expression and effector mechanisms in ischemic stroke remain to be elucidated and current insights in TREM2 function are mainly derived from different disease models and cell types. Nonetheless, the role of TREM2 in ischemic stroke is mainly considered beneficial, and the same holds for its role in AD and demyelinating disease although few studies report a detrimental effect of TREM2 signaling in these neural diseases (83). Moreover, the effect of sTREM2 in ischemic stroke remains unexplored to date and given its implications in AD pathobiology, sTREM2-signaling provides an attractive new target in the stroke research field. Despite the great promise for TREM2 in ischemic stroke, TREM2 signaling has opposite effects in peripheral nerve injury including motor nerve injury (84) and neuropathic pain (85), and after traumatic brain injury (86). This stresses the importance of additional diseasespecific understanding of the TREM2-DAP12 signaling effects. Nonetheless TREM2 provides an attractive target for microglial modulation in the treatment of ischemic stroke and it offers additional information on microglial effector functions in this devastating disease.

### AUTHOR CONTRIBUTIONS

PG conceived and wrote the manuscript. IL revised and edited the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.

#### FUNDING

PG is a postdoctoral researcher of Fonds Wetenschappelijk Onderzoek Vlaanderen, supported by grant 12U7718N.

#### ACKNOWLEDGMENTS

**Figure 1** was created using Servier Medical Art licensed under a Creative Common Attribution 3.0 Generic License, available online at: http://smart.servier.com/.

by promoting M2 macrophage polarization. Stroke. (2015) 46:1929–37. doi: 10.1161/STROKEAHA.115.009426

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

Copyright © 2019 Gervois and Lambrichts. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Antigenic Targets of Patient and Maternal Autoantibodies in Autism Spectrum Disorder

Rut Mazón-Cabrera† , Patrick Vandormael † and Veerle Somers\*

*Biomedical Research Institute, Faculty of Medicine and Life Science, Hasselt University, Diepenbeek, Belgium*

Autism spectrum disorder (ASD) is a neurodevelopmental disorder whose behavioral symptoms become apparent in early childhood. The underlying pathophysiological mechanisms are only partially understood and the clinical manifestations are heterogeneous in nature, which poses a major challenge for diagnosis, prognosis and intervention. In the last years, an important role of a dysregulated immune system in ASD has emerged, but the mechanisms connecting this to a disruption of brain development are still largely unknown. Although ASD is not considered as a typical autoimmune disease, self-reactive antibodies or autoantibodies against a wide variety of targets have been found in a subset of ASD patients. In addition, autoantibodies reactive to fetal brain proteins have also been described in the prenatal stage of neurodevelopment, where they can be transferred from the mother to the fetus by transplacental transport. In this review, we give an extensive overview of the antibodies described in ASD according to their target antigens, their different origins, and timing of exposure during neurodevelopment.

Keywords: autism, immunoglobulins, brain antigens, neurodevelopement, placental transfer

# INTRODUCTION

Autism spectrum disorder (ASD) is a complex and highly heterogeneous neurodevelopmental disorder characterized by persistent deficits in social communication and social interaction (1). The patients also show restricted and repetitive patterns of behavior, interests and activities. Together with these symptoms, various comorbidities can occur, such as intellectual and language impairment, catatonia, epileptic seizures or attention-deficit/hyperactivity disorder (ADHD). These deficits can cause difficulties for the patient to understand, maintain or develop relationships, creating a barrier to their integration in social life.

ASD has become one of the most common neurodevelopmental disorders. The prevalence of ASD among 8-year-old children in the United States has more than doubled since 2000, increasing from 1 in 150 to 1 out of 59 children (2), and varies by sex, race/ethnicity and geographic area. It is four times higher among males than females and most common in non-Hispanic white children.

The diagnosis of ASD, usually made in early childhood, is based on observation of the atypical behaviors using questionnaires such as the Autistic Diagnostic Observation Schedule (ADOS) or Autism Diagnostic Interview-Revised (ADI-R). In each patient, the symptoms can have different manifestations caused by multiple etiologies, sub-types and developmental trajectories, often in combination with the different comorbidities, creating a complex condition (3).

#### Edited by:

*Fabienne Brilot, University of Sydney, Australia*

#### Reviewed by:

*Enrico Castroflorio, Medical Research Council Harwell (MRC), United Kingdom Anna Fogdell-Hahn, Karolinska Institute (KI), Sweden*

\*Correspondence:

*Veerle Somers<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology*

Received: *08 April 2019* Accepted: *13 June 2019* Published: *19 July 2019*

#### Citation:

*Mazón-Cabrera R, Vandormael P and Somers V (2019) Antigenic Targets of Patient and Maternal Autoantibodies in Autism Spectrum Disorder. Front. Immunol. 10:1474. doi: 10.3389/fimmu.2019.01474*

ASD is considered a polygenic hereditary disorder, whose genetic component mostly comprises of common variant singlenucleotide polymorphisms (SNP) and copy number variations (CNV) which can occur both in coding and non-coding regions (4). Moreover, the presence of autistic behaviors in other monogenic disorders such as Angelman syndrome and Fragile X syndrome only shows part of the complexity of this developmental disorder (5). Besides a genetic predisposition, ASD is also strongly influenced by environmental factors, which mostly play their specific roles before, during and after pregnancy (6). Factors as diverse as parental age, prematurity, pollution or geographic area have been found to contribute to increased risk of ASD (2, 7–9).

An overarching understanding of the etiology of the disorder is still lacking, but many areas and processes in the brain have been implicated. Disturbances in activation and regulation of the posterior insula, limbic system or the cortico-striato-thalamocortical circuit, contribute to the picture of a complex disease, not restricted to a single anatomical location (10–12). At the cellular level, alterations in synaptogenesis, synaptic plasticity and pruning, add another dimension of complexity (13–15).

In the last decade, an important role of the immune system has been described in the development of ASD. The activation of microglia, the resident immune cells of the brain, and increased levels of pro-inflammatory cytokines in brain tissue, cerebrospinal fluid and blood give evidence for the presence of ongoing neuroinflammation in brains of some ASD patients (16–19). Moreover, in utero exposure to an inflammatory environment during pregnancy caused by maternal autoimmune disease or maternal immune activation by infections, is sufficient to impart lifelong neuropathology and altered behaviors in offspring (20, 21).

Although ASD and other neurodevelopmental disorders are not typically considered as autoimmune diseases, autoantibodies, or antibodies that bind to self-antigens, have been detected in the sera of patients with ASD, ADHD, bipolar affective disorder and schizophrenia (22). Autoantibodies against known targets have been linked to the development of behavioral symptoms in these disorders, for example anti-dopamine transporter autoantibodies have been related with severity and recovery in ADHD, antibodies against myelin basic protein (MBP) have higher titers in patients with negative symptoms in schizophrenia or N-methyl-D-aspartate receptor (anti-NMDAR) and glutamic acid decarboxylase (anti-GAD) antibodies have been associated with acute maniac episodes in bipolar disorder (23–25). The beststudied example are autoantibodies directed against the NMDAR in a condition known as anti-NMDAR encephalitis, characterized by the development of psychosis, cognitive problems and seizures. The pathogenic role of anti-NMDAR autoantibodies in this disorder has been demonstrated in cultured neurons and in animal models (26–28). Moreover, patients have even been shown to respond to immunotherapy (29).

Besides the formation of autoreactive antibodies in ASD patients themselves, such antibodies have also been described in the blood of mothers whose child will develop ASD (**Figure 1**) (30, 31). These maternal autoantibodies can be transferred from the mother to the child during pregnancy via transplacental transport and might play an etiological role in ASD as they can pass the immature fetal blood-brain barrier (BBB) which is actively changing and semi-permeable during neurodevelopment (32, 33).

In this review, we explore the current evidence of antibodies detected in ASD patients and in mothers of ASD patients, their possible role as biomarkers to support the disease diagnosis and their contribution in the understanding of disease mechanisms.

# AUTOANTIBODIES IN ASD PATIENTS

Autoantibodies in an ASD patient were first described by Todd et al. (31) in a study on hyperserotonemia. Antibodies in the blood and cerebrospinal fluid (CSF) of an ASD patient could block binding of radioactive serotonin to a human cortical brain homogenate. Although the presence of these autoantibodies was not supported by follow-up studies (34, 35), the idea that a subset of ASD patients may have antibodies against specific selfantigens in the brain started to gather much interest. Since then, autoantibodies in ASD patients have been mostly studied by looking at known, mostly brain-related autoantigens which have also been described in other autoimmune conditions (described below and in **Table 1**). Whether these specific autoantibodies can cross the BBB, bind to their antigens in the brain and contribute to disease worsening, has not been demonstrated experimentally so far. Nonetheless, an increased general permeability of the BBB has been described for several psychiatric disorders, including ASD [reviewed in Kealy et al. (52)] which could provide a mechanism that would allow these autoantibodies to reach the brain. Moreover, most of the autoantibody targets described in ASD patients are localized intracellularly. It has long been assumed that large biomolecules such as antibodies cannot traverse the plasma membrane. However, for some anti-DNA antibodies in systemic lupus erythematosus (SLE) patients, membrane translocation and nuclear targeting has been demonstrated (53). Still, for the ASD-related autoantibodies described below, such mechanisms have not been investigated.

# Protein Targets Myelin Basic Protein (MBP)

Myelin basic protein (MBP) is, together with the proteolipid protein (PLP), the most abundant protein in the myelin membrane (54). It is present on the cytoplasmic side of the membrane in oligodendrocytes of the central nervous system (CNS) and peripheral nervous system (PNS) (55). Its main function is structural, participating in membrane stabilization required for the formation of the myelin sheath, which insulates axons and allows saltatory action potential transmission (56). During brain development, myelination starts in the last weeks of the third trimester of pregnancy, and continues in adult life (57). Myelin integrity is essential for proper cognition, as damage in sheath formation can have severe consequences in the integrity of the white matter networks (58). Alterations in white matter development have been associated with autism manifestations, making MBP a highly interesting protein in the understanding of this effect (10, 59). Moreover, MBP damage can lead to induction

of inflammatory and chemotactic mediators which are able to alter the permeability of the BBB (60).

Based on the first descriptions of autoantibodies and abnormal immune function in autistic children (31, 61, 62), Sing et al. (36) investigated the presence of immunoglobulin G (IgG) antibodies against MBP in ASD. MBP autoantibodies had already been described in the CSF of patients with multiple sclerosis (MS) (63).  MBP-reactive antibodies were found using Western blot in 58% of ASD children, while reactivities in the different control populations were much lower, ranging between 0 and 22% (**Table 1**). In several follow-up studies, similar or even higher IgG reactivities were reported in autistic children using enzyme-linked immunosorbent assays (ELISA) against MBP (38–40). On the other hand, a study by Libbey et al. (37) could not replicate the presence of these anti-MBP antibodies.

Besides anti-MBP antibodies of the IgG isotype, IgA and IgM antibodies against MBP have also been reported in ASD patients (38, 40). Moreover, autoantibodies of all isotypes were also found against myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), showing the diversity of the immune reactivity against key myelin proteins in ASD patients. Therefore, these autoantibodies might be informative on the underlying processes of autoimmunity in ASD patients.

However, since anti-MBP antibodies have already been described in several other diseases such as multiple sclerosis, Parkinson's disease, rheumatoid arthritis or schizophrenia (24, 58, 60, 64, 65), it seems unlikely that they would be useful as specific biomarkers for the diagnosis of ASD. Autoantibodies against myelin proteins might still have prognostic value for ASD patients, since serum levels of anti-MBP and anti-MAG antibodies were shown to be increased in severe autism patients compared to patients with mild or moderate disease severity (66).

The reported studies on autoantibodies against MBP in ASD children all have used small patient and control cohorts. Therefore, studies in multiple, independent large cohorts are required using similar detection assays to settle the reported differences in anti-MBP reactivity and to confirm a possible link with disease severity.

#### Neurofilament Protein

Neurofilaments are a class of intermediate filaments, which are part of the neural cytoskeleton, especially in axons of asymmetric, myelinated neurons involved in nerve conduction (67). They are composed of a triplet of proteins with different molecular weights (neurofilament light, 68 kDa; middle, 160 kDa and heavy, 200 kDa) (67). Of these filament proteins, the neurofilament heavy protein (NF-H), also called neuron-axon filament protein (NAFP), is considered as a stable marker for neuron-axon filaments. It has an important role in the development of axons and the transport of vesicles via binding of kinesins, dynein complexes and kinases (68–71). Neurofilament subunit variants have been reported in the development of motor disorders and axonal neuropathies such as amyotrophic lateral sclerosis and Frontiers in Immunology | www.frontiersin.org

#### TABLE 1 | Overview of ASD patient autoantibodies.

  *not*  Patient and

Maternal Autoantibody Targets

*aSensitivity is the percentage of seropositive ASD patients, the amount of seropositive patients over the total tested patient cohort is indicated between brackets. bSpecificity is the percentage of seronegativity in the control populations(TD, MR, A, DS, OND, NNI, HC, AD, ADHD, LKSV), the amount of seropositive controls over the total amount of each control cohort is indicated between brackets. cAge range of study population; NA, not applicable; NR,reported; MR, mental retardation; TD, typically developing; A, adults; DS, Down syndrome; OND, other neurological disorders; NNI, non-neurological illnesses; HC, healthy children; ADHD, attention-deficit/hyperactivity disorder; LKSV,Landau-Kleffner syndrome variant; AD, autoimmune disorder; DSM-III, Diagnostic and statistical manual of mental disorders third edition; DSM-IV, Diagnostic and statistical manual of mental disorders fourth edition; ADOS, AutisticDiagnostic Observation Schedule; ADI-R, Autism Diagnostic Interview-Revised; CARS, Chilhood autism rating scale; ABC, Autism behavior checklist.*

Charcot-Marie-Tooth disease (72, 73). Autoantibodies against NF-H have been described in normal aging, but also in degenerative disorders such as Alzheimer's disease, multiple sclerosis and prion diseases (74–77). In ASD, IgG antibodies reactive against bovine NF-H (NAFP) were found using Western blot in nearly 55% of the patients studied (**Table 1**) (41). However, the percentage of seronegativity in control populations (i.e., specificity) of these autoantibodies is lower than 80% in controls with a typical development and idiopathic mental retardation. A study by Vojdani confirmed the increased presence of IgG, IgM, and IgA autoantibodies against NAFP in autistic patients (40).

#### Glial Fibrillary Acidic Protein (GFAP)

The glial fibrillary acidic protein (GFAP) is a class III intermediate filament cytoskeletal protein. This protein allows the distinction between astrocytes and other glial cells, and is a common astroglial marker in the CNS. Furthermore, it is present in non-myelinating Schwann cells in the PNS and in enteric glial cells (78). In case of acute brain injury, astrocytes become activated, strongly upregulate GFAP expression and increase in size and number, a state called astrogliosis (79). After stroke, traumatic brain injury and spinal cord injury, GFAP, and GFAP breakdown products have been found in CSF and serum (80). These breakdown products trigger an autoantibody response against GFAP in a large subset of patients (81, 82). Elevated GFAP levels have also been described in brain tissue, CSF and serum of ASD patients (83–86), as is the associated autoimmune reactivity. IgG antibodies against GFAP were detected using Western blot in 32% of ASD patients with a specificity of 91% in typically developing subjects, but only 79% in idiopathic mental retarded subjects (**Table 1**) (41). However, in a small study by Kirkman et al. (87), the average titer of anti-GFAP antibodies in autistic patients was not found to be significantly higher than in controls, raising questions about the role of this antibodymediated response in this developmental disorder.

#### Ribosomal P Proteins