TRANSLATIONAL SCIENCE

Pathogenic neuropsychiatric effect of stress-­induced
microglial interleukin 12/23 axis in systemic
lupus erythematosus
Nobuya Abe ‍ ‍,1,2 Masato Tarumi,1,2 Yuichiro Fujieda ‍ ‍,2 Nobuhiko Takahashi,1Kohei Karino,2 Mona Uchida,1 Michihito Kono ‍ ‍,2 Yuki Tanaka,1,3 Rie Hasebe,4,5Masaru Kato ‍ ‍,2 Olga Amengual,2 Yoshiyuki Arinuma ‍ ‍,6 Kenji Oku,2,6
Wakiro Sato,7 Khin Khin Tha,8,9 Miwako Yamasaki,10 Masahiko Watanabe,10
Tatsuya Atsumi,2 Masaaki Murakami ‍ ‍1,3,5
Handling editor Josef S
Smolen► Additional supplemental
material is published online
only. To view, please visit
the journal online (http://​dx.​
doi.​org/​10.1​ 136/​ard-​2022-​
222566).

For numbered affiliations see
end of article.
Correspondence toProfessor Masaaki Murakami,
Division of MolecularPsychoimmunology, Institute for
Genetic Medicine, Graduate
School of Medicine, Hokkaido
University, Sapporo, Japan;​murakami@​igm.h​ okudai.​ac.​jp
and Professor Tatsuya Atsumi,
Department of Rheumatology,
Endocrinology and Nephrology,
Faculty of Medicine, Hokkaido
University, Sapporo, Japan;​at3tat@​med.h​ okudai.​ac.​jp
Received 29 March 2022
Accepted 14 June 2022

© Author(s) (or theiremployer(s)) 2022. Nocommercial re-­use. See rights
and permissions. Publishedby BMJ.
To cite: Abe N,
Tarumi M, Fujieda Y, et al.
Ann Rheum Dis Epub aheadof print: [please include Day
Month Year]. doi:10.1136/
annrheumdis-2022-222566

ABSTRACT
Objectives The central nervous system disorderin systemic lupus erythematosus (SLE), calledneuropsychiatric lupus (NPSLE), is one of the most
severe phenotypes with various clinical symptoms,
including mood disorder, psychosis and delirium asdiffuse neuropsychological manifestations (dNPSLE).
Although stress is one of the aggravating factors forneuropsychiatric symptoms, its role in the pathogenesisof dNPSLE remains to be elucidated. We aimed toinvestigate stress effects on the neuropsychiatricpathophysiology in SLE using lupus-­prone mice andpatients’ data.
Methods Sleep disturbance stress (SDS) for 2 weeks
was placed on 6–8-­week-­old female MRL/lpr andcontrol mice. Behavioural phenotyping, histopathologicalanalyses and gene and protein expression analyses were
performed to assess SDS-­induced neuroimmunologicalalterations. We also evaluated cytokines of thecerebrospinal fluid and brain regional volumes in patientswith dNPSLE and patients with non-­dNPSLE.
Results SDS-­subjected MRL/lpr mice exhibited lessanxiety-­like behaviour, whereas stressed control mice
showed increased anxiety. Furthermore, stress strongly
activated the medial prefrontal cortex (mPFC) in SDS-­
subjected MRL/lpr. A transcriptome analysis of the PFC
revealed the upregulation of microglial activation-­related
genes, including Il12b. We confirmed that stress-­induced
microglial activation and the upregulation of interleukin(IL) 12/23p40 proteins and increased dendritic spines
in the mPFC of stressed MRL/lpr mice. IL-­12/23p40
neutralisation and tyrosine kinase 2 inhibition mitigated
the stress-­induced neuropsychiatric phenotypesof MRL/lpr mice. We also found a higher level of
cerebrospinal fluid IL-­12/23p40 and more atrophy in themPFC of patients with dNPSLE than those with non-­dNPSLE.
Conclusions The microglial IL-­12/23 axis in the mPFC
might be associated with the pathogenesis and apromising therapeutic target for dNPSLE.

INTRODUCTION

Systemic lupus erythematosus (SLE) is an autoimmune disease with a predilection for young
women of childbearing age, leading to a profound

WHAT IS ALREADY KNOWN ON THIS TOPIC⇒ Neuropsychiatric lupus with diffuse
neuropsychological manifestations (dNPSLE)
is attributed to a variety of factors, including
vascular occlusions, blood–brain barrier
impairment, cytokines, autoantibodies anddirect neuronal cell damage.
⇒ Stress affects the activation status of central
neurons and glial cells, probably leading toneuroinflammation.
⇒ Stress, particularly chronic stress, is involvedin the development of autoimmune diseases,
including lupus, and has adverse effects on
the disease activity with neuropsychiatric
symptoms.
WHAT THIS STUDY ADDS
⇒ Stress-­subjected MRL/lpr mice showeddisinhibited behaviour, microglial activation
with IL-­12/23p40 upregulation and neuronal
activation in the medial prefrontal cortex(mPFC).
⇒ Anti-­IL-­12/23p40 neutralising antibody ortyrosine kinase 2 inhibitor ameliorated thesestress-­elicited neuropsychiatric phenotypes in
MRL/lpr.
⇒ A higher level of IL-­12/23p40 in the
cerebrospinal fluid and more atrophic changes
in the mPFC were observed in patients withdNPSLE than those with non-­dNPSLE.
HOW THIS STUDY MIGHT AFFECT RESEARCH,
PRACTICE AND/OR POLICY⇒ An association of the stress-­elicited IL-­12/23
axis in the mPFC with the pathogenesis of
disinhibited agitative behaviour in patients withdNPSLE was suggested.
⇒ Blockade of IL-­12/23 signalling in the mPFC
may be a novel therapeutic target for dNPSLE.
impact on their lives. Patients with SLE manifest
multiple organ disorders such as skin rash, arthritis,
nephritis, haematologic abnormality and inflammation in the central nervous system (CNS).1 Amongthem, CNS diseases occur in up to 50% of patients

Abe N, et al. Ann Rheum Dis 2022;0:1–12. doi:10.1136/annrheumdis-2022-222566   1

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Systemic lupus erythematosus

and demonstrate a wide range of symptoms, including headache,stroke, anxiety, depression, cognitive dysfunction, seizures,
psychosis and acute confusional state (ACS). The CNS disorder,
neuropsychiatric SLE (NPSLE), is one of the most severe manifestations and is further classified into diffuse neuropsychological syndrome (dNPSLE) and focal neurological syndrome(fNPSLE). Although a variety of factors, including vascularocclusions, blood–brain barrier impairment, cytokines, autoantibodies and direct neuronal cell damage, have been suggested forthe development of dNPSLE,2 3 its pathogenesis remains poorly
understood. In addition, neuropsychiatric symptoms often occur
independently of the systemic disease activity among patientswith SLE, making it difficult to predict the development ofdNPSLE.4 Rheumatologists have been using glucocorticoids andpancytotoxic immunosuppressants for treatment, but there is
no available therapeutic strategy that targets the disease-­specificpathogenesis.1 5
Stress as emotional and physiological challenges can affect theneural activation status in the CNS. In the short term, it promotes
allostasis for adaptation to the surrounding environment, but inthe long term, it exhausts the body physically and mentally. It
has been known that stress affects the functions of several physiological systems via the neuroendocrine pathway.6 Stress also
targets and remodels the CNS itself structurally and functionally, contributing to alterations in behavioural and physiologicalresponses.7 In animal models, stress induced by sleep deprivation atrophies hippocampal and cortical neurons, resulting
in impaired retention and memory.7–9 Also, sleep deprivation
increases cytokines and oxidative stress markers in neurons.7
We previously reported sleep disturbance stress (SDS) induced
brain microinflammation in the presence of CNS-­specific autoreactive CD4-­positive T cells, exacerbating clinical symptoms
of experimental autoimmune encephalomyelitis (EAE).10 Thus,
stress together with immune cells would be involved in the exacerbation of autoimmune diseases. Indeed, SDS is reported to
impair metabolism and upregulate proinflammatory cytokine inhumans.11 12 Stress exposure also increases the risk of developing
autoimmune diseases, including lupus, and has adverse effects
on the disease activity with neuropsychiatric manifestations in
SLE.13 14 However, the molecular mechanisms of stress on thedNPSLE pathogenesis are still unknown. Therefore, we here
investigated whether SDS promotes neuropsychiatric symptoms
and the molecular pathogenesis using lupus-­prone mice and its
association with patients with dNPSLE.

atrophic brain regions using voxel-­based morphometry (VBM)
in the patients.

Statistical analysis

Experimental data using mice were analysed with an unpaired
Student’s t-­test and two-­way analysis of variance with Tukey-­
Kramer post-­hoc multiple comparisons test or paired t-­test for
the unpaired or paired values of continuous variables, respectively. For the analyses of human sample data, we used the Mann-­
Whitney U test and Kruskal-­Wallis test with post-­hoc Steel-­Dwass
multiple comparison method for the values of continuous variables and χ2 test for the proportions of categorical variables. To
explore differentially expressed genes in the RNA-­seq analysis,
we used adjusted p values with false discovery rate correction by
the Storey method for genes with an absolute fold change (FC)over 1.5 compared with control strains. A receiver operating
characteristics analysis was performed to evaluate the diagnostic
ability of the data for dNPSLE with the area under the curve(AUC). Neuroimaging data were analysed by analysis of covariance with age, sex, disease duration, total intracranial volume
and white matter (WM) volume as confounding factors. In thelinear correlation analysis, Pearson’s product moment correlation coefficients were calculated. P values lower than 0.05 wereconsidered statistically significant. We used JMP Pro V.14 (SASInstitute, USA) for all analyses. All statistical tests were two-­sided, and all experiments were performed at least two times.

RESULTS
Sleep disturbance stress-induced abnormal behaviour inlupus-prone mice

Briefly, we used lupus-­prone mouse models with SDS load. In
animal experiments, 6–8-­week-­old female mice were used foreach experiment. Behavioural phenotyping, histopathologicalanalyses, RNA sequencing (RNA-­seq), flow cytometry and ELISA
were performed to assess SDS-­induced behavioural changes andneuroimmunological alterations. We retrospectively reviewed
medical records of the patients with SLE whose serum and cerebrospinal fluid (CSF) were preserved at Hokkaido University andKitasato University between 2006 and 2020. We also collected
the clinical data of 71 consecutive patients who underwent brain
MRI at Hokkaido University Hospital between 2019 and 2020.
We evaluated serum and CSF cytokine levels using ELISA andWe employed MRL/MpJJmsSlc-­lpr/lpr (MRL/lpr) female mice,
which show SLE-­
like manifestations, including nephritis and
CNS symptoms.15 For the stress load, we used SDS loading cages,
in which continuous stress to inhibit regular sleep is imposed onmice on a free rotation wheel for 2 weeks (figure 1A, online
supplemental figure 1A). Mice housed in SDS cages showed
more activity throughout the night and higher levels of serum
corticosterone than those housed in a normal cage (online
supplemental figure 1B-­
F). We then performed behavioural
phenotyping of SDS-­
subjected MRL/lpr mice and of control
MRL/MpJJmsSlc-+/+ (MRL/MpJ) mice. We employed anelevated plus maze test (EPM) and open field test (OF) to assess
risk-­taking agitation-­like behaviours, which are often observedin patients with dNPSLE with ACS as vigilance and psychomotor overactivity.16 In the EPM, SDS-­subjected control micetended to show more anxiety-­like behaviour than mice without
stress, which is commonly seen in chronic stress-­exposed mice.
In contrast, SDS-­
subjected MRL/lpr showed significantly lessanxiety-­like behaviour than control (figure 1B,C). There wasa slight but significant difference in total travelling distancein the EPM between the SDS-­subjected MRL/lpr and control
mice (figure 1D). Consistently, SDS-­
subjected MRL/lpr mice
showed less anxiety-­like behaviour than SDS-­free MRL/lpr mice
without any decrease in general locomotor activity in the OF(figure 1E–G). To exclude the possibility that systemic inflammation makes SDS-­subjected mice disinhibited on the behaviour, weevaluated the SDS effect in NOD/ShiJcl, which is a mouse modelof chronic inflammatory autoimmune disease like Sjögren’s
syndrome (SS). In contrast to SDS-­
subjected MRL/lpr, SDS-­
receiving NOD/ShiJcl demonstrated similar behaviour to SDS-­
free mice in the EPM (online supplemental figure 1G,H). Thus,
stress had disinhibitory effects on the behaviours of MRL/lpr
mice.

2

Abe N, et al. Ann Rheum Dis 2022;0:1–12. doi:10.1136/annrheumdis-2022-222566

PATIENTS, MATERIALS AND METHODS

Detailed information about each experiment and statistical analysis is described in online supplemental patients, materials andmethods.

Study design

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Systemic lupus erythematosus

Figure 1 Stress-­elicited abnormal behaviour and mPFC activation in lupus-­prone mice. (A) The study protocol for chronic SDS. (B–D) EPM in
SDS-­subjected or SDS-­free MRL/lpr and control mice (MRL/MpJ). (B) representative trajectories. (C) Percentage of time spent in the open arms and(D) total travelled distance to assess anxiety-­like behaviour and general locomotor activity, respectively (n=12–17 per group). (E–G) OF results.
(E) representative tracking images. (F) Percentage of time spent in the centre zone and (G) total distance travelled to assess anxiety-­like behaviour
and general locomotor activity, respectively (n=14–20 per group). Data are means±SEM. *p<0.05, **p<0.01 and ****p<0.0001 using a two-­way
ANOVA with post-­hoc Tukey-­Kramer multiple comparison test. ANOVA, analysis of variance; EPM, elevated plus maze test; mPFC, medial prefrontal
cortex; OF, open field test; SDS, sleep disturbance stress.

Persistent neuronal activation in medial prefrontal cortex ofstress-subjected MRL/lpr miceover NAc activation induced-­anxious effect in SDS-­
subjected
MRL/lpr mice.

One of the representative vrain regions receiving stress stimulus is the paraventricular nucleus (PVN).17 PVN neurons sendaxonal projections to the ventral tegmental area (VTA) as theorigin of dopaminergic neurons that project to the medial
prefrontal cortex (mPFC) and nucleus accumbens (NAc).18 19
Previous reports demonstrated that acute single stress activatesthe mPFC and represses anxiety-­like behaviour, while chronic
stress causes anxiety by inhibiting the mPFC and activating NAc
neurons.20 21 Therefore, we investigated neuronal activity inthese regions in the presence or absence of SDS (figure 2A,B). In
the PVN, both SDS-­subjected strains showed a higher number ofphosphorylated cFos (p-­cFos)-­positive cells than SDS-­free mice(online supplemental figure 2A-­C). In the VTA, more p-­cFos
positive cells were observed in SDS-­subjected mice than SDS-­free
mice regardless of the mouse strain (figure 2C–E). In contrast,
only SDS-­
subjected MRL/lpr mice showed elevated neuronal
activation in the mPFC (figure 2F–H). Meanwhile, both stressedstrains showed more p-­cFos positive cells in the NAc comparedwith SDS-­free strains (figure 2I–K). In contrast, among NOD/
ShiJcl mouse strain, SDS-­subjected mice showed similar numberof p-­cFos-­positive cells to SDS-­free mice (online supplemental
figure 2D,E). These findings suggest that the mPFC is an
important region for anxiolytic effect for disinhibited behaviour

We hypothesised that the stress load alters gene expression
profiles in the mPFC of MRL/lpr mice. An RNA-­seq transcriptome analysis of the PFC among the strains detected 7952
differentially expressed genes with an absolute FC over 1.5. A
principal component analysis revealed a stress-­
induced effecton the gene expressions corresponding to the mouse strains(figure 3A). Among these genes, 509 genes were significantly
upregulated or downregulated in SDS-­subjected MRL/lpr micecompared with the other mouse groups (figure 3B). Using
these genes, an ingenuity pathway analysis identified pathways
associated with inflammatory and neuronal signalling, suchas phagosome formation, neuroinflammation, Th1 pathway,
protein-­coupled receptor and cAMP response elementG-­binding protein (CREB) signalling (figure 3C, online supplemental figure 3A). Consistently, Gene Ontology and Kyoto
Encyclopedia of Genes and Genomes pathway analyses detected
pathways related to the positive regulation of cytokine production, G protein-­coupled receptor activity and neuroactive ligand
receptor interaction (figure 3D, online supplemental figure 3B).

Abe N, et al. Ann Rheum Dis 2022;0:1–12. doi:10.1136/annrheumdis-2022-222566

3

Enhanced microglia activation signatures in the pFc ofstressed MRL/lpr mice

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Systemic lupus erythematosus

Figure 2 Neuronal activation of stress-­responsive brain regions. (A) The representative stress-­responsive neuronal pathway. The PVN sends axonal
connections to the VTA, which projects to the mPFC and NAc. (B) Representative images of phosphorylated cFos (p-­cFos)-­immunostained cells with
DAPI. Scale bar: 20 µm. (C–K) Evaluation of neuronal activation using immunohistochemistry of p-­cFos-­positive cells in the (C–E) VTA, (F–H) mPFC and(I) to K) NAc between SDS-­subjected or SDS-­free MRL/lpr, and control mice. (C, F and I) Schematic drawings of the VTA, mPFC and NAc were takenfrom Franklin and Paxinos (1997).64 (D, G, J) representative immunohistochemical images of activating p-­cFos-­positive neuronal cells with DAPI in the
VTA, mPFC and NAc. Scale bars: (D) and (J) 50 µm and (G) 100 µm. (H) Quantification of p-­cFos-­positive cells in the VTA (n=7–12 per group), mPFC
(n=10–12 per group), and NAc (n=4–8 per group). Data are means±SEM. *p<0.05, **p<0.01 and ****p<0.0001 using a two-­way ANOVA with thepost-­hoc Tukey-­Kramer multiple comparison test. ANOVA, analysis of variance; DAPI, 4′,6-­diamidino-­2-­phenylindole; mPFC, medial prefrontal cortex;
NAc, nucleus accumbens; p-­cFos, phosphorylated cFos; PVN, paraventricular nucleus; VTA, ventral tegmental area.
Considering the inflammatory nature of lupus, we focused on
635 genes associated with the inflammatory pathways analysed above. SDS-­
subjected MRL/lpr mice demonstrated relatively high expressions for microglia-­
activating genes, suchas H2-­Eb1, Nos2, Il12b and Fcgr4, and low expressions for
microglial-­inactivating genes, including Arg1, Nr4a3 and Treml4(figure 3E). The Il12b gene encodes interleukin 12 (IL-­12) p40
subunit, which is shared by IL-­12 and IL-­23, both of which are
critical for inflammation development. We confirmed that Il12bgene had the highest expression in the PFC of SDS-­subjected

MRL/lpr mice by quantitative PCR (figure 3F). Although theserum IL-­12/23p40 level of SDS-­subjected MRL/lpr mice waslower than that of SDS-­free MRL/lpr mice, the highest CSF
IL-­
12/23p40 protein level was observed in SDS-­
subjected
MRL/lpr mice (figure 3G, online supplemental figure 3C). We
also examined the CSF levels of interferon-γ and IL-­17A, whichare IL-­12/23p40 signaling-­inducible cytokines. The CSF interferon-γ and IL-­17A tended to be higher levels in SDS-­subjected
MRL/lpr mice (online supplemental figure 3D,E). Thus, the4

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Systemic lupus erythematosus

Figure 3 Upregulation of inflammatory gene expression and IL-­12/23p40 level in the PFC of stressed MRL/lpr mice. (A) Principal component
analysis of genes with an absolute FC over 1.5 in SDS-­subjected or -free strains (n=3 per group). (B) Venn diagram showing the number of stress-­affected differentially expressed genes with an absolute FC >1.5 and false discovery rate-­corrected p value <0.2 among the mouse groups. SDS-­
subjected MRL/lpr demonstrated unique expressions of 343 upregulated and 166 downregulated genes compared with SDS-­free MRL/lpr mice andcontrol strains. (C) Ingenuity pathway analysis and (D) GO analysis of the 343 differentially upregulated genes from SDS-­subjected MRL/lpr mice.
Dots represent p values. (E) SDS-­induced changes in inflammation-­related gene expressions in the PFC of MRL/lpr and controls compared with therespective SDS-­free strains. red, SDS-­induced MRL/lpr uniquely expressed genes. (R=0.1097, linear regression, Pearson’s correlation). (F) Il12b mRNA
expression relative to SDS-­free control mice by quantitative PCR (n=6–8 per group). (G) IL-­12/23p40 level in the CSF measured by ELISA (n=10–11
per group). (F, G) data are means±SEM. *p<0.05, **p<0.01 and ****p<0.0001 using a two-­way ANOVA with the post-­hoc Tukey-­Kramer multiple
comparison test. ANOVA, analysis of variance; CSF, cerebrospinal fluid; FC, fold change; GO, Gene Ontology; IL, interleukin; PFC, prefrontal cortex; SDS,
sleep disturbance stress.
upregulation of microglial proinflammatory genes, including
Il12b, in the PFC of SDS-­subjected MRL/lpr mice was observed.

Microglial activation and neuronal alterations in the mPFC of
SDS-subjected MRL/lpr mice

We next investigated which cell types produce IL-­12/23p40 in
the PFC. We first analysed the CNS invasion of CD45high immune
cells, especially T cells. Although peripheral CD45high immune
cells were present in the cortex of 14-­week-­old MRL/lpr mice,as previously described,22 few CD45high cells were detected in
the cortex of 8-­week-­old MRL/lpr mice (online supplemental
figure 4A,B). Therefore, we focused on CNS cells responsible
for IL-­12/23p40 production. Public databases of gene expressions in CNS cells demonstrated that microglia and macrophages
express Il12b gene (online supplemental figure 4C).23 Consistently, SDS-­subjected MRL/lpr mice had the highest number ofIba-­1+-­activated microglia in the mPFC based on the aggregationof a lysosomal activation marker, CD68, without any microglial density changes (figure 4A-­C, online supplemental figures
5A,B). A flow cytometric analysis revealed that IL-­12/23p40-­
positive cortical microglia increased in SDS-­subjected MRL/lpr
mice (figure 4D,E, online supplemental figure 5C,D). Additionally, mPFC microglia in NOD/ShiJcl mouse strain did not
be affected by SDS (online supplemental figure 5E-­G). We theninvestigated which cells receive IL-­
12/23p40 in the mPFC.
Previous reports showed that neurons express IL-­12 receptors
Abe N, et al. Ann Rheum Dis 2022;0:1–12. doi:10.1136/annrheumdis-2022-222566

and IL-­12 enhances neurite outgrowth.24 25 Indeed, mPFC
neurons expressed IL12Rβ1, a subunit of IL-­12/23p40 receptor(online supplemental figure 6A). We further found that Stat4, adownstream molecule of IL-­12/23 signalling, was highly phosphorylated in the PFC neurons of SDS-­subjected MRL/lpr mice(figure 4F,G, online supplemental figure 6B,C). Phosphorylation of Stat3, another downstream molecule of IL-­12/23 signalling, was not significantly affected by SDS in the PFC neuronsof SDS-­
subjected MRL/lpr mice (online supplemental figure
6D-­F), indicating mainly activated IL-­12 signalling in the PFC
neurons. Furthermore, SDS-­
subjected MRL/lpr mice had themost abnormal spines in proximal dendrites to soma of mPFC
neurons (figure 4HI), where few dendritic spines are generally
expressed.26 We also observed that MRL/MpJ control micestereotaxically injected with recombinant IL-­12/23p40 demonstrated more abnormal dendritic spines in mPFC neurons (online
supplemental figure 6G,H). Therefore, we detected IL-­12/23p40
upregulation in activated microglia and Stat4-­mediated neuronalalterations in the mPFC of SDS-­subjected MRL/lpr mice.

Cancelling neuropsychiatric phenotypes in stressed MRL/lpr
mice by IL-12/23p40 neutralisation and tyrosine kinase 2
inhibition
To investigate whether the blockade of IL-­12/23p40 signalling
ameliorates the stress-­elicited phenotypes in MRL/lpr mice, we
5

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Systemic lupus erythematosus

Figure 4 Microglial activation with IL-­12/23p40 upregulation and dendritic alterations in stressed MRL/lpr mice. (A–C) Microglial activation
state analysis in the mPFC. (A) Representative immunohistochemical images of Iba-­1-­positive microglia with a CD68 lysosomal marker.
(B) Percentage of reactive microglia and (C) microglial density (n=4 per group). (D–E) Flow cytometric analysis for IL-­12/23p40 production from live
CD45intCD11bhighCX3CR1+ cortical microglia. (D) Representative gating for IL-­12/23p40-­positive microglia. (E) Percentage of IL-­12/23p40-­producing
microglia (n=4–5 per group). (F and G) Phosphorylated-­Stat4 expression in TUBB3+ neurons measured by flow cytometry. (F) Representative
histogram for phosphorylated-­Stat4 and (G) FC of the mean fluorescence intensity for the phosphorylated-­Stat4 expression relative to SDS-­free control
mice (n=4–5 per group). (H–I) Golgi-­Cox staining of neurons in the mPFC. (H) Representative images of layer 2/3 pyramidal neurons. (I) Quantificationof the dendritic spinal density proximal to the soma of layer 2/3 pyramidal neurons (n=31–62 dendrites per group). Scale bars: (A) 50 µm and (K) 20µm. (B), (C), (E) and (G) Data are means±SEM. or (I) medians (IQR). *p<0.05, **p<0.01 and ****p<0.0001 using a two-­way ANOVA with the Tukey-­
Kramer test. ANOVA, analysis of variance; FC, fold change; IL, interleukin; mPFC, medial prefrontal cortex; SDS, sleep disturbance stress.
intracerebroventricularly infused IL-­12/23p40 neutralising antibody in SDS-­subjected MRL/lpr mice (figure 5A). The infused
immunoglobulin G (IgG) successfully reached the mPFC, andIL-­
12/23p40 antibody reduced the CSF IL-­
12/23p40 level
without any change in the serum level of IL-­12/23p40 or spleen
weight (figure 5B, online supplemental figure 7A–C). Indeed,IL-­12/23p40 blockade in the CNS ameliorated stress-­induced
anxiolytic behaviour (figure 5C–E). Moreover, microglial

activation and phosphorylated Stat4 levels in the mPFC werereduced (online supplemental figure 7D-­
F). The abnormal
increase of proximal dendritic spines was also remedied(figure 5F,G).
We next employed deucravacitinib, a clinically applied selective inhibitor of tyrosine kinase 2 (Tyk2), which is an IL-­12/23-­
downstream intracellular signalling kinase.27 We found that Tyk2
inhibitor treatment reduced the spleen weight (figure 6A–C)

6

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Systemic lupus erythematosus

Figure 5 Cancellation of stress-­elicited phenotypes in MRL/lpr by neutralising antibody-­mediated IL-­12/23 signalling blockade. (A) Schematic
representation of the CNS-­targeted IL-­12/23p40 blockade in SDS-­subjected MRL/lpr mice. (B–G) Successful inhibition of SDS-­induced neuropsychiatricphenotypes by IL-­12/23p40 depletion. (B) ELISA for IL-­12/23p40 concentration in the CSF (n=4–6 per group). (C) Representative tracks, (D) percentageof time spent in the open arm and (E) total travelled distance in the EPM (n=11 to 13 per group). (F) Representative Golgi-­Cox staining of layer 2/3
pyramidal neurons in the mPFC. (G) Quantification of the spinal density of proximal dendrites to soma (n=28–33 dendrites per group). (F) scale bar,
20 µm. (B), (D) and (E) Data are means±SEM or (G) medians (IQR). *p<0.05 and ****p<0.0001 using an unpaired Student’s t-­test. CSF, cerebrospinal
fluid; CNS, central nervous system; EPM, elevated plus maze test; IgG, immunoglobulin G; IL, interleukin; mPFC, medial prefrontal cortex; SDS, sleep
disturbance stress.
and suppressed cortical microglial activation with IL-­12/23p40
production, phosphorylation of Stat4 in cortical neurons, andanxiolytic behaviour without any effects on general locomotor
activity, and increased the number of spines in proximal dendrites(figure 6D-­N, online supplemental figure 8). Thus, blockade of
the IL-­12/23p40-­Tyk2 signalling pathway in the CNS successfully inhibited the stress-­induced neuropsychiatric phenotypes in
SDS-­subjected MRL/lpr mice.

High IL-12/23p40 levels in the CSF and mPFC atrophy inpatients with dNPSLE

patients with anxiety in both cohorts (online supplemental tables
S1 and S2). We investigated the CSF IL-­12/23p40 level of thepatients with primary SS as a disease control. The IL-­12/23p40
levels were significantly higher in the CSF of patients withdNPSLE than that of patients with primary SS (online supplemental figure 9D).
We next investigated morphological changes of the brain inpatients with dNPSLE and patients with non-­dNPSLE by VBM
using another Hokkaido University cohort (figure 7A and online
supplemental table 3). The patients with dNPSLE showed lessvolumes of total grey matter and WM, and more WM lesions
than patients with non-­dNPSLE (figure 7F, online supplemental
figure 9E–H). Based on the results of the SDS mouse model,
we calculated the mPFC volume and atrophic Z-­score using anage-­matched healthy control dataset as a reference, finding alarger mPFC volume reduction with higher Z-­score in patientswith dNPSLE than patients with non-­dNPSLE despite a similar
frontal cortex volume (figure 7H1, online supplemental figure9I). In the Hokkaido University Hospital cohort (online supplemental table 1), patients with dNPSLE received more sleep medications than patients with non-­dNPSLE (20/28, 71% vs 6/19,
32%, p=0.0064) at the perioperative period of the CSF collection without any statistical difference in the glucocorticoid dose
(online supplemental table 1 and 4). These might indicate a highprevalence of sleep disorder on dNPSLE onset. Together, theseresults as patients with dNPSLE have increased IL-­
12/23p40
levels in the CSF and mPFC atrophy, possibly being linked to theresults of our mouse model.

To investigate whether IL-­
12/23p40 in the CNS affects thepathogenesis of dNPSLE, we measured IL-­12/23p40 levels in the
CSF and serum of healthy controls and lupus patients with active
disease (figure 7A). In the Hokkaido University Hospital cohort,including patients with dNPSLE and patients with SLE without
diffuse neuropsychiatric symptoms (non-­dNPSLE), which
include fNPSLE (online supplemental table 1), CSF IL-­12/23p40
levels were highest in patients with dNPSLE, whereas serum
levels were similar among all groups (figure 7B, online supplemental figure 9A). Although statistical difference was notobserved due to the small number of patients with dNPSLE, thepatients without treatment tended to show higher levels of CSF
IL-­12/23p40 than those treated with glucocorticoid combinedwith immunosuppressants, suggesting that high variance of CSF
IL-­12/23p40 levels was likely caused by therapeutic effects ofimmunosuppressants (online supplemental figure 9B). The CSF
IL-­12/23p40 level had high diagnostic utility for dNPSLE, withan AUC of 0.8438 (95% CI 0.7377 to 0.9498, sensitivity 78%
and specificity 88%) (figure 7C). Using the Kitasato University
Hospital cohort (online supplemental table 2), we validated theseresults, as the higher CSF levels of IL-­12/23p40 in patients withdNPSLE than fNPSLE and good diagnostic efficacy for dNPSLE
with an AUC of 0.7346 (95% CI 0.5472 to 0.9220, sensitivity
61% and specificity 78%) (figure 7D-­E, online supplemental
figure 9C). Notably, the patients with ACS or psychosis occupied 70%–80% of the dNPSLE population, and there were few

Previous studies have reported that chronic stress inhibits
PFC function with a decreasing density of dendritic spines,
impairing behavioural flexibility, including more anxiety-­likebehaviours.20 21 28–31 Contrary to these reports, our results
demonstrated that stressed lupus-­prone mice showed PFC
impairment with increasing dendritic spinal density and disinhibited risk-­taking and less anxiety-­like behaviours. We further

Abe N, et al. Ann Rheum Dis 2022;0:1–12. doi:10.1136/annrheumdis-2022-222566

7

DISCUSSION

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Systemic lupus erythematosus

Figure 6 Improvement of stress-­elicited neuropsychiatric phenotypes in lupus-­prone mice with Tyk2 inhibitor. (A) The experimental protocol forthe systemic administration of deucravacitinib in SDS-­subjected MRL/lpr mice. (B–N) SDS-­elicited phenotypes were cancelled by deucravacitinib.
(B) Macroscopic findings of the spleen. (C) Spleen weight comparison (n=3 per group). (D) Representative images of activated microglia.
(E) Percentage of reactive microglia and (F) microglial density (n=3 per group). (G) Representative gating for IL-­12/23p40+ microglia and (H) its
percentage (n=3 per group). (I) Histogram of phosphorylated-­Stat4 expression in TUBB3+ neurons. (J) Representative trajectories, (K) percentage oftime spent in the open arm and (L) total distance in the EPM (n=3–4 per group). (M) Representative images of layer 2/3 pyramidal neurons in themPFC. (N) Quantification of the proximal dendritic spinal density (n=35–44 dendrites per group). Scale bars: (B) 10 mm, (D) 50 µm and (M) 20 µm.
Data are means±SEM except (N) which shows medians (IQR). *p<0.05, **p<0.01 and ****p<0.0001 using an unpaired Student’s t-­test. DMSO,
dimethyl sulfoxide; EPM, elevated plus maze test; IL, interleukin; mPFC, medial prefrontal cortex; SDS, sleep disturbance stress; Tyk2, tyrosine kinase 2.
showed that the stress-­induced activation of microglia in themPFC plays a key role.
It is well known that activated microglia induce neuroinflammation, predisposing susceptible individuals to neuropsychiatric diseases,32–34 even though microglia engage with neurons
to maintain homeostasis in the CNS.32–34 In other words, in apathological state, activated microglia potentially exacerbate
neurological diseases by dysregulating neural circuits.35 36 For
instance, homeostatic microglia drive negative feedback mechanisms to inhibit neuronal activation via extracellular ATP toprotect the brain from excessive activation, while activated proinflammatory microglia do not trigger these negative feedback
mechanisms.37 In addition, activated microglia alter neuronal
functions and eliminate dendritic spines in pyramidal neuronsof the mPFC.38 39 Moreover, microglia from lupus-­prone mice
exhibit an activated phenotype with upregulated inflammation-­
related genes, including Il12b, and cause neuronal dysfunction
leading to neuroinflammation and microglial phagocytosis.40 41
Although some microglia populations protect lupus model micefrom neurodegeneration,42 43 activated microglia also induce

impairment of the blood–brain barrier to cause neuroinflammation development.44 Thus, it is reasonable that activatedmicroglia can induce the abnormal activation of neuronal circuitsin the mPFC to cause neuropsychiatric phenotypes in stressed
MRL/lpr mice. Although stress is known to induce microglial
activation via the norepinephrine-β adrenergic receptor signalling pathway,45 46 the molecular mechanisms causing the microglial activation in SDS-­subjected MRL/lpr mice are unknown atpresent. In addition, the behavioural testing results had relatively
high variance in stressed-­MRL/lpr mice, possibly being derived
from the variance of disease penetrance. Further research forunderstanding the molecular mechanisms of stress-­
inducedmicroglial activation and neuronal alteration in lupus-­
prone
condition is required.
IL-­
12/23p40 is a subunit that dimerises with IL-­
12p35 orIL-­23p19 to form IL-­12 and IL-­23.47 In autoimmune disorders,IL-­12 and IL-­23 are associated with the pathogenesis predominantly through T cell-­mediated immunity. While some studies
reported that IL-­12 can relieve neuroinflammation in an EAE
model,48 49 previous research also demonstrated that IL-­12 and

8

Abe N, et al. Ann Rheum Dis 2022;0:1–12. doi:10.1136/annrheumdis-2022-222566

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Systemic lupus erythematosus

Figure 7 IL-­12/23p40 upregulation and mPFC atrophy in patients with NPSLE with diffuse manifestations (dNPSLE). (A) Schema for the evaluationof human clinical samples and data. (B) and (C) The derivation cohort of patients with SLE and HCs at Hokkaido University Hospital. (B) CSF IL-­
12/23p40 level measured by ELISA (n=13 HCs, n=19 non-­dNPSLE and n=28 dNPSLE). (C) An ROC analysis of CSF IL-­12/23p40 levels for dNPSLE
diagnosis. (D) and (E) The validation cohort of patients with NPSLE at Kitasato University hospital. (D) CSF IL-­12/23p40 levels (n=9 fNPSLE and n=18
dNPSLE). (E) An ROC analysis of the CSF IL-­12/23p40 level for dNPSLE classification. (F) Representative brains with atrophy rendered on the reference
anatomical brain view against the IXI reference dataset. (G) Used brain ROI mask of the mPFC, rendered as red. (H) Correlation between the mPFC
volume and TIV (blue, n=57 non-­dNPSLE; red, n=18 dNPSLE). (I) Z-­scores of mPFC atrophy against the IXI reference dataset. (B), (D) and (I) Data are
medians (IQR). *p<0.05, **p<0.01 and ****p<0.0001 using (B) the Kruskal-­Wallis test with the post-­hoc Steel-­Dwass method, (D) and (I) Mann-­
Whitney U test and (H) ANCOVA adjusting for TIV, age, sex and disease duration of SLE. ANCOVA, analysis of covariance; CSF, cerebrospinal fluid; CNS,
central nervous system; dNPSLE, diffuse neuropsychiatric systemic lupus erythematosus; EPM, elevated plus maze test; HCs, healthy controls; IgG,
immunoglobulin G; IL, interleukin; mPFC, medial prefrontal cortex; NPSLE, neuropsychiatric systemic lupus erythematosus; ROC, receiver operating
characteristic; ROI, region of interest; SDS, sleep disturbance stress; SLE, systemic lupus erythematosus; TIV, total intracranial volume.
IL-­23 can exacerbate neuroinflammation in EAE.50 51 They also
exacerbate the pathology of Alzheimer’s disease.52 We showedthat IL-­
12/23p40 mainly expressed from activated microglia
alter mPFC neurons, modifying the neuronal structure as previously demonstrated.24 A phase 3 clinical trial for ustekinumab,

an IL-­12/23p40 neutralisation antibody, in patients with SLE wasdiscontinued due to poor efficacy (Ustekinumab Press Release).53
However, considering that patients with NPSLE were excluded
from the trial, whether this antibody or the Tyk2 inhibitor has
efficacy in patients with dNPSLE should be explored.

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9

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Systemic lupus erythematosus

Figure 8 Graphical abstract. SDS-­subjected lupus-­prone MRL/lpr mice demonstrated disinhibited anxiolytic behaviour. Mechanistically, microglial
activation, IL-­12/23p40 upregulation and neuronal activation with increasing dendritic spines in the medial prefrontal cortex were observed.
These stress-­induced neuropsychiatric phenotypes were reversed by blockade of the IL-­12/23 axis using IL-­12/23p40 neutralising antibody or Tyk2
inhibitor. Patients with neuropsychiatric lupus showing diffuse neuropsychological manifestations demonstrated elevated levels of IL-­12/23p40 in the
cerebrospinal fluid and medial prefrontal cortical atrophy. These results suggest a pathological link between the stress-­induced microglial IL-­12/23p40
axis with neuronal activation and the development of neuropsychiatric lupus with diffuse manifestations. IL, interleukin; SDS, sleep disturbance stress;
Tyk2, tyrosine kinase 2.
As a possible molecular mechanism of dNPSLE, we hypothesised that stress-­
induced microglial activation, which led toan overactivation of neurons in the mPFC; this overactivationis critical for disinhibited symptoms like agitation, psychosis
and ACS. Our study demonstrated that chronic stress induced
a risk-­taking behaviour with mPFC overactivation in SDS-­
subjected MRL/lpr mice, which would be similar behaviours to
the hyperactivity and psychomotor agitation in patients withNPSLE. Indeed, a specific type of delirium with increased vigilance manifesting agitation, overactivity and hallucinations isobserved in patients with NPSLE with ACS. Our mouse model
could at least in part explain some of these ACS manifestations
as stress-­induced and inflammation-­induced mPFC overactivation in patients with dNPSLE. Consistently, patients with SLEshow an altered mPFC status in decision-­making tasks,54 reduced
metabolism and atrophy,55 and decreased cerebral blood flow.56
An increase of dendritic spines in PFC neurons is also observedin autism-­spectrum disorder and in patients taking antidepressants or N-­methyl-­D -­aspartic acid, which manifest anxiolytic/
depressive hyperactive behaviour.57–60
Patients with SLE often show more exasperated neuropsychiatric symptoms just after receiving glucocorticoid therapy
compared with patients with other autoimmune disorders. These
post-­
glucocorticoid neuropsychiatric symptoms are similar tothe diffuse manifestations in NPSLE.61 Because glucocorticoids
sometimes induce mPFC impairment via neuronal alterations,62 63
IL-­12/23 signalling in the altered microglia-­neuronal axis in themPFC demonstrated here may contribute to the developmentof neuropsychiatric symptoms in patients with SLE undergoing
glucocorticoid treatment. Compared with glucocorticoid monotherapy, we thus hypothesise that immunosuppressants such as
cyclophosphamide, which would induce microglial apoptosis,
combined with glucocorticoid possibly works better for dNPSLE
symptoms through inhibiting the interaction between activatedmicroglia and neurons described here. Moreover, JAK/Tyk2-­Stat

inhibitors would be beneficial for dNPSLE through inhibiting
activated microglia-­inducing neuronal alteration.
Taken together, we demonstrate that stress has pathogenic
neuropsychiatric effects on a lupus rodent model by activating
microglia and altering neurons in the mPFC via the IL-­12/23
signalling pathway. We also found similar phenomena as anelevated level of CSF IL-­12/23p40 and mPFC atrophy in patientswith dNPSLE (figure 8). Therefore, our findings suggest thatIL-­12/23p40 in the mPFC is a therapeutic target for dNPSLE.

10

Abe N, et al. Ann Rheum Dis 2022;0:1–12. doi:10.1136/annrheumdis-2022-222566

Author affiliations
1
Division of Molecular Psychoimmunology, Institute for Genetic Medicine, Graduate
School of Medicine, Hokkaido University, Sapporo, Japan2
Department of Rheumatology, Endocrinology and Nephrology, Faculty of Medicine
and Graduate School of Medicine, Hokkaido University, Sapporo, Japan
3
Group of Quantum Immunology, Institute for Quantum Life Science, National
Institute for Quantum and Radiological Science and Technology, Inage, Japan
4
Center for Infectious Cancers, Institute for Genetic Medicine, Hokkaido University,
Sapporo, Japan
5
Division of Molecular Neuroimmunology, National Institute for Physiological
Sciences, National Institute of Natural Sciences, Okazaki, Japan
6
Department of Rheumatology and Infectious Diseases, School of Medicine, Kitasato
University, Sagamihara, Japan
7
Department of Immunology, National Institute of Neuroscience, National Center of
Neurology and Psychiatry, Kodaira, Japan
8
Department of Diagnostic and Interventional Radiology, Hokkaido University
Hospital, Sapporo, Japan
9
Global Center for Biomedical Science and Engineering, Faculty of Medicine,
Hokkaido University, Sapporo, Japan10
Department of Anatomy, Faculty of Medicine, Hokkaido University, Sapporo, Japan
Acknowledgements We thank Ms Yumiko Kaneko, Ms Yui Ishikura, Mr Yusuke
Fujita and Ms Chiemi Nakayama for their experimental support, and Dr Yasuhiro
Hasegawa (Kitasato University) for collecting the clinical data. We are grateful to Dr
Peter Karagiannis (Center for iPS Cell Research and Application, Kyoto University,
Kyoto) for reading the manuscript. We also appreciate ​BioRender.​com for the supportof drawing figures.
Contributors Conceptualisation: NA and MM. Methodology: NA, NT, YF, YT, RH
and KKT. Investigation: NA, NT, MT, KK, MU and KKT. Visualisation: NA and YF.
Resources: YA, WS, KKT and MY. Funding acquisition and project administration: KO,

Ann Rheum Dis: first published as 10.1136/ard-2022-222566 on 11 July 2022. Downloaded from http://ard.bmj.com/ on August 10, 2022 by Tatsuya Atsumi Hokka Kita-Ku. Protected bycopyright.

Systemic lupus erythematosus

TA and MM. Supervision: MKono, MKato, MY, MW, TA and MM. Writing—originaldraft: NA, YF and KO. Writing—review and editing: OA, KKT, MW, TA and MM.
Guarantors: TA and MM.

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Abe N, et al. Ann Rheum Dis 2022;0:1–12. doi:10.1136/annrheumdis-2022-222566

11

Funding This work was funded by the grant from Bristol Myers Squibb Foundation
to TA, and grants from JSPS KAKENHI, Q-­Leap (JPMXS0120330644), AMED, The
Joint Usage/Research Center Institute for Genetic Medicine, Hokkaido University, thePhoto-­excitonix Project at Hokkaido University and the Promotion Project for Young
Investigators at Hokkaido University to MM.
Competing interests None declared.
Patient and public involvement Patients and/or the public were not involved in
the design, or conduct, or reporting, or dissemination plans of this research.
Patient consent for publication Not applicable.
Ethics approval This study involves human participants. Ethical approval for theclinical studies was granted by institutional review board of Hokkaido University
Hospital (approval numbers: 019-­0055 and 020-­0110) and that of Kitasato
University School of Medicine (approval number: B20-­231) and for the animal
experiments and clinical studies was granted by the Institutional Animal Care and
Use Committees of Hokkaido University (approval number: 14–0083). This study
complied with the Declaration of Helsinki. Participants gave informed consent toparticipate in the study before taking part.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available in a public, open accessrepository. Data are available upon reasonable request. In detail, RNA-­seq datasetsthat support the observations of this study have been deposited in the Gene
Expression Omnibus with the series accession number GSE176429. MRI data havebeen stored locally following national and Japanese laws on the protection ofindividuals with regard to the processing of personal data. Other data are available
in the main text or the supplementary materials and are available upon reasonable
request to the corresponding author.
Supplemental material This content has been supplied by the author(s).
It has not been vetted by BMJ Publishing Group Limited (BMJ) and may nothave been peer-­reviewed. Any opinions or recommendations discussed aresolely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all
liability and responsibility arising from any reliance placed on the content.
Where the content includes any translated material, BMJ does not warrant theaccuracy and reliability of the translations (including but not limited to local
regulations, clinical guidelines, terminology, drug names and drug dosages), andis not responsible for any error and/or omissions arising from translation andadaptation or otherwise.
ORCID iDs
Nobuya Abe http://orcid.org/0000-0001-8644-1007
Yuichiro Fujieda http://orcid.org/0000-0003-4705-341X
Michihito Kono http://orcid.org/0000-0002-7663-534X
Masaru Kato http://orcid.org/0000-0002-2023-6585
Yoshiyuki Arinuma http://orcid.org/0000-0003-1305-991X
Masaaki Murakami http://orcid.org/0000-0001-7159-7279

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56 Papadaki E, Kavroulakis E, Bertsias G, et al. Regional cerebral perfusion correlateswith anxiety in neuropsychiatric SLE: evidence for a mechanism distinct fromdepression. Lupus 2019;28:1678–89.
57 Li N, Lee B, Liu R-­J, et al. mTOR-­Dependent synapse formation underlies the rapid
antidepressant effects of NMDA antagonists. Science 2010;329:959–64.
58 Forrest MP, Parnell E, Penzes P. Dendritic structural plasticity and neuropsychiatricdisease. Nat Rev Neurosci 2018;19:215–34.
59 Moda-­Sava RN, Murdock MH, Parekh PK, et al. Sustained rescue of prefrontal
circuit dysfunction by antidepressant-­induced spine formation. Science
2019;364:eaat8078.
60 Holmes SE, Scheinost D, Finnema SJ, et al. Lower synaptic density is associated withdepression severity and network alterations. Nat Commun 2019;10:1529.
61 Shimizu Y, Yasuda S, Kako Y, et al. Post-­steroid neuropsychiatric manifestations
are significantly more frequent in SLE compared with other systemic autoimmune
diseases and predict better prognosis compared with de novo neuropsychiatric SLE.
Autoimmun Rev 2016;15:786–94.
62 Barsegyan A, Mackenzie SM, Kurose BD, et al. Glucocorticoids in the prefrontal cortex
enhance memory consolidation and impair working memory by a common neuralmechanism. Proc Natl Acad Sci U S A 2010;107:16655–60.
63 Butts KA, Weinberg J, Young AH, et al. Glucocorticoid receptors in the prefrontal
cortex regulate stress-­evoked dopamine efflux and aspects of executive function. Proc
Natl Acad Sci U S A 2011;108:18459–64.
64 Franklin KBJ, Paxinos G. The mouse brain in stereotaxic coordinates. Academic Press,
1997. ISBN: 9780122660702.

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Ann Rheum Dis: first published as 10.1136/ard-2022-222566 on 11 July 2022. Downloaded from http://ard.bmj.com/ on August 10, 2022 by Tatsuya Atsumi Hokka Kita-Ku. Protected bycopyright.

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Supplementary Materials for
Pathogenic neuropsychiatric effect of stress-induced microglial interleukin12/23 axis in systemic lupus erythematosus
Nobuya Abe, Masato Tarumi, Yuichiro Fujieda, Nobuhiko Takahashi, Kohei Karino,
Mona Uchida, Michihito Kono, Yuki Tanaka, Rie Hasebe, Masaru Kato, Olga Amengual,
Yoshiyuki Arinuma, Kenji Oku, Wakiro Sato, Khin K. Tha, Miwako Yamasaki,
Masahiko Watanabe, Tatsuya Atsumi, Masaaki Murakami
Corresponding to:
Masaaki Murakami
Email: murakami@igm.hokudai.ac.jp
Tatsuya Atsumi
Email: at3tat@med.hokudai.ac.jp
This file includes:
Supplementary Patients, Materials and Methods
Supplementary References
Supplementary Figures 1 to 9
Supplementary Tables 1 to 4

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Supplementary Patients, Materials and Methods
Mice
MRL/MpJJmsSlc-lpr/lpr (MRL/lpr) and MRL/MpJJmsSlc-+/+ (MRL/MpJ) mice werepurchased from Japan SLC (Tokyo, Japan). MRL/lpr mice demonstrate central nervous
system (CNS) symptoms by 6-8 weeks of age but subsequently develop systemic
inflammation with nephritis after 10 weeks of age.1 To avoid the effect of systemic
inflammation and to consider the frequent occurrence for the development ofneuropsychiatric lupus (NPSLE) in women, 6-8-week-old female mice were analyzed forall experiments. NOD/ShiJcl mice were purchased from CLEA Japan (Tokyo, Japan) andwere used as disease control mice.
All mice were maintained under specific pathogen-free conditions on a 13-h light, 11-hdark cycle, and food and water were provided ad libitum in accordance with the protocolsof Hokkaido University.
Sleep disturbance stress loading
To expose mice to sleep disturbance stress (SDS) for the induction of sleep disorder, themice were set into SDS cages equipped with a running wheel (140 cm in diameter, SW15-SD, Melquest Co., Japan) for two weeks, which caused the mice to run on the wheel
almost all day and receive long-term stress. Food and water were provided ad libitum,and paper-chip bedding was replaced every other day. From the seventh to ninth day
during the SDS experiment, the rotation frequencies of the running wheel for each mousewere continuously recorded every 1 hour as actograms using a counting interface (CIF4M, Melquest, Japan) to evaluate circadian rhythm disruption. The running wheel activity
was calculated as the ratio of the number of wheel rotations during the activity and rest
states. Normal circadian rhythm was assessed using SDS-free control mice set in thenormal cage with a running wheel (SW-15S, Melquest, Japan) for the same period as
SDS-subjected mice.
Mouse samples including serum and cerebrospinal fluid (CSF)
Upon brain tissue collection from anesthetized mice before phosphate-buffered saline(PBS) perfusion, 0.5 mL of blood was collected from the right submandibular vein andput on ice. The blood was centrifuged at 800 g for 15 minutes at 4 °C, and thesupernatant was collected as serum. In the CSF collection, the scalp of PBS-perfusedmice was removed, and superficial neck muscles were cut. The atlanto-occipital
membrane above the cisterna magna was dried with paper towels and tapped using a 30G
needle (BD) to aspirate the CSF. Blood contamination in the CSF was evaluated byplacing a white background below the CSF-collected tube, and blood-contaminated CSF
was discarded. The serum and CSF were collected and stored at −30 °C until further
processing. To evaluate the stress degree, serum from mice before SDS load was alsocollected.
Immunohistochemistry
Brains were collected from mice after transcardial perfusion with cold PBS and then 10%
neutral buffered formalin. Tissue was then immersed in 10% formalin overnight at 4 °C
followed by cryoprotection in 30% sucrose for 48 hours at 4 °C. The processed tissue
was cryosectioned coronally in 40-50 μm thick slices and collected in PBS. The sections
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were blocked with 5% bovine serum albumin (BSA) with 0.3% Triton X-100 for 1 hourat room temperature and then incubated overnight at 4 °C with the following primary
antibodies diluted in PBS with 2% BSA and 0.3% Triton X-100: anti-p-cFos (1:1000,
D82C12, Cell Signaling Technology), anti-Iba-1 (1:400, 013-27691, Wako, and NB1001028, Novus Biologicals), anti-CD68 (1:300, MCA1957GA, Serotec), anti-TCRβ (1:200,
109208, BioLegend), anti-I-A/I-E (1:200, 107614, BioLegend), anti-IL12Rβ1 (1:100,
A1886, ABclonal) and anti-NeuN (1:500, ABN90, Merck). After three washes,
secondary Alexa Fluor dye-conjugated antibodies (Invitrogen) were added at 1:200 for 2
hours at room temperature. Slides were mounted in ProLong Diamond Antifade
Mountant with DAPI (4',6-diamidino-2-phenylindole) (ThermoFisher Scientific) andimaged using a Keyence inverted fluorescence microscope, BZ-9000 (Keyence, Japan).
Z-stack images were processed by full focus image analysis.
RNA sequencing analysis
After perfusion with 20 mL of cold PBS, the PFC region from mice was mechanicallydissected and homogenized using a polytron homogenizer with the ISOSPIN Cell &Tissue RNA Kit (Nippon Gene, Japan) to isolate total RNA. RNA quantity and quality
were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). All samples
whose RNA Integrity Number as RNA quality was 7.5 or higher were proceeded to thesubsequent library preparation. PolyA RNA purified from total RNA using oligo(dT) 25
magnetic beads was transcribed into full-length complementary DNA (cDNA) using a
SureSelect Strand Specific RNA Library Preparation Kit (G9691A, Agilent
Technologies). The processed library was purified using AMPure XP beads (A63880,
Beckman Coulter). Sequencing was performed with a HiSeq 2500 (single-read, 2 × 50bp) sequencer (Illumina). Strand NGS software (Agilent Technologies) was used to alignthe RNA-seq reads to the mouse reference genome (mm10). Quality control was also
assessed using Strand NGS. Aligned reads were then filtered based on a quality threshold(≥20), and reads failing the quality control threshold were removed. After filtering, thealigned reads including partial reads were normalized and quantified using the DESeq2
algorithm. Matlab (MathWorks, USA) was used to visualize various outputs including
the gene expressions, primary component analysis, and Venn diagrams. The gene sets ofinterest were analyzed by Ingenuity Pathway Analysis (IPA) (QIAGEN, CA), Gene
Ontology (GO) analysis (geneontology.org), and KEGG pathway analysis(www.genome.jp/kegg/). Gene sets associated with the inflammatory pathway include
genes in the neuroinflammation signaling pathway in IPA, positive regulation of cytokine
production in the GO biological process, and cytokine-cytokine receptor interaction
pathway in the KEGG pathway.
Quantitative real-time PCR (qPCR)
cDNA was synthesized from isolated RNA using SuperScript VILO (Invitrogen). The
transcript levels were measured with the Applied Biosystems 7500 Real-Time PCR
System and TaqMan Minor Groove Binder probes specific for the genes Il12b(Mm01288989_m1) and Actb (Mm02619580_g1). The Il12b expression level wascompared using the ΔΔCt method normalized to Actb.

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Enzyme-linked immunosorbent assay (ELISA)
Mouse serum and CSF samples were used to measure interleukin (IL)-12/23p40 with the
Mouse IL-12/IL-23p40 ELISA Kit (BioLegend) according to the manufacturer’s
instructions. To assess the degree of physiopsychological stress, sera were collected frommice before and after SDS load to measure the serum corticosterone level using a
Corticosterone ELISA Kit (Cayman Chemical, USA). IL-12/23p40 was measured inhuman serum and CSF samples with the BD OptEIA Human IL-12 (p40) Kit following
the recommendations of the manufacturer.
Multiplex cytokine beads assay
Mouse CSF was analyzed using the Bio-Plex Pro Mouse Cytokine Assay (Bio-Rad) withthe analytes including interferon-γ and IL-17A. Data were acquired on a Luminex 200
xPONENT System (Merck KGaA).
Single cell separation
Mice were anesthetized with pentobarbital sodium and transcardially perfused with 20
mL of sterile PBS to dissect the cortex. Cortical tissue was rinsed with Hanks’ balanced
salt solution (HBSS), mechanically dissociated, and enzymatically digested with papain
using a Neural Tissue Dissociation Kit (P) (Miltenyl Biotec) in accordance with the
instruction manual. For myelin removal, cells were resuspended in PBS with 0.9 M
sucrose and centrifuged for 10 minutes at 400 g. The myelin-containing supernatant wasremoved, and the pelleted glial cells were washed with HBSS and processed for further
analysis. To detect cytokine production in microglia, mice were intraperitoneallyadministered with 200 μg of lipopolysaccharide (L8274, Sigma-Aldrich) 18 hours before
the analysis. Prior to flow cytometric analysis, isolated glial cells were incubated for 4
hours with Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture, 10%
fetal bovine serum (FBS) and Golgi plug containing brefeldin A (BD).
For the separation of neuronal cells, single cells separated from cortical tissue weretreated with the Adult Mouse Neuron Isolation Kit (Miltenyl Biotec). Using a magnetic
cell sorting system, negatively-selected neurons were collected.
Flow cytometry
Cells were treated with the Zombie Fixable Viability Kit (BioLegend) to discriminate
between live and dead cells and then with anti-CD16/CD32 antibody (clone: 2.4G2) toblock Fc receptors for 10 minutes before staining. Cell surface staining was performed
using antibodies in PBS with 5% FBS and 5 mM ethylenediaminetetraacetic acid
(EDTA). For intracellular staining, cells were fixed and permeabilized(Fixation/Permeabilization Solutions, BD) and then stained. Monoclonal antibodies forCD11b (clone: M1/70, BioLegend), CD45 (30-F11, BioLegend), CX3CR1 (SA011F11,
BioLegend), CD140a (APA5, BioLegend), ACSA-2 (REA969, Miltenyi Biotec), MBP(2H9, Novus Biologicals), IL-12/IL-23p40 (C15.6, BioLegend), TUBB3 (TUJ1,
BioLegend), phospho-Stat4 (4LURPIE, eBioscience) and phospho-Stat3 (13A3-1,
BioLegend) were used. Stained cells were acquired on a CytoFLEX S (Beckman
Coulter), and data were analyzed using FlowJo software (BD).

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Golgi-Cox staining
Golgi-Cox staining was performed using a superGolgi Kit (Bioenno Lifesciences) inaccordance with the manufacturer’s instructions. Brains were collected from mice after
transcardial perfusion with 20 mL of cold normal saline. The dissected brain was cut into
a block on the anterior side at approximately 1×2×1 cm3 to include the bilateral mPFC
region. Stained brain blocks were cut into 200 μm sections with a vibratome (Leica). The
sections were mounted on gelatin-coated slides, stained, and then covered with Mount
Quick (Daido Sangyo). Z-stacked images were acquired with the full focus function on a
fluorescence microscope, BZ-X800 (Keyence, Japan).
Image quantification
The images were analyzed using ImageJ software ver. 1.53c (National Institutes of
Health, USA).
Neuronal activation analysis
Two immunofluorescent images of brain regions of interest (ROIs) with a 20× field for pcFos staining were acquired per mouse. Activating neuronal cells were defined as p-cFospositive DAPI-stained cells, and the number of cells were manually counted and thenaveraged to calculate the density per mouse.
Microglial activation state analysis
Two 50-μm coronal sections containing the medial prefrontal cortex (mPFC) region were
immunostained for Iba-1 and CD68. For each section, two 20× fields of full focused view
were collected per mouse. The activation status of microglia was scored from 0 (lowest
activation) to 4 (highest activation) depending on the CD68 expression pattern, which
was analyzed and scored as 0-1 (no/scarce expression), 2 (punctate expression), 3(multiple punctate expression), and 4 (aggregated expression). Activated microglia withan activation score of 3 or 4 were categorized as reactive microglia. The average
percentage of reactive microglia was calculated for each mouse group.
Dendritic spine countFour 100× coronal views of Golgi-Cox-stained sections containing mPFC layer 2/3
pyramidal neurons were obtained. For each view, 1-2 full focused neurons were included,
and 5-7 dendrites from each neuron were recognized per mouse. The number of dendritic
spines 40 μm proximal to soma of impregnated pyramidal neurons in the mPFC, where
the spinal density is generally lower,2 were counted per each dendrite. Since layer 2/3
pyramidal neurons are reported to be susceptible to chronic physiopsychological stress,
we selected layer 2/3 pyramidal neurons for this analysis.
In vivo pharmacological intervention
Osmotic pump intracerebroventricular administration of anti-IL-12/23p40 antibody
A monoclonal anti-IL-12/23p40 antibody (C17.8, BioLegend, 1 mg/mL diluted in saline)
or mouse IgG2a isotype control antibody (MG2a-53, BioLegend, 1 mg/mL diluted insaline) was loaded into 200-μL osmotic pumps (Alzet) with a 14-day infusion rate of 0.5
μL per hour. Mice were implanted with osmotic pumps that were connected to a cannula(Brain Infusion Kit III, Alzet) inserted at +1.1 mm lateral, −0.5 mm anterior-posterior,and −2.5 mm deep from the bregma to target the right lateral ventricle. The pump was
subcutaneously placed.

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Systemic administration of Tyk2 inhibitor
Mice were intraperitoneally injected with 0.12 mg/kg of deucravacitinib diluted indimethyl sulfoxide (10 mM, MedChemExpress, USA) or an equal volume of dimethyl
sulfoxide daily for two weeks, followed by further analysis. The dosage of
deucravacitinib was determined based on the clinical dose for psoriasis.
Elevated plus maze test
The maze consisted of two open and two closed arms that extended out from a central
square. Each arm was 30 cm long and 5 cm wide. The platform was 50 cm above thefloor. Mice were placed in the center area of the maze facing an open arm and allowed to
explore the maze for 5 minutes. A video-tracking system Smart 3.0 (Panlab-Harvard
Apparatus, USA) was used to record the time spent in open and closed arms. The
proportion of the time spent in the open arm was used as a measure of anxiety-likebehavior. The total distance mice travelled was a surrogate measurement of generallocomotor activity.
Open field test
The open field chamber consisted of a square black base (50 cm × 50 cm) with opaque
white walls 50 cm in height. Mice were allowed free exploration for 10 minutes andvideo-tracked. The proportion of time spent in the center zone was used to evaluate
anxiety-like behavior. The total travelled distance was used as a measure of generallocomotor activity.
Brain stereotaxic microinjection
An anesthetized 8-week-old MRL/MpJ control mouse was placed in a stereotaxic
micromanipulator instrument (Narishige, Japan). Fur above the skull was shaved, and theskin was wiped with 70% ethanol. A 26s-gauge needle attached to a Hamilton syringe
was pushed toward the mPFC region at +0.3 mm lateral, +1.8 mm anterior-posterior and−2.0 mm deep relative to the bregma. Recombinant mouse IL-12/23p40 (150 ng/0.5 μL,
BioLegend) or PBS as vehicle were injected for over 8 minutes into the mPFC. After 48
hours, the mice were analyzed.
Clinical data of patients with systemic lupus erythematosus (SLE)The patients included in this study met the 1997 update of the 1982 American College of
Rheumatology (ACR) revised criteria or 2012 Systemic Lupus International
Collaborating Clinics (SLICC) criteria for the classification of SLE.3,4 Clinical data wereextracted from medical records as follows: demographics such as sex, disease durationand disease activity index; complications including hypertension, diabetes mellitus anddyslipidemia; manifestations including non-neuropsychiatric symptoms such as skin rash,arthritis, nephritis and antiphospholipid syndrome as well as neuropsychiatricmanifestations; laboratory findings such as anti-DNA antibody, anti-Ro/SSA antibody,
antiphospholipid antibodies and complements; and treatments including glucocorticoids,
immunosuppressants, and antithrombotic therapy. Disease activity was assessed by SLE
disease activity index 2000 (SLEDAI-2K) 5 and SLICC/ACR damage index (SDI).6
Patients with NPSLE were diagnosed based on clinical and laboratory assessments.7
Diffuse manifestations in NPSLE are defined to include psychosis, acute confusional
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state, mood disorder, and cognitive impairment, and focal manifestations are defined asneurological syndromes such as headache, seizure, cerebrovascular disease,demyelinating disease, and peripheral neuropathy. In this study, patients with meningitis
and myelopathy were excluded to avoid too strong an effect on CSF cytokine levels bydirect inflammation in the meninges and to evaluate inflammation inside the CNS.
Human samples including serum and CSF
We reviewed medical records to extract the clinical data of patients with SLE including
NPSLE, whose serum and CSF were preserved at Hokkaido University (S-Table 1) orKitasato University (S-Table 2) between 2006 and 2020. At the time of the new onset or
relapse, serum and CSF samples were collected from the SLE patients. A lumbar
puncture was performed with the patients in a lateral position, and the CSF was collectedin polypropylene tubes. The samples were centrifuged at 1,000 g for 15 minutes at 4 °C,
and the supernatants were aliquoted to avoid a freeze–thaw cycle and stored at −80 °C
until further use. The serum and CSF samples of healthy controls were provided by the
National Center of Neurology and Psychiatry Biobank, a member of the National Center
Biobank Network (Tokyo, Japan). The CSF samples of patients with primary Sjögren’s
syndrome (SS) were collected at Hokkaido University. Informed consent for the study
was obtained from all patients included in this study or their relatives.
Brain magnetic resonance imaging acquisition
We obtained brain magnetic resonance (MR) images from 71 SLE patients at Hokkaido
University Hospital from 2019 to 2020 (Table S3). All brain imaging data were obtainedon a 3.0 T MRI scanner (Achieva TX, Philips Medical Systems) and a standard 32channel radiofrequency head coil (Philips Medical Systems, Best, the Netherlands) at
Hokkaido University Hospital. 3D-T1 magnetization-prepared rapid gradient echo
(MPRAGE) images of the head were acquired with the following parameters: TR(repetition time) 6 ms, TE (echo time) 3 ms, flip angle 8°, field of view 24 cm × 24 cm,
matrix size 240 × 240, slice thickness 1.0 mm, interslice gap 1.0 mm, and 180 sagittal
slices. FLAIR (fluid-attenuated inversion recovery) image acquisition parameters were as
follows: TR 10000 ms, TE 100 ms, TI (inversion time) 2700, ETL (echo train length) 20,
field of view 20 cm × 20 cm, frequency encoding 256, phase encoding 192, slice
thickness 5 mm, interslice gap 6.5 mm, and 23 axial slices.
Voxel-based morphometric analysis
MRI scans were processed by Brain Anatomical Analysis using Diffeomorphic
deformation (BAAD) software (version 4.3, www.shiga-med.ac.jp/∼
hqbioph/BAAD(English)/BAAD.html). The brain was extracted from 3D-T1 MPRAGE
images by a revised graph-cut-based skull-stripping, segmented into gray matter, whitematter and CSF, and warped into Montreal Neurological Institute (MNI) space. Images
were set around the anterior commissure-posterior commissure lines and re-sampled witha voxel size of 1 mm3. Tissue segmentation and bias correction of intensity nonuniformities were performed using an Adaptive Maximum A Posterior (AMAP)
technique with Computational Anatomy Toolbox 12 (The Structural Brain Mapping
Group at the University of Jena, Germany) to avoid a priori information on the tissue
probabilities. Tissue Probability Maps were used for spatial normalization, initial skull7

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stripping and initial segmentation estimates. Before gray matter and white matter
segmentation, an automated white matter lesion (WML) correction was conducted with
the Lesion Segmentation Toolbox on FLAIR images by masking the averaged signal
intensities of surrounding normal white matter. WML was divided into two regions, theperiventricular area and deep white matter area, and both volumes were calculated using
FLAIR images. For denoising, a spatial-adaptive Non-Local Means noise reduction filter
and Markov Random Field approach were applied during AMAP segmentation.
Coordinate transformation from native space to MNI space was conducted using theDiffeomorphic Anatomical Registration Through Exponentiated Lie Algebra (DARTEL)
algorithm, resulting in voxel volume conversion into a voxel signal. ROI-wise volumetricanalysis was performed with the MarsBar Toolbox (marsbar.sourceforge.net) using ROIs
preset for the LONI Probabilistic Brain Atlas (LPBA40). An additional ROI of thebilateral mPFC was generated by combining brain masks of the Brainnetome Atlas(atlas.brainnetome.org). Local volume calculation was adjusted by total intracranial
volume (TIV), age, sex and WML. For the Z-score estimation, age group-matchedsubjects in the Information eXtraction from Images (IXI) dataset were used as areference, with age, sex, TIV and WM volume as confounding factors. Z-scores were
calculated using the following equation.
𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑚𝑒𝑎𝑛 − 𝐼𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙 𝑣𝑎𝑙𝑢𝑒
𝑍 𝑠𝑐𝑜𝑟𝑒 =𝐶𝑜𝑛𝑡𝑟𝑜𝑙 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛

Supplementary References
1. Jeltsch-David H, Muller S. Neuropsychiatric systemic lupus erythematosus andcognitive dysfunction: the MRL-lpr mouse strain as a model. Autoimmun Rev
2014;13:963-73.
2. Spruston N. Pyramidal neurons: dendritic structure and synaptic integration. Nat Rev
Neurosci 2008;9:206-21.
3. Kaul A, Gordon C, Crow MK, et al. Systemic lupus erythematosus. Nat Rev Dis
Primers 2016;2:16039.
4. Petri M, Orbai A-M, Alarcón GS, et al. Derivation and validation of the Systemic
Lupus International Collaborating Clinics classification criteria for systemic lupus
erythematosus. Arthritis Rheum 2012;64:2677-86.
5. Gladman DD, Ibañez D, Urowitz MB. Systemic lupus erythematosus disease activity
index 2000. J Rheumatol 2002;29:288-91.
6. Gladman D, Ginzler E, Goldsmith C, et al. The development and initial validation of
the Systemic Lupus International Collaborating Clinics/American College of
Rheumatology damage index for systemic lupus erythematosus. Arthritis Rheum
1996;39:363-9.
7. Schwartz N, Stock AD, Putterman C. Neuropsychiatric lupus: new mechanistic
insights and future treatment directions. Nat Rev Rheumatol 2019
8. Zhang Y, Chen K, Sloan SA, et al. An RNA-sequencing transcriptome and splicing
database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci
2014;34:11929-47.

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Supplementary Figures

Supplementary Figure 1. The effects of sleep disturbance stress (SDS).
(A) The appearance of the SDS cage equipped with a running wheel, water supply and chow feeder.
(B) Representative images of the time-corresponding activity and rest in SDS-subjected, -free
MRL/lpr and control mice. Black indicates wheel rolling sensation and is defined as activity; white
indicates rest. SDS-subjected mice of both strains demonstrated more activity than SDS-free mice atnight time. Each line corresponds to mouse activity for three days. (C) The activity (black) / rest
(white) ratio (n = 6 per group). (D, E) Descriptive images of wheel rolling counts to evaluate thecircadian rhythm for daily activity in the presence or absence of SDS in (D) MRL/lpr and (E) control
mice (n = 10 to 11 per group). (F) Ratio of the serum corticosterone level in mice after 14 days of
SDS relative to before SDS (n = 6 per group). (G) Percentage of time spent in the open arms and (H)total travelled distance in SDS-subjected or -free NOD/ShiJcl mice (n = 5 to 7 per group). (C, F) Data
are means. *p < 0.05, ****p < 0.0001 using a paired t-test for paired data. (D, E) Data are means(line) ± s.e.m. (band). (G, H) Data are means ± s.e.m. An unpaired Student’s t-test.
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Supplementary Figure 2. Neuronal activation of stress-responsive brain regions.
(A-E) Evaluation of neuronal activation using immunohistochemistry of phosphorylated cFos (pcFos)-positive cells in the paraventricular nucleus (PVN) between sleep disturbance stress (SDS)subjected or -free MRL/lpr, MRL/MpJ control, and NOD/SchiJcl mice. (A) Schematic drawing of
the PVN was taken from Paxinos and Watson (2007). (B) Representative immunohistochemical
images of neuronal activating p-cFos-positive cells with 4',6-diamidino-2-phenylindole (DAPI) in the
PVN of MRL/lpr and MRL/MpJ control. Scale bar, 100 μm. (C) Quantification of p-cFos-positive
cells in the PVN (n = 6 to 13 per group). (D) Representative immunohistochemical images ofneuronal activating p-cFos-positive cells with DAPI in the mPFC of NOD/SchiJcl. Scale bar, 100
μm. (E) Quantification of p-cFos-positive cells in the mPFC (n = 3 per group). Data are means ±s.e.m. *p < 0.05 using a two-way ANOVA with the post-hoc Tukey-Kramer multiple comparison testor an unpaired Student’s t-test.

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Supplementary Figure 3. Additional results of pathway analysis using public databases.
(A, B) (A) Ingenuity Pathway Analysis using the uniquely downregulated gene set and (B) KEGG
(Kyoto Encyclopedia of Genes and Genomes) analysis using the uniquely upregulated anddownregulated gene set in the prefrontal cortex of sleep disturbance stress (SDS)-subjected MRL/lpr
mice. Bars show inhibition Z-scores or observed gene counts, and dots represent p-values. (C) Seruminterleukin (IL)-12/23p40 levels measured by ELISA (n = 8 per group). (D) CSF interferon (IFN)-γand (E) IL-17 A levels measured by multiplex beads assay (n = 5 per group). Data are means ± s.e.m.
*p < 0.05, **p < 0.01 using a two-way ANOVA with the Tukey-Kramer method.

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Supplementary Figure 4. Scarce infiltration of peripheral CD45high cells and specificity of Il12b
expression in the CNS microglia/macrophages.
(A) Representative images of immunohistochemistry for TCRβ and/or MHC Class II (I-A/I-E)positive cells in the cerebral cortex of SDS-subjected, -free MRL/lpr, and control mice. Scale bar,
100 μm. (B) Representative gating images for the isolated cortex to evaluate CD45high cell
infiltration. CD45high cells, especially with TCRβ positivity, infiltrated the CNS of MRL/lpr mice inan age-dependent manner independently of the SDS load, confirming the scarce CNS infiltration of
CD45high cells in 6-to-8-week-old MRL/lpr mice. (C) Il12b gene expression in mouse CNS cells
shows prominent expression in microglia and macrophages. RNA-seq data from the Barres lab(www.brainrnaseq.org/).8 Data are means ± s.e.m.
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Supplemental material

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Supplementary Figure 5. Activation state analysis and gating strategy in microglia.
(A) Representative images of Iba-1 and CD68 staining. Scale bar, 50 μm. (B) Microglial activation
state scores in microglial activation analysis. Microglia falling into the category of 3 or higher were
defined as reactive microglia. (C) Representative images of the gating strategy for identifying
microglia. After doublet discrimination, Zombie-positive (dead) cells and dump cells including
ACSA-2 (astrocyte), CD140a (oligodendrocyte precursor cell) and MBP (myelin basic protein:oligodendrocyte) positive cells were excluded. Gated CD45intCD11bhigh cells were selected as live
microglia. (D) Flow cytometric gating of (right) fluorescence minus one (FMO) negative control and(left) fully stained samples from SDS-subjected MRL/lpr mice. (E) Representative images of mPFC
microglia of the NOD/ShiJcl. (F) Percentage of reactive microglia and (G) microglial density of the
NOD/ShiJcl (n = 3 per group). Data are means ± s.e.m. An unpaired Student’s t-test.

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Supplementary Figure 6. Neuronal expression analysis and stereotaxic microinjection with IL-12/23p40.
(A) Co-expression analysis of IL12Rβ1 by immunohistochemistry for microglia as Iba-1-positive cells andneurons as NeuN-positive cells. Scale bar, 50 μm. (B) Representative images of the gating strategy to identify
neurons. After doublet discrimination, Zombie-positive (dead) cells and dump cells including astrocytes,
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Supplemental material

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microglia, oligodendrocytes and oligodendrocyte precursor cells were excluded. Gated TUBB3+ cells wereselected as live neurons. (C) Flow cytometric histogram of (gray) FMO negative controls and (red) fully stained
samples from SDS-subjected MRL/lpr mice. (D) Representative histogram for phosphorylated-Stat3 in themPFC neurons. (E) Flow cytometric histogram of FMO negative controls and fully stained samples from SDSsubjected MRL/lpr mice. (F) Fold-change of the mean fluorescence intensity for the phosphorylated-Stat3
expression relative to SDS-free control mice (n = 3 per group). Data are means ± s.e.m. A two-way ANOVA
with the Tukey-Kramer method. (G) Experimental design for the evaluation of the IL-12/23p40 effect on mPFC
neuronal cells by the stereotaxic microinjection model. (H) Golgi-Cox staining of neurons in the mPFC of IL12/23p40-injected or vehicle-injected MRL/MpJ mice. Lower panels are magnified images of the rectangularareas in the upper panels. Scale bars, (upper panel) 50 μm, and (lower panel) 20 μm.

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Supplementary Figure 7. The effects of IL-12/23 signaling blockade therapy in the CNS of stresssubjected MRL/lpr mice.
(A) Representative images of the mPFC in IgG-treated or PBS-treated mice with osmotic pumps indicate
successful antibody distribution throughout the mPFC. (B) No effect of anti-IL-12/23p40 blockade in the
CNS on the serum IL-12/23p40 level was detected by ELISA (n = 4 to 6 per group). (C) Macroscopic
findings of the spleen in anti-p40 antibody- or IgG-treated SDS-subjected MRL/lpr mice. (D)Representative images of activated microglia. (E) Representative histogram of the phosphorylated-Stat4
expression in TUBB3+ neurons. (F) Fold-change in the mean fluorescence intensity of the phosphorylatedStat4 expression in TUBB3+ neurons relative to controls (n = 3 per group). Scale bars, (A) 100 μm, (C) 10mm, and (D) 50 μm. Data are means ± s.e.m. ***p < 0.001 using an unpaired Student’s t-test.

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BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceplaced on this supplemental material which has been supplied by the author(s)Ann Rheum Dis

Supplementary Figure 8. Additional information for the results of experiments
using Tyk2 inhibitor.
Fold-change in the mean fluorescence intensity of phosphorylated-Stat4 expression in
TUBB3+ neurons of deucravacitinib-treated SDS-subjected MRL/lpr mice relative to
vehicle-treated mice (n = 3 per group). Data are means ± s.e.m. **p < 0.01 using an
unpaired Student’s t-test.

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Supplemental material

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Supplementary Figure 9. Specificity of CSF IL-12/23p40 and brain atrophy in NPSLE patients withdiffuse manifestations (dNPSLE).
(A) Serum IL-12/23p40 levels in the derivation cohort at Hokkaido University Hospital (n = 12 healthy controls,n = 17 non-dNSPLE, n = 26 dNPSLE). (B) CSF IL-12/23p40 levels in dNPSLE patients (n = 8 no treatment, n =6 glucocorticoid (GC), n = 14 GC combined with immunosuppressants (IS)). (C) Serum IL-12/23p40 levels inthe validation cohort at Kitasato University Hospital (n = 9 NSPLE with focal manifestations (fNPSLE), n = 18
dNPSLE). (D) CSF IL-12/23p40 levels at Hokkaido University Hospital (n = 8 primary Sjögren’s syndrome(SS), n = 26 dNPSLE). (E) Representative serial axial brain images on which the Z-scores of brain atrophy wererendered in a non-dNPSLE and a dNPSLE patient. (F-I) Voxel-based morphometric analysis of patients with
non-dNPSLE (blue, n = 56) and dNPSLE (red, n = 15) to compare (F) total gray matter volume, (G) total whitematter volume (WMV) corresponding to total intracranial volume (TIV), (H) WM lesions volume corresponding
to age, and (I) the bilateral frontal cortex volume corresponding to TIV. (A-E) Data are medians (interquartile
range). Statistical analysis was performed using a Kruskal-Wallis test with post-hoc Steel-Dwass method or a
Mann-Whitney U test. (F-I) ANCOVA adjusting for age, sex, disease duration of SLE and TIV.

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Supplemental material

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Supplementary Tables
Supplementary Table 1. Demographic and clinical characteristics of SLE patients at Hokkaido
University Hospital for IL-12/23p40 measurements.
Characteristics
Demographics
Age at lumbar puncture, years
Sex, female
Disease duration, years
SLEDAI-2K

All SLE (n=47)

Non-dNPSLE (n=19) dNPSLE (n=28)

30 [23-41]
40 (85%)
6 [0-15]
14 [10-19]

29 [21-37]
18 (95%)
2 [0-9]12 [8-16]

31 [25-44]
22 (79%)
11 [0-17]
15 [11-22]

Disease classification at the time of sampling
New onset
18 (38%)
Relapse29 (62%)12 (63%)7 (37%)

6 (21%)
22 (89%)Complications
Hypertension
Dyslipidemia
Diabetes

4 (21%)
2 (11%)0

9 (32%)
5 (18%)
2 (7%)

8 (42%)
4 (21%)
6 (32%)
1 (5%)
4 (21%)
7 (37%)
1 (5%)
2 (11%)

8 (29%)
3 (11%)
5 (18%)0
1 (4%)
5 (18%)
00

8 (42%)
3 (16%)
7 (37%)
2 (11%)
1 (5%)

6 (21%)
3 (11%)
4 (14%)0
2 (7%)

13 (28%)7 (15%)
2 (4%)

Symptoms except diffuse neuropsychiatric manifestations
Rash
16 (34%)
Oral ulcer
7 (15%)
Arthritis
11 (23%)
Myositis
1 (2%)
Serositis
5 (11%)
Nephritis
12 (26%)
Headache
1 (2%)
Peripheral neuropathy
2 (4%)
Seizure0
Leucopenia
14 (30%)Hemolytic anemia
6 (13%)
Thrombocytopenia
11 (23%)
Hemophagocytic syndrome
2 (4%)
Antiphospholipid syndrome
3 (6%)Classification of diffuse neuropsychiatric manifestations
Anxiety
Mood disorder
Cognitive dysfunction
Psychosis
Acute confusional state0
8 (29%)
1 (6%)
4 (14%)
15 (54%)

Laboratory findings
Serum
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Supplemental material

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceplaced on this supplemental material which has been supplied by the author(s)Immunoglobulin G (IgG), mg/dL
Anti-DNA antibody (RIA), IU/mL
Anti-dsDNA antibody (ELISA), IU/mL
Anti-Ro/SSA antibody, U/mL
Anti-cardiolipin (CL) IgG
Anti-CL/β2-glycoprotein complex
antibody
Lupus anticoagulant (DRVVT)
Complement C3, mg/dL
Complement C4, mg/dL
CH50, IU/mL
Cerebrospinal fluid
cell count, /μL
mononuclear cell count, /μL
Protein, mg/dL
Glucose, mg/dL
IgG index, ratio
Interleukin-6 (IL-6), pg/mL
IL-8, pg/mL
Treatment
Glucocorticoid- glucocorticoid dose at CSF collection
Cyclophosphamide
Mycophenolate mofetil
Azathioprine
Hydroxychloroquine
Tacrolimus
Cyclosporine
Belimumab
Warfarin
Direct oral anticoagulant
AspirinClopidogrel
Cilostazol
Sleep medication

1599 [983-2016]
9.8 [0-170.2]
8 [0-92.1]
39.7 [0-126.9]
10/16 (62%)

1599 [981-2212]
21 [0-278.1]
26.4 [0-93.3]
59.4 [10.2-136.5]
3/5 (60%)

1609 [998-1912]
9.8 [0-42.3]
0 [0-147.7]
29.9 [0-117.5]
7/11 (64%)

5 (11%)
13 (37%)57 [32-87]
10 [3-17]
26.1 [10.1-48.1]

2 (13%)
6 (46%)
47 [30-107]
6 [3-27]
16.4 [9.6-55.7]

3 (11%)
7 (32%)
70 [40-82]
11 [2.3-16]
29.8 [10.9-45.8]

1 [0-3]
1 [0-3]30 [23-46]
55 [52-68]
0.82 [0.73-0.97]
5.9 [2.5-15.5]
48.9 [4.98-160.8]

1 [0-3]
1 [0-3]26 [21.8-41.5]
55 [53-66.8]
0.76 [0.68-0.93]
2.9 [1.9-5.6]
23.5 [1-45.9]

1 [0-3]
1 [0-2.8]
35.5 [23.8-55.8]
54 [50.3-70.3]
0.85 [0.73-0.98]
8.9 [3.2-26.3]
99.9 [17.7-186.3]

33 (70%)12 (63%)30 [14-40]
2 (11%)
2 (11%)
00
5 (26%)0
1 (5%)
1 (5%)0
3 (16%)0
2 (11%)
6 (32%)

21 (75%)20 [10-56]
4 (14%)
2 (7%)
3 (11%)
2 (7%)
2 (7%)
4 (14%)00
0
4 (14%)
1 (4%)
1 (4%)
20 (71%)6 (13%)
4 (9%)
3 (6%)
2 (4%)
7 (15%)
4 (9%)
1 (2%)
1 (2%)0
7 (15%)
1 (2%)
3 (6%)
26 (55%)

Ann Rheum Dis

Values are presented as n (%) or medians (interquartile range).
Abbreviations: SLE, systemic lupus erythematosus; NP, neuropsychiatric; dNPSLE, NPSLE with diffuse
manifestations; SLEDAI-2K, Systemic Lupus Erythematosus Disease Activity Index 2000; DRVVT, dilute
Russel's viper venom time; CH50, 50% hemolytic complement activity

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Supplemental material

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Supplementary Table 2. Demographic and clinical characteristics of NPSLE patients at Kitasato
University Hospital for IL-12/23p40 measurements.
Characteristics
Demographics
SLEDAI-2K

All NPSLE (n=27) fNPSLE (n=9)

dNPSLE (n=18)

13 [10-18]

6 [0-16]

15 [12-19]

Disease onset
Initial onset
Relapse12 (44%)15 (56%)

3 (33%)
6 (67%)

9 (50%)
9 (50%)

Complications
Hypertension
Dyslipidemia
Diabetes

3 (11%)0
2 (7%)0

3 (17%)0

2 (11%)0
1 (11%)
1 (11%)
00
2 (22%)
1 (11%)
6 (67%)
2 (22%)
2 (22%)
00
1 (11%)
4 (44%)

7 (39%)
2 (11%)
3 (17%)
1 (6%)
5 (28%)
8 (44%)000
1 (6%)
2 (11%)
3 (17%)
1 (6%)
6 (33%)

Symptoms except diffuse neuropsychiatric manifestations
Rash
7 (26%)
Oral ulcer
3 (11%)
Arthritis
4 (15%)
Myositis
1 (2%)
Serositis
5 (19%)
Nephritis
10 (37%)
Headache
1 (2%)
Peripheral neuropathy
6 (22%)
Seizure2 (7%)
Leucopenia
2 (7%)
Hemolytic anemia
2 (7%)
Thrombocytopenia
3 (11%)
Hemophagocytic syndrome
2 (7%)
Antiphospholipid syndrome
10 (37%)
Classification of diffuse neuropsychiatric manifestations
Anxiety
Mood disorder
Cognitive dysfunction
Psychosis
Acute confusional state
Laboratory findings
Serum
Immunoglobulin G (IgG), mg/dL
Anti-DNA antibody (RIA), IU/mL
Anti-dsDNA antibody (ELISA), IU/mL
Anti-Ro/SSA antibody

1654 [1159-2004]
8.9 [0-44]
8 [0-92.1]
16 (59%)1 (6%)
2 (11%)0
3 (17%)
12 (67%)1691 [1270-2915]
3.8 [0-32]
23 [23-23]
6 (67%)

1602 [1120-1891]
9.4 [3.93-76.8]
23 [10-56]
10 (56%)21

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Supplemental material

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceplaced on this supplemental material which has been supplied by the author(s)Anti-NR2 antibody
Anti-cardiolipin (CL) IgG
Anti-CL/β2-glycoprotein complex
antibody
Lupus anticoagulant (DRVVT)
Complement C3, mg/dL
Complement C4, mg/dL
CH50, IU/mL

8 (33%)
9 (33%)

4 (50%)
4 (44%)

4 (25%)
5 (28%)

4 (15%)
6 (22%)
65 [40-79]
17 [5-23]
31 [12-40]

2 (22%)
2 (22%)
78 [59.5-127]
18 [9-36]
40 [20.5-49.5]

2 (11%)
4 (22%)
58 [37-76.8]
13 [5-20]
27.5 [11.5-36]

Cerebrospinal fluid
cell count, /μL
mononuclear cell count, /μL
Protein, mg/dL
Glucose, mg/dL
IgG index, ratio
Interleukin-6 (IL-6), pg/mL
Anti-NR2 antibody

2 [0-12]
2 [0-7]64 [37-104]
58 [46-72]
0.56 [0.49-0.74]
18.5 [4.1-150.8]
17 (74%)

3 [0-23]
3 [0-23]
46 [34.5-74]
59 [47-71]
0.53 [0.44-0.69]
30.7 [2.2-941]
6 (86%)

2 [0-13]
2 [0-9]83.5 [48.3-124]
54 [46-74]
0.60 [0.49-0.76]
15.1 [4.85-110.1]
11 (69%)Treatment
Glucocorticoid
Cyclophosphamide
Mycophenolate mofetil
Azathioprine
Hydroxychloroquine
Tacrolimus
Cyclosporine
Belimumab
Warfarin
Direct oral anticoagulant
AspirinClopidogrel
Cilostazol

27 (100%)
10 (37%)2 (7%)
1 (4%)
3 (11%)
4 (15%)
4 (9%)
1 (4%)
5 (19%)
0
6 (22%)
1 (4%)
1 (4%)

9 (100%)2 (22%)
00
1 (11%)0
1 (11%)0
1 (11%)
0
3 (33%)00

18 (100%)
8 (44%)
2 (11%)
1 (6%)
2 (11%)
3 (17%)
3 (17%)
1 (6%)
4 (22%)0
3 (17%)
1 (6%)
1 (6%)Ann Rheum Dis

Values are presented as n (%) or medians (interquartile range).
Abbreviations: NPSLE, neuropsychiatric systemic lupus erythematosus; fNPSLE, NPSLE with focal
manifestations; dNPSLE, NPSLE with diffuse manifestations; SLEDAI-2K, Systemic Lupus
Erythematosus Disease Activity Index 2000; NR2, N-Methyl-D-Aspartic Acid Receptor 2; DRVVT, dilute
Russel's viper venom time; CH50, 50% hemolytic complement activity

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Supplementary Table 3. Demographic and clinical characteristics of SLE patients at Hokkaido
University Hospital used in VBM analysis.
Characteristics
Demographics
Age at image acquisition, years
Sex, female
Disease duration, years
SLICC Damage index

All SLE (n=71)

Non-dNPSLE (n=56) dNPSLE (n=15)

43 [36-49]
65 (92%)
11 [7-19]
1 [0-1]45 [37-49]
53 (95%)
11 [6-18]
0.5 [0-1]

41 [31-52]
12 (80%)16 [7-21]
1 [1-2]Complications
Hypertension
Dyslipidemia
Diabetes

24 (34%)28 (39%)2 (3%)

18 (32%)20 (36%)2 (4%)

6 (40%)
8 (53%)
0

Disease classification
NPSLE- Focal manifestations (fNPSLE)- Diffuse manifestations (dNPSLE)
Antiphospholipid syndrome

17 (24%)- 2 (12%)- 15 (88%)
38 (54%)2 (11%)- 2 (100%)-030 (54%)15 (100%)-0- 15 (100%)
8 (53%)

Voxel based morphometric parameters
Stroke or white matter lesions

35 (49%)24 (43%)

Total intracranial volume, mL
Cerebrospinal fluid, mL

1469 [1430-1571] 1466 [1418-1570]
380 [345-435]
366 [340-424]

11 (73%)1482 [14401632]
445 [380-501]

Laboratory findings
Serum
Antinuclear antibody
Anti-DNA antibody (RIA), IU/mL
Anti-dsDNA antibody (ELISA), IU/mL
Anti-Ro/SSA antibody
Anti-cardiolipin (CL) IgG
Anti-CL/β2-glycoprotein complex antibody
Lupus anticoagulant (DRVVT)
Complement C3, mg/dL
Complement C4, mg/dL
CH50, IU/mL

67 (94%)2.75 [0.35-11.4]
0.6 [0-14.7]
19.6 [0-122]
12/24 (50%)
23 (41%)27 (38%)91 [72-112]
19 [11-24]
48.8 [37.3-54.6]

53 (95%)
3.9 [0-19.3]
0.6 [0-18.7]
11.5 [0-121]
10/19 (53%)
18 (32%)20 (36%)90 [72.3-112]
18 [11-24]
49.1 [37.2-55.9]

14 (93%)
2.5 [1-4.5]
0.7 [0-5.8]
56 [1.4-138]
2/5 (40%)
5 (33%)
7 (47%)
99 [65-112]
20 [12-27]
46.8 [38.5-54.1]

Treatment
Glucocorticoid
Mycophenolate mofetil
Azathioprine
Hydroxychloroquine
Tacrolimus

58 (82%)13 (18%)5 (7%)
23 (32%)29 (41%)43 (77%)8 (14%)
4 (7%)
18 (32%)21 (38%)15 (100%)
5 (33%)
1 (7%)
5 (33%)
8 (53%)
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Supplemental material

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any relianceplaced on this supplemental material which has been supplied by the author(s)Belimumab
Sleep medication
Post-treatment history of cyclophosphamide

2 (3%)
15 (21%)
17 (24%)

1 (2%)
10 (18%)7 (13%)

1 (7%)
5 (33%)
10 (67%)

Anticoagulation- Warfarin- Direct oral anticoagulant

14 (20%)- 11 (79%)- 3 (21%)

10 (18%)- 9 (90%)- 1 (18%)

4 (27%)- 2 (50%)- 2 (50%)

Antiplatelets- Aspirin- Clopidogrel- Cilostazol

31 (44%)- 25 (81%)- 11 (35%)- 2 (7%)25 (44%)- 20 (80%)- 9 (38%)- 2 (8%)6 (40%)- 5 (83%)- 2 (33%)-0

Ann Rheum Dis

Values are presented as n (%) or medians (interquartile range).
Abbreviations: NPSLE, neuropsychiatric systemic lupus erythematosus; dNPSLE, NPSLE with diffuse
manifestations; fNPSLE, NPSLE with focal manifestations; SLICC, Systemic Lupus International
Collaborating Clinics; DRVVT, dilute Russel's viper venom time; CH50, 50% hemolytic complement activity

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Supplementary Table 4. Sleep medications prescribed to the SLE patients in the Hokkaido
University Hospital cohort
Characteristics
Benzodiazepines
Brotizolam
Clonazepam
Flunitrazepam
Etizolam
Lormetazepam
Lorazepam
Imidazopyridine
Zolpidem
Cyclopyrrolone
Zopiclone
Melatonin receptor agonist
Ramelteon

All SLE (n=26)

Non-dNPSLE (n=6)

dNPSLE (n=20)

12 (46%)2 (8%)
3 (12%)
2 (8%)
2 (8%)
3 (12%)

4 (67%)
1 (17%)000
0

8 (40%)
1 (5%)
3 (15%)
2 (10%)
2 (10%)
3 (15%)

4 (15%)

1 (17%)

3 (15%)

1 (4%)0

1 (5%)

1 (4%)0

1 (5%)

Values are presented as n (%).

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