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. Author manuscript; available in PMC: 2025 Feb 15.
Published in final edited form as: J Neuroimmunol. 2024 Jan 3;387:578282. doi: 10.1016/j.jneuroim.2023.578282

Dysregulated Autotaxin Expression by T Cells in Multiple Sclerosis

Cora L Petersen-Cherubini 1,2, Yue Liu 2, Joshua L Deffenbaugh 2, Shawn P Murphy 2, Matthew Xin 2, Christina N Rau 2, Yuhong Yang 3, Amy E Lovett-Racke 2,4
PMCID: PMC10923181  NIHMSID: NIHMS1959216  PMID: 38183947

Abstract

Multiple sclerosis (MS) is a demyelinating disease characterized by infiltration of autoreactive T cells into the central nervous system (CNS). In order to understand how activated, autoreactive T cells are able to cross the blood brain barrier, the unique molecular characteristics of pathogenic T cells need to be more thoroughly examined. In previous work, our laboratory found autotaxin (ATX) to be upregulated by activated autoreactive T cells in the mouse model of MS. ATX is a secreted glycoprotein that promotes T cell chemokinesis and transmigration through catalysis of lysophoshphatidic acid (LPA). ATX is elevated in the serum of MS patients during active disease phases, and we previously found that inhibiting ATX decreases severity of neurological deficits in the mouse model. In this study, ATX expression was found to be lower in MS patient immune cells during rest, but significantly increased during early activation in a manner not seen in healthy controls. The ribosomal binding protein HuR, which stabilizes ATX mRNA, was also increased in MS patients in a similar pattern to that of ATX, suggesting it may be helping regulate ATX levels after activation. The proinflammatory cytokine interleukin-23 (IL-23) was shown to induce prolonged ATX expression in MS patient Th1 and Th17 cells. Finally, through ChIP, re-ChIP analysis, we show that IL-23 may be signaling through pSTAT3/pSTAT4 heterodimers to induce expression of ATX. Taken together, these findings elucidate cell types that may be contributing to elevated serum ATX levels in MS patients and identify potential drivers of sustained expression in encephalitogenic T cells.

Keywords: autotaxin, autotaxin/LPA axis, CD4 T cells, multiple sclerosis, CNS autoimmunity

Graphical Abstract

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1. INTRODUCTION

Multiple sclerosis (MS) is a debilitating neurodegenerative disease caused by autoreactive T cells that attack the myelin sheath surrounding neurons. During MS, these encephalitogenic T cells are able to cross the blood brain barrier (BBB) and initiate robust neuroinflammation that leads to lesions in the brain and spinal cord(Chitnis, 2007; Kaskow & Baecher-Allan, 2018). There is no cure for MS, and current therapies vary in efficacy, but can slow disease progression(J. H. Yang et al., 2022). In order to develop better treatment options for MS patients, the molecular pathways facilitating the central nervous system (CNS) infiltration by T cells need to be better understood.

Previous work by our lab utilized the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), to study differential signaling pathways in the T cells that induce neuroinflammation(P. W. Lee et al., 2017). We found that encephalitogenic T cells differentiated with IL-12 + IL-23 or IL-6 + IL-23 express unique molecules that may be facilitating their passage into the CNS. Recently, we have investigated the role of the secreted glycoprotein autotaxin (ATX) in enabling T cell trafficking in the context of CNS autoimmunity(Petersen-Cherubini et al., 2023). This was the first known observation of ATX being expressed on the protein level directly by T cells. We found that murine encephalitogenic T cells not only increase ATX expression but treating with an ATX inhibitor in vivo decreases T cell trafficking to the CNS during EAE. Paired with other studies showing ATX is increased in the serum and cerebral spinal fluid of MS patients(Schmitz et al., 2017; Zahednasab et al., 2014), we hypothesized that ATX may be playing a role in pathogenic human T cells as well.

ATX is responsible for the catalyzation of lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA)(van Meeteren & Moolenaar, 2007). LPA binds to surface receptors, LPAR1–6, to facilitate downstream growth factor-like signaling, including migration, survival, or proliferation depending on the type and specificity of receptors present(Xiang et al., 2020). While the catalytic properties of ATX can affect a variety of cell types, it known that ATX expression by endothelial cells is important to T cell trafficking(Bai et al., 2013; Nakasaki et al., 2008). ATX signaling in high endothelial venules (HEVs) promotes T cell transmigration from the blood stream into the lymphatic system(Nakasaki et al., 2008), and blocking this signaling traps T cells in the endothelium (Bai et al., 2013). Thus, endothelial-driven ATX is vital to naïve T cells, that do not express ATX, to enter and populate lymphoid tissues. ATX expression is also increased in mature dendritic cells(J. H. Lee et al., 2021), activated myeloid cells (Ninou et al., 2020), B cells(Lin et al., 2019), and CNS-resident glial cells during inflammatory conditions(Bitar et al., 2022; Joshi et al., 2021; Savaskan et al., 2007).

Dysregulated ATX signaling has also been implemented in a variety of inflammatory diseases including pulmonary fibrosis(Maher et al., 2019; Ninou et al., 2018), cancer(Azare et al., 2011; David et al., 2010; Noh et al., 2006; Peyruchaud et al., 2020; Tang et al., 2020), and MS(Schmitz et al., 2017; Zahednasab et al., 2014). Schmitz et al showed that ATX is increased in the serum of MS patients during the first documented attack and during relapse(Schmitz et al., 2017), but the source of this ATX remains unclear. Importantly, both of these time-points are characterized by increased presence of activated immune cells(Kaskow & Baecher-Allan, 2018). Augmented serum LPA levels have also been associated with increased BBB permeability(Bhattarai et al., 2021, 2023; Bitar et al., 2022), and in vitro demyelination of axons(Nagai et al., 2010). Thus, better understanding how ATX may be contributing to MS pathology, by investigating what cell types are promoting amplified levels during disease, would be beneficial for improved therapeutic strategies.

Since ATX is expressed by so many cell types, in this study, we focused on elucidating changes to ATX expression in activated T cells by MS patients since it has not previously been demonstrated that T cells are a source of ATX prior to our studies. Our studies found that polyclonal activation of peripheral blood mononuclear cells (PBMCs) is sufficient to increase ATX expression in human CD4 T cells. While MS patients have lower levels of ATX at rest, they increase ATX expression faster than healthy controls after activation, potentially identifying one source for ATX fluctuations in the serum during active stages of disease. Lastly, this study identified the proinflammatory cytokine IL-23 as a potential contributor to dysregulated ATX signaling in MS patients through pSTAT3/pSTAT4.

2. METHODS

2.1. Human samples

Most healthy donor lymphocyte samples were obtained from The American Red Cross or Versiti as buffy coats. The demographics of these healthy donors consisted of males and females aged 18–50 years, defined as “healthy” by these institutions at the time of blood draw. Samples from untreated MS patients [both male and female with primary progressive MS (PPMS), secondary progressive MS (SPMS), or relapsing remitting MS (RRMS)] and some healthy control samples were obtained after informed consent via leukapheresis. PBMCs were isolated from all samples via Ficoll gradient (GE Healthcare), followed by cryopreservation in freezing media consisting of 50% RPMI 1640, 40% heat-inactivated human serum, and 10% DMSO, and stored in liquid nitrogen until resuspension for in vitro experiments. Due to limited numbers of healthy donor PBMCs available via leukapheresis, we used outside vendors to secure additional healthy control samples. In both cases, the white blood cells are concentrated using centrifugation with citrate as the anti-coagulant and processed similarly with the exception that the buffy coats from outside sources were not processed and frozen until the next day. The inclusion of a few healthy control samples from leukapheresis allowed us to verify that slight differences in PBMC acquisition was not impacting the data.

2.2. Cell culture

Frozen peripheral blood mononuclear cells (PBMCs) were recovered from liquid nitrogen, washed with PBS, resuspended in RPMI1640/10% FBS, and placed in tissue culture flasks for 2hr at 37°C with 5% CO2 to remove adherent cells. Non-adherent cells, which included T cells, were collected and activated with anti-CD3/CD28 (1μg/mL each) coated plates, or with Dynabeads™ Human T-Activator CD3/CD28 beads (Gibco) at a ratio of 1 bead:10 cells. UV-inactivated influenza A H1N1 antigen was also used in indicated experiments at 10ug/mL without removal of adherent antigen presenting cells. During activation, cells were incubated for at 37°C for time-points specified, and cytokines IL-12 (0.5ng/mL, R&D Systems), IL-6 (25ng/mL), and IL-23 (5ng/mL, R&D Systems) were added to cultures when specified. Supernatants were collected and used for Human ENPP-2/Autotaxin DuoSet ELISA (R&D Systems). Cells were then collected, washed once with PBS, and processed for flow cytometry, or saved in Trizol for RT-qPCR. CD4+ T cells were isolated after washing resting or activated cells once with PBS. Miltenyi CD4+ T cell isolation kit, or MojoSort human CD4 T cell isolation kits were used according to manufacturer protocol.

2.3. Autotaxin ELISA

Human ENPP-2/Autotaxin DuoSet ELISA (R&D Systems) was used to measure secreted ATX from in vitro human cultures according to manufacturer protocol. Briefly, Immulon 2 HB Flat Bottom Microtiter Plates (ThermoFisher) were coated with 0.48μg/mL of capture antibody and left at room temperature overnight. Plates were washed twice with 0.05% Tween 20 in PBS and blocked for 1hr with 200μL 1% BSA/PBS. Supernatants were collected from samples at indicated time points. Standard ATX was diluted and 100μL added to plates in triplicate at concentrations between 5–0.08ng/mL. Supernatant samples were vortexed and 100μL was added to each well in duplicate. Plates were incubated overnight at 4°C and then washed four times with 0.05% tween in PBS. Detection antibody was added to plate at 50ng/mL in 100uL and incubated at room temperature for 2 hr. Plates were washed four times and 40-fold dilution of Streptavidin-HRP was added for 20mins at room temperature in the dark. Plates washed four times and 100μL Tetramethylbenzidine (Millipore Sigma, T0440) added and incubated in dark for 10mins. Reaction was stopped using 100μL 1M sulfuric acid, and plate was read on Molecular Devices Emax Precision Plate Reader at 450nm. Softmax Pro version 7.0 (Molecular Devices) was used to process plate and data was then analyzed in Excel. GraphPad Prism 7 was used to generate graphs and run statistical analysis. Values were defined as significant if p≤0.05.

2.4. Flow cytometry

Samples collected at indicated timepoint and washed once with PBS. FcR block (Miltenyi Biotec) in flow buffer (1%BSA/PBS) added to cells for 10mins prior to the addition of surface antibodies, and then cells were incubated in antibodies for 30 mins at 4°C. Samples were washed three times Fixation/Permeabilization solution (BD Biosciences) added to cells for 20 mins on ice. Cells were washed with 1X BD Perm/Wash™ buffer before added FC block. Primary ATX antibody (LS Biosciences) was added at 1μg/mL and incubated for 1hr at RT. Samples were washed thrice and then secondary antibody (Rabbit IgG APC-conjugated secondary antibody, R&D Systems) added for 30 mins at RT. Cells washed three times and resuspended in 0.5% formaldehyde. Samples run on BD FACSymphony™ A3 Cell Analyzer with 100,000 events collected per sample and processed with FlowJo™ v10.8.1 Software (BD Life Sciences). Data visualization and statistical analysis performed with GraphPad Prism, version 9.3.0 for Mac OS, GraphPad Software, San Diego, California USA, www.graphpad.com.

2.5. RT-qPCR

RNA was isolated by Trizol extraction and concentrations were measured with NanoDrop (Thermo Scientific). Superscript™ III kit protocol (Invitrogen) was used to generate cDNA. RT-qPCR was run in 384-well plates loaded with 80–100ng cDNA, 5μL Universal Taqman® PCR Master Mix (Applied Biosystems), 0.5μL human Enpp2 TaqMan® Primer (Thermo Fisher Scientific, Hs00905117_m1), and 0.5μL water. Samples were plated in duplicate with the endogenous loading control Hprt1 (TaqMan® Primer, Thermo Fisher Scientific, Hs01003267_m1). Plate was processed in the OSU Cancer Genomics Core Facility. RQ values represent 2−ΔΔCT values calculated from endogenous loading control and the average of each control group. Data analysis was conducted with Excel, and visualized with GraphPad Prism.

2.6. ChIP and re-ChIP Assays

Healthy human CD4+ T cells were activated with plate bound anti-CD3/28 in the presence of IL-12 (0.5ng/mL) for 72hr. Cells were stimulated with IL-23 (5ng/mL) for 2hr prior to collection. SimpleChIP® Plus Enzymatic Chromatin IP Kit with Magnetic Beads (Cell Signaling Technology) was used according to the manufacturer’s protocol. Briefly, DNA was cross-linked, nuclei were pelleted and digested, and then the chromatin was immunoprecipitated overnight at 4°C using pSTAT3 Rabbit mAb (Cell Signaling Technology, #9145), Bcl-6 rabbit pAb (Santa Cruz Biotechnology, sc-858) or Normal Rabbit IgG (Cell Signaling Technology, #2729). If re-ChIP was performed, 10mM DTT was added to samples and incubated at 37°C for 30mins before addition of pSTAT4 rabbit mAb (Cell Signaling Technology, #4134) overnight at 4°C. Chromatin was then eluted from magnetic beads, and cross-link reversed. DNA was purified using spin columns and PCR was run using two Enpp2 primers sequences (Supplemental Table 1). Presence of immunoprecipitated DNA was then visualized with gel electrophoresis.

2.7. Statistics

Statistical analysis was performed using GraphPad Prism (version 9.3.0 for Mac OS, GraphPad Software, San Diego, California USA, www.graphpad.com) as indicated in the figure legends. Welch’s T test was used to compare two data sets, and one or two-way ANOVA tests were used to compare samples of three or more groups. Pairing was applied when appropriate. P-values p≤0.05 were considered significant, and level of significance is represented by asterisks between compared groups.

3. RESULTS

3.1. Activation increases autotaxin expression in CD4+ T Cells

In our previous studies, we found ATX to be expressed by activated murine T cells (Petersen-Cherubini et al., 2023). To determine if T cell activation induces ATX expression in human T cells, PBMCs were polyclonally activated and screened for ATX by flow cytometry. Since ATX is a secreted protein and can be produced by several different immune cell types, intracellular ATX was assessed to measure ATX produced by cells prior to secretion (Fig. 1a–b). There was a significant increase in the percent of ATX+CD25+ CD4 T cells after activation (Fig. 1c,d), as well as an increase in ATX mean fluorescent intensity (MFI) that peaked at 48hr, indicating that the amount of ATX produced per CD4 T cell was highest at this time (Fig. 1e).

Figure 1: ATX is increased after activation in CD4+ T cells.

Figure 1:

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PBMCs from healthy individuals (n=4) were activated with polyclonal activation for 24, 48, or 72hr. Resting cells were stained the day of cell seeding after a 4hr incubation with golgi plug. A) Gating strategy showing lymphocytes, single cells, live cells, and CD3+ cells. B) Representative flow plots from one healthy control after 48hr of polyclonal activation with anti-CD3/CD28 coated beads. Flow plots show control staining (secondary antibody only), surface staining with ATX antibody, and intracellular staining with ATX antibody. Cells gated on CD3+ live singlets, and the frequency of cells in each quadrant is indicated. C) Representative histograms from 3 healthy controls showing increased ATX staining in activated CD4 T cells. Secondary antibody only staining (control, white), resting CD4 T cells (light grey), or polyclonally activated cells at 72hr (dark grey). Histograms normalized to mode. D) Percent ATX+CD25+ CD4 T cells at different timepoints of activation. E) Quantification of ATX median fluorescence intensity (MFI) in resting or activated CD4+ T cells. F) ATX MFI in CD4 T cells at rest or after influenza (H1N1) activation. G) Secreted ATX in supernatants of polyclonally activated cells at 24, 48, or 72hr activation. Two-way ANOVA with Tukey’s post-hoc test. Representative of two independent experiments. * denotes p<0.05, ** p<0.005, *** p <0.0005.

Polyclonal activation mimics interactions with antigen presenting cells to activate T cells irrespective of their T cell receptor specificity. To better simulate physiological conditions, ATX MFI was analyzed after exposure to influenza antigen in the presence of antigen presenting cells. Like with the polyclonal activation, peak ATX expression was seen in CD4+ T cells at 48hr (Fig. 1f) verifying that this was not an artifact of polyclonal activation alone.

To validate the changes seen by flow cytometry, secreted ATX was also assessed in the supernatants of polyclonal activated cells by ELISA. ATX significantly increased after 72hr of activation (Fig. 1g). Since the cells were cultured in FBS containing medium, we also validated that the ELISA kit did not cross-react with bovine ATX (Supplemental Fig. 1). No ATX protein was detected in FBS media, confirming that the ATX observed was produced by the cultured human cells and can be detected by both ELISA and through intracellular flow cytometry staining.

3.2. Autotaxin expression is dysregulated in MS PBMCs

Next, we sought to determine if MS patient PBMCs differentially express ATX. At rest, CD4 T cells from MS patients have decreased ATX expression on both the transcript (Fig. 2a) and the protein (Fig. 2b) levels compared to healthy controls. This difference seemed to be coming from mostly the memory population, rather than naïve cells which had a low frequency of ATX+ CD4+ T cells (Fig. 2c). There was also a trend towards lower a frequency of CD8 T cells expressing ATX in MS patients, but it was not significant (Supplemental Fig. 2a).

Figure 2: Resting T cells from MS patients have dysregulated ATX expression.

Figure 2:

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A) Relative quantification (2−ΔΔCT) of ATX mRNA in resting CD4+ T cells from healthy (n=10) or MS patients (n=10). Mean ± SEM, Welch’s unpaired T test. B) Representative flow plots of ATX staining in resting CD4+ naïve (CD45RO−) or memory (CD45RO+) T cells, pre-gated on CD4+CD3+ live singlets. Control shows secondary antibody only staining. Numbers indicate frequency of CD4+ T cells in each quadrant. C) Quantification of %ATX+ CD4 T cells in the memory or naïve populations. Healthy (n=6), MS (n=8); Welch’s unpaired T test between each group. Data representative of two independent experiments. * denotes p<0.05, ** p<0.005.

Since ATX expression is upregulated after activation, we compared levels of ATX between MS and healthy controls after 24 and 48hr of polyclonal activation. Contrary to resting conditions, MS patients had significantly more ATX+ cells (gated only on live singlets) after 24hr of activation, but this difference was not observed at 48hr (Fig. 3a,b), suggesting that ATX overexpression in MS may be time-dependent. Importantly, there was no significant difference in the frequency of activated cells as determined by CD25+ staining (Suppl. Fig. 3a), indicating that this increase in ATX is not due to differences in activation levels alone. Secreted ATX was also assessed from cultured supernatants, and MS patients showed significantly more ATX after 48hr than healthy controls (Fig. 3c). The frequency of ATX+ cells was also quantified within the CD3− population, which includes B cells, and any non-adherent myeloid populations. While there was no difference between MS and healthy at rest, after the polyclonal activation of T cells, the frequency of ATX+CD3− cells significantly increased, suggesting that crosstalk between activated T cells and resting antigen presenting cells is sufficient to increase ATX expression in multiple cell types (Supplementary Fig. 3b). Taken together, these findings suggest that after activation, MS patient PBMCs begin expressing ATX faster than healthy controls, and this leads to increased levels of secreted ATX over time.

Figure 3: Polyclonal activation increases ATX expression in MS patients faster than healthy controls.

Figure 3:

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PBMCs from healthy (n=6) or MS (n=9) were plated for two hours in flasks to remove adherent cells. Non-adherent cells were collected and replated at 3 million cells/well in 48-well plates. T cells were activated with anti-CD3/anti-CD28 coated beads for 24 or 48hr and then collected and stained for flow cytometry. A) Representative flow plots and quantification of ATX staining in all live singlets, gated based on secondary antibody only control. B) Quantification of ATX+ live cells. Mean ± SEM, Welch’s unpaired T test. C) Secreted ATX measured from cultured supernatants shown in ng/mL. Mean ± SEM, Welch’s unpaired T test. D-F) ATX MFI in D) resting naïve or memory CD4 T cells, E) 24hr polyclonally activated, or F) 48hr polyclonally activated CD4 T cells. N=4/group, mean ± SEM, Welch’s T test. Data representative of two independent experiments. * denotes p<0.05

CD4 T cell-specific ATX levels were also assessed in activated cells. At 24hr, MS patients showed a trend toward an increased ATX+ CD4+ population, though due to high variability amongst healthy controls, this was not statistically significant (Fig. 3d). However, we did notice that MS patients show an increase in ATX MFI in the naïve population at 24hr of activation (Fig. 3e). No differences were observed in activated naïve or memory cells at 48 hr (Fig. 3f). Taken together, these data suggest that MS patient CD4 T cells have intrinsically dysregulated ATX expression, which leads to under-production at rest, and over production during early activation.

3.3. Changes to HuR levels correlate with altered autotaxin expression

To better understand what may be causing these differences in ATX levels in MS patients, HuR expression was analyzed. HuR is an RNA binding protein that regulates the stability of ATX mRNA(Sun et al., 2016) and is increased in activated cells(Atasoy et al., 1998; Yu et al., 2021). Recently, MS patient PBMCs were found to have decreased HuR expression(Pistono et al., 2020). To determine if changes in HuR expression may be contributing to changes in ATX levels, we analyzed HuR by flow cytometry. Resting MS patients had decreased HuR expression in both CD3+ (Fig. 4a) and CD3− (Fig. 4b) subsets, supporting previous work that HuR is decreased in total PBMCs in MS patients(Pistono et al., 2020), and elucidating that this reduction occurs across multiple immune cell subsets and is not merely representing the phenotype of the most numerous cell population. MS patients’ resting naïve CD4+ T cells, however, had increased frequencies of HuR+ cells, whereas the memory population had a lower frequency and only increased for a short time after activation (Fig. 4c–e). HuR+ populations in CD8 T cells were also quantified, and while they were also significantly decreased HuR+ population in MS patients at rest, there was no difference after activation (Supplemental Fig. 2d–f). It appears that increases in HuR+ cells and ATX expression in MS patients occur early after activation and begin to return to baseline levels within 48hr. Considering HuR stabilizes ATX mRNA, it is logical that having higher baseline levels of HuR in resting naïve CD4 T cells may be contributing to more ATX production in these cells during early activation.

Figure 4: MS patient CD4+ T cells differentially express HuR.

Figure 4:

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A,B) HuR MFI histograms and quantification in CD3+ (A) or CD3− (B) cells gated on live singlets. Histograms show data from one representative sample per group, (white = isotype control, grey= healthy, purple= MS). Healthy (n=6), MS (n=8), Welch’s unpaired T test, mean ± SEM. C-E) Percent of CD4+ T cells that are HuR+ at rest (C), or after 24hr (D), or 48hr (E) of polyclonal activation. Healthy (n=4), MS (n=4), Welch’s unpaired T test, mean ± SEM. Data representative of two independent experiments. * denotes p<0.05, ** p<0.005, *** p <0.0005.

3.4. Th1 and Th17 differentiated T cells increase autotaxin expression

We have previously shown that ATX mRNA is significantly increased in murine Th1 and Th17 cells in response to IL-23(P. W. Lee et al., 2017). To assess conservation across species and determine if IL-23 may be contributing to overexpression in MS patients, healthy controls and MS patients’ CD4 T cells were differentiated into Th1 and Th17 effector phenotypes in the presence of IL-23 before analyzing ATX expression. On the transcript level, healthy controls showed no difference in ATX expression with differentiation, but ATX remained significantly upregulated in MS patient Th1 and Th17 cells after 72hr of activation (Fig. 5a). The same result was also observed in the levels of secreted ATX (Fig. 5b), suggesting that encephalitogenic cytokine signaling enhances ATX expression in MS CD4 T cells. Since IL-23 was the common factor between the two effector phenotypes, we hypothesized that downstream signaling may be playing a role in the perpetuation of ATX signaling in these cells.

Figure 5: ATX expression is increased in MS Th1 and Th17 cells differentiated with IL-23.

Figure 5:

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A) Relative quantification of Enpp2 (ATX) mRNA shown as 2−ΔΔCT. Healthy (n=11) or MS (n=9) T cells were activated with anti-CD3/anti-CD28 for three days in the presence of IL-12 (0.5ng/mL) + IL-23 (5ng/mL) or IL-6 (25ng/mL) + IL-23 (5ng/mL). CD4 T cells were then isolated and ATX mRNA was quantified using reverse transcription real-time qPCR. Relative quantification is shown as delta CT values normalized to the average of the activated condition for each group. Two-way ANOVA with Sidak’s multiple comparisons test, mean ± SEM. B) Secreted ATX determined by ELISA after activation and differentiation as described in A. Healthy (n=6), MS (n=7). Two-way ANOVA with Sidak’s multiple comparisons test, mean ± SEM. Data representative of two independent experiments. * denotes p<0.05, ** p<0.005.

3.5. pSTAT3 and pSTAT4 bind to autotaxin promoter regions downstream of IL-23 signaling

We have have previously shown that IL-23 signals through pSTAT3 and pSTAT4 to promote encephalitogenicity(P. W. Lee et al., 2017). The IL-23 receptor is also increased by both IL-6 and IL-12 signaling(Ghoreschi et al., 2010; P. W. Lee et al., 2017). Therefore, we hypothesized that ATX may be transcriptional regulated by IL-23 receptor signaling via pSTAT3 and/or pSTAT4 in Th1 and Th17 cells. Chromatin immunoprecipitation (ChIP) assays were used to determine if pSTAT3 and pSTAT4 bind to the ATX promoter region. ChIP analysis was run on human Th1 differentiated cells after stimulation with IL-23. Two possible ATX-binding promoter regions were identified as potential binding sites for pSTAT3 and pSTAT4 (using gtrd.biouml.org) (Suppl. Table 1) and PCR was used to determine amplification of these regions after ChIP. pSTAT3 and pSTAT4 both bound to the two promotor regions analyzed while the IgG and irrelevant transcription factor antibody controls were negative (Fig. 6a), indicating that IL-23 resulted in pSTAT3 and pSTAT4 binding to the same general areas within the ATX promoter. ChIP re-ChIP was utilized to investigate the potential presence of pSTAT3/pSTAT4 heterodimers which are uniquely observed downstream of IL-23 receptor signaling. Following pSTAT3 ChIP, the pSTAT3-bound DNA was immunoprecipitated with pSTAT4 antibody or control IgG. The same promotor sequences were bound in the re-ChIP with pSTAT4, but not control IgG (Fig. 6b), suggesting the formation of pSTAT3/pSTAT4 heterodimers, downstream of IL-23 signaling, may modulate ATX expression. This data would indicate that enhanced expression of IL-23R by CD4 T cells in MS patients would result in more pSTAT3 and pSTAT4 transcriptional regulation of ATX and may be responsible for the dysregulated expression of ATX in MS patients. 4,33

Figure 6: pSTAT3 and pSTAT4 bind the Enpp2 promoter.

Figure 6:

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Human CD4 T cells were differentiated into Th1 cells with anti-CD3/CD28+IL12 for 72hr and stimulated with IL-23 for 2 hr. A) ChIP and B) ChIP re-ChIP assays were done with IgG, Bcl6 antibody, pSTAT3 antibody, and pSTAT4 antibody. PCR amplified 2 segments in the Enpp2 (ATX) promoter. Data representative of three independent experiments.

4. DISCUSSION

Several reports have shown that ATX is dysregulated in the context of MS, but the source of this increased ATX had not been elucidated. In this study, we identify activated PBMCs, specifically CD4+ T cells, as a potential cause of differential serum ATX levels in MS patients. Activation alone is sufficient to increase intracellular ATX levels in both healthy and MS patients. However, while resting T cells from MS patients have decreased levels of ATX, they more quickly upregulate ATX expression after 24hr of activation, cumulating in higher secreted levels compared to healthy controls. Furthermore, the proinflammatory cytokine IL-23 sustains elevated ATX expression in activated Th1/Th17 cells from MS patients, likely through the formation of pSTAT3 and pSTAT4 heterodimers that bind to the ATX promoter.

LPA, the main product of ATX signaling, increases BBB permeability in several disease models(Masago et al., 2018; On et al., 2013). LPA catalysis is also positively correlated with ATX levels(Watanabe et al., 2007), so it would be logical to assume that increased serum ATX levels would have a negative effect on CNS permeability. ATX also induces T cell chemokinesis at sites of T cell transmigration into the lymphatic system(Nakasaki et al., 2008). In a recent study by our lab, we found that inhibiting the ATX generated in the context of T cell activation was enough to significantly decrease T cell motility in vitro, and in vivo treatment with an ATX inhibitor decreased neurological deficits and limited the number of CNS infiltrating T cells in EAE studies(Petersen-Cherubini et al., 2023). Thus, it is possible that inhibiting ATX in MS would be a valuable therapeutic option. Several current therapies for MS indirectly affect ATX signaling, further supporting this hypothesis. Fingolimod competitively inhibits ATX activity(van Meeteren et al., 2008), and similarly, natalizumab binds to the same surface integrins as ATX, which may prevent ATX from binding to cells to modulate local LPA production(Fulkerson et al., 2011; Kanda et al., 2008). Further study needs to be conducted to determine the extent to which these drugs regulate aberrated serum ATX and how this may be affecting BBB in MS patients.

RRMS patients have significantly elevated ATX in their serum compared to healthy controls (Schmitz et al 2017; Zahednasab et al 2014). In addition, ATX is elevated during the initial documented MS attack (Schmitz et al 2017), indicating that ATX levels are associated with active inflammation. Our observation that in vitro activation of T cells increases ATX rapidly with peak expression at 48hr is consistent with T cells potentially being a major source of elevated ATX in RRMS patients and consistent with the observation that activated T cells are more prevalent during active stages of disease (Kaskow & Baecher-Allen, 2018). Interestingly, in our study, polyclonal activation of T cells alone is enough to increase ATX expression in CD3− cells, showing that antigen presenting cells may increase ATX expression in response to T cell activation. In breast cancer cells, ATX transcript is associated with increased motility and invasiveness(S. Y. Yang et al., 2002), so it is possible that ATX produced by multiple cell types after T cell activation may be contributing to not only augmented serum levels, but pathogenic immune cell motility during MS. More detailed investigation in vivo is required to support this hypothesis.

HuR expression is decreased in PBMCs of MS patients and is correlated with disease severity(Pistono et al., 2020). However, until now it was unclear which cell populations were responsible for dysregulated HuR signaling, as previously only total PBMC populations were analyzed. Here, we show that HuR is significantly decreased in several types of MS patient immune cells, but it is increased in naïve CD4 T cells, the population we have found to most significantly increase ATX expression after activation. Since HuR positively regulates ATX expression(Sun et al., 2016), it is conceivable that ATX may be increased faster in activated naïve CD4 T cells due to higher resting levels of HuR, thus elucidating a possible mechanism of ATX dysregulation. It is our theory that aberrant ATX in naïve CD4 T cells after activation could then lead to increased T cell motility and potentially support infiltration into the CNS, though this is a topic for future study.

Aberrations in STAT3 and STAT4 signaling have both been linked to increased MS pathology(Frisullo et al., 2006; Lu et al., n.d.; Nageeb et al., 2018). These are the major transcription factors associated with the differentiation of Th1 and Th17 cells, both of which are cell types increased in the circulation of MS patients(Segal, 2014). In previous work, we found that IL-23 can signal through pSTAT3 and pSTAT4 in Th1 and Th17 cells(P. W. Lee et al., 2017). Here, we expand on that finding to show that IL-23 potentially signals through pSTAT3/pSTAT4 heterodimers that bind to the ATX promoter to modulate expression. Paired with the previous observation, from our group and others, that the IL-23 receptor is upregulated by MS patient T cells(Khalifa et al., 2023; P. W. Lee et al., 2017), our finding that ATX only remains significantly upregulated by Th1/Th17 cells of MS patients illuminates another mechanism through which IL-23 may be promoting encephalitogenicity in MS.

The present study finds dysregulated ATX expression occurs in MS patient T cells, both at rest and shortly after activation, and increased levels can be prolonged in Th1 and Th17 cells through IL-23 signaling. These findings support the notion that activated immune cells may be a source of increased ATX levels seen during active stages of disease in MS patients, and thus may make ATX a potential therapeutic target.

Supplementary Material

1

Highlights.

  • Autotaxin is associated with activated encephalitogenic CD4 T cells, and MS patients increase autotaxin faster after activation compared to healthy controls.

  • Dysregulation in HuR levels coincide with patterns in autotaxin expression, suggesting this protein may play a role in regulating autotaxin in human CD4 T cells.

  • IL-23 signaling increases autotaxin expression in Th1 and Th17 cells of MS patients.

  • pSTAT3/4 heterodimers may bind to the autotaxin promoter to increase expression after IL-23 stimulation in Th1 and Th17 cells.

Acknowledgment:

This work was funded by a grant from the National institute of Allergy and Infectious Disease, RO1AI140741.

Abbreviations:

ATX

autotaxin

EAE

experimental autoimmune encephalomyelitis

BBB

blood brain barrier

CNS

central nervous system

CSF

cerebrospinal fluid

HEVs

high endothelial venules

IL

interleukin

LPARs

LPA receptors

LPA

lysophosphatidic acid

MS

multiple sclerosis

Footnotes

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