SRI-42127, a novel small molecule inhibitor of the RNA Regulator HuR, potently attenuates glial activation in a model of lipopolysaccharide-induced neuroinflammation

Abstract

Glial activation with the production of pro-inflammatory mediators is a major driver of disease progression in neurological processes ranging from acute traumatic injury to chronic neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer’s disease. Posttranscriptional regulation is a major gateway for glial activation as many mRNAs encoding pro-inflammatory mediators contain adenine- and uridine-rich elements (ARE) in the 3’ untranslated region which govern their expression. We have previously shown that HuR, an RNA regulator that binds to AREs, plays a major positive role in regulating inflammatory cytokine production in glia. HuR is predominantly nuclear in localization but translocates to the cytoplasm to exert a positive regulatory effect on RNA stability and translational efficiency. Homodimerization of HuR is necessary for translocation and we have developed a small molecule inhibitor, SRI-42127, that blocks this process. Here we show that SRI-42127 suppressed HuR translocation in LPS-activated glia in vitro and in vivo and significantly attenuated the production of pro-inflammatory mediators including IL1β, IL-6, TNF-α, iNOS, CXCL1 and CCL2. Cytokines typically associated with anti-inflammatory effects including TGF-β1, IL-10, YM1 and Arg1 were either unaffected or minimally affected. SRI-42127 suppressed microglial activation in vivo and attenuated the recruitment/chemotaxis of neutrophils and monocytes. RNA kinetic studies and luciferase studies indicated that SRI-42127 has inhibitory effects both on mRNA stability and gene promoter activation. In summary, our findings underscore HuR’s critical role in promoting glial activation and the potential for SRI-42127 and other HuR inhibitors for treating neurological diseases driven by this activation.

1. INTRODUCTION

Activated microglia and astroglia are the “first responders” to trauma, infection, or stroke in the central nervous system (CNS) and they drive acute inflammatory responses through production of cytokines, chemokines and other inflammatory mediators (Hamby & Sofroniew, 2010; Ransohoff, Schafer, Vincent, Blachère, & Bar-Or, 2015). In the early phases, activated glia can promote secondary tissue injury through release of inflammatory mediators such as TNF-α, IL-6, and IL-1β or nitric oxide (NO), the product of iNOS. Secretion of chemokines further amplifies the inflammatory response by drawing in peripheral immune cells such as neutrophils or monocytes (Garry, Ezra, Rowland, Westbrook, & Pattinson, 2015; Gaudet & Fonken, 2018; Murphy, 2000; Murray, Parry-Jones, & Allan, 2015; Takano, Oberheim, Cotrina, & Nedergaard, 2009). In more chronic neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson’s or Alzheimer’s, these same mediators can accelerate disease progression (Kwon & Koh, 2020). A critical control point for production of inflammatory mediators in glia is at the posttranscriptional level involving adenine- and uridine-rich elements (ARE) in the 3’ untranslated region (UTR) of the mRNA (Anderson, 2008, 2010; Kwan, Floyd, Kim, & King, 2017; Li et al., 2012; Matsye et al., 2017; Wang, Leavenworth, et al., 2019). Through interactions with RNA-binding proteins (RBP), the ARE governs the stability and translational efficiency of mRNAs which directly impacts protein production. HuR is an RBP with three RNA recognition motifs (RRM) and binds AREs in the 3’ UTRs of cytokines, chemokines and other inflammatory mediators such as iNOS and COX-2 (Brennan & Steitz, 2001; Dixon et al., 2001; Srikantan & Gorospe, 2012). HuR is a major positive regulator of mRNAs with AREs, and we have previously reported that it plays a key role in promoting a pro-inflammatory gene signature in glia activated by glioma tumor conditioned media, lipopolysaccharide (LPS) or stretch-injury (Kwan, Floyd, Kim, et al., 2017; Matsye et al., 2017; Wang, Leavenworth, et al., 2019). This signature encompasses major components of the inflammatory cascade in many CNS diseases. Our work has further demonstrated that glial HuR can impact CNS disease including spinal cord injury (SCI), stroke, glioblastoma, multiple sclerosis, and amyotrophic lateral sclerosis (Ardelt et al., 2017; Kwan, Floyd, Kim, et al., 2017; Kwan, Floyd, Patel, Mohaimany-Aponte, & King, 2017; Matsye et al., 2017; Wang, Leavenworth, et al., 2019; Wheeler et al., 2012). On the one hand, genetic deletion of HuR in microglia attenuates glioblastoma growth through an alteration of the tumor immune microenvironment. On the other hand, transgenic overexpression of HuR in astroglia increases vasogenic edema in acute stroke and SCI, worsens outcome in the former and neuronal loss in the latter (Ardelt et al., 2017; Kwan, Floyd, Patel, et al., 2017; Wang, Leavenworth, et al., 2019). This background provides a strong rationale for targeting HuR therapeutically where secondary inflammatory responses accelerate tissue injury or disease progression.

HuR is predominately a nuclear protein, and key components for its ability to enhance mRNA stabilization and translational efficiency are RNA binding, cytoplasmic translocation, and association with polysomes (Brennan & Steitz, 2001; Filippova et al., 2021; Galban et al., 2003; Keene, 1999; Keene, 2001). In the first report identifying small molecule inhibitors of HuR, Meisner et al. discovered that HuR homodimerization was necessary for subcellular trafficking and RNA binding (Meisner et al., 2007). In their study, mathematical modeling predicted that the inhibitors they identified, including MS-444, interacted with RRMs 1 and 2 to block dimerization. Our group recently performed a high throughput screen using a split luciferase assay which can detect HuR dimerization in cultured cells (Filippova et al., 2021; Filippova et al., 2017). We identified a novel class of dimerization inhibitors with SRI-42127 being one of the prototypes. A computational docking analysis of SRI-42127 suggests that it contacts key amino acids in RRMs 1 and 2 of the HuR monomer to induce a conformational change that interferes with dimerization. In this report, we use liposaccharide (LPS) as a simple model of inducing neuroinflammation to test the efficacy of SRI-42127 in attenuating HuR shuttling in glial cells and blocking neuroinflammatory responses linked to the mediators it regulates.

2. Materials and METHODS

2.1. Cell Culture

Primary microglial cells (PMG) and astrocytes were isolated from 1–3-day-old mice pups as previously described (Giulian & Baker, 1986; Qin, Niyongere, Lee, Baker, & Benveniste, 2008). PMG were maintained in DMEM-F12 (Corning) with 20% FBS and reduced to 10% for experiments. BV2 cells were a gift from Dr. Tika Benveniste (University of Alabama at Birmingham) and were maintained in cultures as described previously (Petrova, Akama, & Van Eldik, 1999). Astrocytes were prepared as previously described (Li et al., 2012) and maintained in maintained in Oligo Media (High glucose 1x DMEM, 16mM HEPES buffer, 1X non-essential amino acids, 2 mM L-glutamine, 10% fetal bovine serum, 1 mM sodium pyruvate, 50 μg/ml gentamicin). For in vitro inhibitor studies, PMG, astrocytes, and BV2 cells were seeded at a density of 1 × 10⁶ cells per well in 6-well-plates, treated with LPS at 1 μg/ml (InvivoGen), and varying doses of a small-molecule inhibitor SRI-42127 or vehicle consisting of 4% dimethyl sulfoxide, (DMSO). After 24 h, conditioned media and cell lysates were collected for analysis. Viability assays were performed at 50% confluency of PMG (~1 × 10⁵ cells) in a 48-well-plate by adding the PrestoBlue Cell Viability Reagent (Thermofisher) and incubating at 37° C for 15 min. Four independent cell culture samples were analyzed for each drug dose. The plate was read in an Infinite M200 plate reader (Tecan) at 590nm. For hypoxia-related experiments, 5 × 10⁵ cells were seeded in 35 mm dishes and exposed to 1% O₂ for 18h with or without SRI-42127. Three independent cell culture samples were analyzed. Control cells were maintained under normoxic conditions.

2.2. Animal Studies

All animal procedures were approved in compliance with National Research Council Guide for the Care and Use of Laboratory Animals. C56/Bl6 mice (Jackson Laboratory) were used for in vivo studies. Microglial HuR^−/− mice were generated in our laboratory previously (Wang, Leavenworth, et al., 2019). Experiments were performed in mice between ages of 8 to 12 weeks with equivalent numbers of females and males. For in vivo studies, LPS was injected intraperitoneally (i.p.) at a dose of 2 mg/kg. SRI-42127 (30mg/ml in DMSO) was diluted 1:10 in a 20% solution of 2-hydroxypropyl-β- cyclodextrin (Sigma) and mice were given 15mg/kg i.p. or the equivalent volume of vehicle (2% 2-hydroxypropyl-β- cyclodextrin and 10% DMSO) at different time points depending on the analysis. For flow cytometry and assessment of microglial activation, mice were given three doses 6 h apart and then euthanized at 24 h. For qPCR analysis of cytokine induction, mice were given a dose of SRI-42127 within 10–15 minutes after LPS and then euthanized at 2 h post LPS. Brains were harvested, and 2–3 mm slices from anterior, middle, and posterior brain (AB, MB, and PB) were excised using Precision Brain Slicer (Braintree Scientific Inc) for analyses. For immunohistochemical analysis, some mice were perfused transcardially with ice-cold PBS and 4% PFA, and brains were dissected out and snap frozen in powdered dry ice. Using a sliding microtome, 40 micron sagittal free-floating sections were made and stored in PBS:Glycerol mix (50:50) at −20C. For qPCR analysis, total RNA was extracted from these tissues.

2.3. Flow Cytometry

For single cell flow cytometry, PMG (1 × 10⁶ cells) were treated with LPS and SRI-42127 or vehicle for 24 h and stained with Zombie NIR Viability Kit (Biolegend) for 20 min at 4° C. For intracellular staining, fixation was performed using Fixation/Permeabilization Solution (BD Biosciences) and permeabilized with Perm/Wash Buffer overnight at 4C. On the following day, cells were stained with fluorescent-conjugated antibodies for HuR (Biolegend) and nuclear Hoechst fluorescent dye for 30 min at 4C. 5 × 10³ cells per sample were captured and analyzed as single cells in an Imagestream Mk II (Luminex) using Extended Depth of Field (EDF). Data were analyzed using IDEAS software (Luminex) to quantify the number of cells with high cytoplasmic HuR (subtracting nucleus mask from whole cell mask) in cells co-stained with HuR and Hoechst. The experiment was repeated once on an independent biological sample. For flow analysis of brain tissues, single cell suspensions of AB, MB, and PB regions were prepared using gentleMACS dissociator (Miltenyi) in Hank’s Balanced Salt Solution with DNAse I from bovine pancreas (Sigma). RBC lysis solution (Miltenyi) was applied to the samples and Percoll gradients of 90% and 70% were used to remove myelin debris on the surface. Cells from the interface were collected for staining (30 min at 4° C) with a fluorescent antibody cocktail mix consisting of Zombie NIR viability (Biolegend) at 1:500, CD45 e450 (eBioscience) at 1:500, CD11b BV605 (Biolegend) at 1:200, Ly6G APC (Biolegend) at 1:500 and Ly6C PE/Cy7 (Biolegend) at 1:400 to identify neutrophils and monocytes. After fixation with 2% PFA these cells were passed through Attune Flow Cytometer (Thermofisher) and analyzed with FlowJo software (FlowJo).

2.4. Immunohistochemistry, western blotting and ELISA

For immunocytochemistry, 1 × 10⁴ PMG or astrocytes were cultured in a Nunc Chamber Slide System (Thermofisher) and fixed with 2% PFA at 24 h after LPS stimulation and treatment with vehicle or SRI-42127 (0.5 μM). The following antibodies were used: Iba1 (Wako) at 1:1,000, GFAP (Dako) at 1:4,000, HuR (Sigma) at 1:2,000, or HMGB1 (Fabgennix) at 1:1,000 and DAPI at 1:30,000. Ten random 20x fields were captured by fluorescent imaging on a BX41 microscope (Olympus). Intracellular localization of HuR and HMGB1 was quantitated using Fiji, an open-source software (see Supporting Information Fig. S2). Outlines of the cell were traced based on Iba1 (PMG) or GFAP (astrocyte) immunofluorescence. Nuclei were traced based on DAPI staining. HuR fluorescence intensity (FI) was quantified in nucleus and the whole cell, subtracting background signal measured in the vicinity of the cell. Cytoplasmic FI was calculated by subtracting the nuclear FI from the whole cell FI. A nuclear/cytoplasmic ratio was then calculated by dividing the nuclear FI by the cytoplasmic FI (see Supporting Information Fig. S2). Twelve to fifteen cells were analyzed per condition. For immunohistochemistry, free floating sagittal sections were stained with anti-Iba1 (1:1,000; Abcam) and anti-HuR (1:2,000; Sigma) antibodies followed by DAPI staining. Methods for quantitating HuR FI and calculation of N/C ratio were the same as for immunocytochemistry described above. For microglial activation, Iba1 FI was quantified in the hippocampal region 24 h after LPS stimulation in three vehicle and three SRI-42127-treated mice. In total, five random 20x fields (~5–10 microglia per field) were analyzed per section from the hippocampal region. Images were captured by confocal imaging (z stacks consisting 10 steps of 1um) on a Ti2-C2 microscope (Nikon). Two sections were analyzed per brain from groups of three mice treated with SRI-42127 or vehicle.

For western blotting, whole cells lysates were prepared using M-PER kit (Thermofisher) and quantification was done with BCA protein assay kit (Thermofisher). Fifty ug of protein per sample was electrophoresed in 4–20% mini-PROTEAN gel (Biorad) and transferred to a nitrocellulose membrane. The following antibodies were used: HuR (Santa Cruz) at 1:1000, KSRP (KSRP antibodies were provided by Dr. C. Chen) at 1:100, iNOS (Sigma) at 1:500, Arg1 (Santa Cruz) at 1:1,000, YM1 (Abcam) at 1:1,000 and GAPDH (Cell Signaling) at 1:3,000. For nuclear and cytoplasmic extract preparation, cells were gently lysed using cytoplasmic extraction buffer (10mM HEPES, 60mM KCl, 1mM EDTA, 0.05% (v/v) Triton X-100, 1mM DTT, 1x Protease inhibitor cocktail and 1x Phosphatase inhibitor cocktail). Cytoplasmic lysates were collected using brief centrifugation (10,000 rpm for 2 min). Nuclei were washed twice gently with cytoplasmic extraction buffer without detergent. Washed nuclei were lysed using nuclear extraction buffer (20mM Tris-Cl, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 1x Protease inhibitor cocktail, 1x Phosphatase inhibitor cocktail and 25% (v/v) glycerol) and nuclear lysates were collected using centrifugation (10,000rpm for 10 min). For protein quantification by ELISA, conditioned media from microglia and astrocytes at 24 h post-LPS were assayed using the U-PLEX (Meso Scale Discovery) platform as per the manufacturer’s instructions. Each sample was analyzed in duplicate and the mean value was used for analysis.

2.5. RNA preparation, qPCR, RNA kinetics and luciferase assay

For brain tissues, AB, MB, and PB regions were harvested at 2 h post-LPS plus SRI-42127 or vehicle treatment and lysed with Trizol (Thermofisher). RNA was purified from lysates using the RNAspin Mini isolation kit (Cytiva). In cultured cells, the buffer included with the RNA isolation kit was used for lysis. cDNA was prepared using a high-capacity cDNA reverse transcription kit (Thermofisher). Quantitative PCR was performed as previously described using commercially available primers with a ViiA7 Real-Time PCR system (Thermo Fisher) (Matsye et al., 2017). Each sample was analyzed in duplicate. For RNA kinetics, BV2 microglial cells were stimulated with LPS (1 μg/ml) and treated with vehicle or SRI-42127 (1.0 μM). At 20 h post treatment, cells were treated with actinomycin D (5 μg/ml) for up to 4 hours. RNA half-lives were estimated using methods described elsewhere (Matsye et al., 2017). For luciferase experiments, BV2 cells were co-transfected with different luciferase reporter constructs along with a β-galactosidase control plasmid (King, 1996) using TurboFect Transfection Reagent (Thermo Fisher Scientific). The following promoter fragments were analyzed: TNF-α (provided by Dr. Economou and modified as previously described (Li et al., 2012; Rhoades, Golub, & Economou, 1992)), IL-1β (provided by Dr. Dong, University of Pittsburgh (Su et al., 2009)), TREM1 (provided by Dr. Nagaoka, Juntendo University, Tokyo, Japan (Hosoda, Tamura, Kida, & Nagaoka, 2011)), and Hel-N1 (King, 1996). After 24 h post transfection, cells were washed with PBS and treated LPS (1 μg/ml) and vehicle or SRI-42127 (1 μM). At 3 h post treatment, cells were washed with PBS and lysed using 1X reporter lysis buffer from a luciferase assay system kit (Promega). Luciferase activity was measured in a Synergy 2 multimode microplate reader (Bio-Tek) using the manufacturer’s protocol. B-galactosidase activity was measured as described previously (King, 1996), and data were normalized to this activity as a transfection control.

2.6. Migration Assay

Bone Marrow from long bones of wild-type mice was centrifuged to isolate cells as described elsewhere and treated with RBC Lysis Solution (Miltenyi) (Amend, Valkenburg, & Pienta, 2016). Cells were incubated in Zombie NIR Viability Kit (Biolegend) along with fluorescent-conjugated surface markers for neutrophils (CD45+ Ly6G+ Ly6C^lo) and monocytes (CD45+ Ly6C+ Ly6G−) for 20 min at 4° C. Sorting was done using a FACS Melody Cell Sorter (BD Biosciences). Transwell Migration assay was performed using 3 μm PET inserts (Corning Life Sciences), in which 1 × 10⁵ neutrophils or 5 × 10⁴ monocytes were added to the top chamber and conditioned media from LPS-stimulated PMG or astrocytes treated with vehicle or SRI-42127 (at different doses) to the bottom chamber. After 1.5 h, meshes from the inserts were stained using Hema Manual Staining and Stat Pack (Thermo Fisher) and mounted for manual counting from 20 random 10x fields per replicate with five replicates per condition.

2. 7. Statistics

Statistics were calculated using Graphpad v. 9.0 (Graphpad Prism, Inc). One way ANOVA was used for single cell flow cytometry, immunochemistry-based HuR localization, western blot analyses, and in vitro inhibitor experiments. Unpaired t tests were used for in vivo tissue flow cytometry, qPCR microglial activation and counting, and densitometry.

3. RESULTS

3.1. SRI-42127 blocks HuR cytoplasmic translocation in activated microglial cells

Cultured primary microglial cells (PMG) were treated with LPS at 1 μg/ml and varying doses of SRI-42127 and assessed by single cell flow cytometry to track the intracellular location of HuR (Fig. 1A). Hoechst dye was used as a marker for nuclear localization. For each cell, the intensity of HuR immunofluorescence that localized to the cytoplasmic compartment was assessed (representative gating shown in Supporting Information Fig. S1). In unstimulated cells (DMSO), the characteristic nuclear predominance of HuR was observed as reflected by a low percentage of cells with high HuR intensity in the cytoplasm. With LPS stimulation, there was a ~6-fold increase in percentage of cells with high cytoplasmic HuR consistent with translocation (~60%). SRI-42127 treatment attenuated this shift in a dose-dependent pattern, and at 1 μM the percent of cells with high cytoplasmic HuR was similar to unactivated PMG (~10%). This dose range did not cause toxicity to PMG as determined by a cell viability assay (Fig. 1B). To determine the specificity of SRI-42127, we assessed its effect on cytoplasmic translocation of HMGB1, which like HuR, is nuclear predominant and translocates with microglial activation (Paudel, Angelopoulou, Piperi, Othman, & Shaikh, 2020). PMG were treated with LPS and 0.5 μM of SRI-42127 for 24 h. Cells were fixed and co-immunostained with either HuR or HMGB1 antibodies in combination with Iba1 antibodies and DAPI staining to calculate a HuR nuclear/cytoplasmic (N/C) ratio (see Materials and Methods and Supporting Information Fig. S2 for details). In unactivated PMG, both HuR and HMGB1 were nuclear predominant with little to no merged signal with cytoplasmic Iba1 (Fig. 1C). The calculated N/C ratio was between ~ 1.4 for HuR and 2.1 for HMGB1 (Fig. 1D). With LPS stimulation, both HuR and HMGB1 translocated to the cytoplasm with the N/C ratio lowering to ~0.5. For HuR, treatment with SRI-42127 reversed the N/C ratio back to the unstimulated state, but had no effect on HMGB1. As an alternative approach to assessing the impact of SRI-42127 on HuR localization, we performed western blotting on nuclear and cellular extracts from PMG and BV2 microglial cells treated with LPS and SRI-42127 (0.5 μM for PMG and 1.0 μM for BV2) or vehicle for 24 h (Supporting Information Fig. S3). With LPS stimulation there was nearly a 3-fold increase in cytoplasmic HuR (N/C ratio decreased from 4.5 to 1.6). This was reversed by SRI-42127 (N/C ratio of 2.6). For BV2 cells the N/C ratio was similarly reversed with SRI-42127 treatment (from 0.4 to 1.5) which was similar to unstimulated conditions (N/C ratio of 1.3). Since hypoxia/ischemia induces HuR activation and translocation (Ardelt et al., 2017; Levy, Chung, Furneaux, & Levy, 1998; Schultz, Preet, Dhir, Dixon, & Brody, 2020), we assessed the effect of SRI-42127 in preventing cytoplasmic accumulation of HuR in PMG under hypoxic (1% O₂) conditions (Supporting Information Fig. S4). We observed a 50% increase in cytoplasmic HuR under hypoxic conditions compared to normoxia which was blunted by SRI-42127 by ~70% with SRI-42127 treatment. Taken together, these findings indicate that SRI-42127 attenuated cytoplasmic translocation of HuR in activated PMG with LPS and hypoxia, and the effect was specific for HuR.

Figure 1: SRI-42127 blocks HuR cytoplasmic translocation in primary microglia.

(A) primary microglia (PMG) were stimulated with LPS, treated with varying doses of SRI-42127 or vehicle (V) for 24 h, and then assessed for intracellular HuR localization by single cell flow cytometry using Hoechst dye as a marker of nuclear localization. Representative images of single cells are shown with the nuclei outlined. LPS activates PMG and induces HuR translocation (pseudocolored yellow) which is reversed with SRI-42127 treatment. The percent of cells with high cytosolic HuR was determined at each dose (gating strategy shown in Supporting Information Fig. S1). Each dosing group is the mean ± SD of two independent samples with 5,000 cells individually analyzed per sample. (B) Prestoblue viability assay shows that SRI-42127 is not toxic to PMG in the dose range shown. (C) SRI-42127-does not block translocation of HMGB1 in PMG. Cells were stimulated with LPS (1 μg/ml) and treated with 0.5 μM of SRI-42127 for 24 h. Both HMGB1 and HuR are predominantly nuclear in the resting state (LPS −), but translocate to the cytoplasm with LPS activation (LPS + vehicle) as indicated by a merged (yellow) signal with Iba1 immunofluorescence. Treatment with SRI-42127 blocks HuR, but not HMGB1, cytoplasmic translocation as indicated by the loss of merged yellow signal. (D) HuR and HMGB1 nuclear and cytoplasmic localization was quantified using Fiji software in 12–15 microglial cells for each condition (see Methods and Supporting Information Fig. S2) and a nuclear/cytoplasmic ratio was calculated. SRI-42127 completely reversed the ratio back to pre-LPS conditions for HuR but did not affect HMGB1. P values: *< 0.05, ** < 0.01, ***< 0.001, ****< 0.0001. Scale bar, 10 μm.

3.2. SRI-42127 suppresses production of pro-inflammatory mediators in activated glia

PMG were treated with LPS and varying doses of SRI-42127 or vehicle for 24 h, and mRNAs of pro- and anti-inflammatory factors were quantified by qPCR (Fig. 2). Consistent with microglial activation, there were large mRNA inductions of inflammatory cytokines, chemokines and other mediators including IL-1β, IL-6, TNF-α, iNOS, IL-10, CXCL1, CXCL2, CXCL10, CCL2, CCL3, and CCL4. These inductions were as high as ~2300-fold for iNOS and ~3500-fold for IL-6. Treatment with SRI-42127 led to a significant and dose-dependent suppression of these inflammatory mediators with the largest effects on iNOS (8-fold), IL-6 (6.8-fold), and IL-1β (5.9-fold) at 1 μM of inhibitor. Major chemokines CXCL1, CXCL2 and CCL3 were suppressed by more than 2-fold. Other mRNAs had modest although significant declines at 1.0 μM (TNF-α, CXCL10, CCL2, and CCL4). NOX2 and TLR4 were suppressed with LPS activation, and SRI-42127 treatment partially reversed this at higher doses (0.5 and 1.0 μM). There was a mixed effect on anti-inflammatory markers, with TGF-β1 and YM1 modestly increasing and IL-10 decreasing by ~2.5-fold at 1 μM. Since many of these factors are secreted, we quantified them by ELISA in the culture medium (Fig. 3A). Most were minimally or not detected in unstimulated (control) cells but were markedly induced upon LPS stimulation. IL-6 and TNF-α increased by ~25,000- and 160-fold respectively and IL-1β went from being undetected to ~350 pg/ml. Chemokines were likewise induced by as much as 220-fold for CCL3. Treatment with SRI-42127 attenuated most inflammatory factors in a dose-dependent pattern, with the greatest effect on IL-6 which was suppressed even at a lower dose of SRI-42127 (1.7-fold at 0.1 μM and nearly 10-fold at 1.0 μM). IL-1β and TNF-α were suppressed by 5-fold at 1.0 μM. Interestingly, mRNA levels for TNF-α were only modestly suppressed (1.6-fold) indicating a relative dissociation between effects on mRNA and protein levels. Chemokines were suppressed by 2 to 6-fold with CCL3 showing the greatest effect. Anti-inflammatory factors were mixed with IL-10 being suppressed by 3-fold at 1.0 μM of inhibitor and TGF-β1 not being affected at all. Western blot analysis of microglial lysates showed a 16-fold induction of iNOS with LPS stimulation which was suppressed in a dose-dependent manner up to ~4.5-fold at 1.0 μM of SRI-42127 (Fig. 3B, 3C and Supporting Information Fig. S5). HuR and another RBP, KH-type splicing regulatory protein (KSRP), were induced by 1 to 2-fold by LPS and suppressed by nearly 50% (back to pre-LPS levels) at both 0.5 and 1.0 μM doses. Arginase (Arg)1, as with TGFβ1 in the ELISA experiment, was not affected, indicating that the effect of SRI-42127 was target-specific.

Figure 2: SRI-42127 suppresses inflammatory cytokine and chemokine mRNA induction in activated PMG.

Cultured PMG were stimulated with LPS and treated with varying doses of SRI-42127 for 24 h and assessed for cytokine and chemokine mRNA expression by qPCR. Each data point is the mean ± SD of 3 independent culture samples. P values: *< 0.05, ** < 0.01, ***< 0.001, ****< 0.0001.

Figure 3: SRI-42127 suppresses inflammatory cytokine and chemokine proteins in activated PMG.

PMG were stimulated with LPS (1 μg/ml) and treated with varying doses of SRI-42127 for 24 h. (A) Secreted cytokines and chemokines in the conditioned medium were quantitated by ELISA. Each data point is the mean ± SD of three 3 individual culture samples. P values: *< 0.05, ** < 0.01, ***< 0.001, ****< 0.0001. (B) Western blot of PMG lysates (from two independent cell culture samples) probed with antibodies to targets shown on the left of the blot. Protein levels were quantitated by densitometry using GAPDH as a loading control. All values were expressed relative to the average of LPS-stimulated cells which was set at 1.0. (C) Densitometry summary of western blot experiments which includes data points from a third independent experiment shown in Supporting Information Fig. S5. Values represent the mean ± SEM *P < 0.05.

Because HuR plays a key role in promoting cytokine induction in activated astroglia (Kwan, Floyd, Kim, et al., 2017), we assessed the effect of SRI-42127 on LPS-treated astrocytes in vitro. As with PMG, LPS induced cytoplasmic translocation of HuR which was reversed with SRI-42127 (Fig. 4A and Supporting Information Fig. S2). Western blot of extracts from nuclear and cytoplasmic compartments showed an increase in the N/C ratio with SRI-42127 (26.8 versus 13.2 for vehicle) that actually exceeded the ratio of non-LPS conditions (Supporting Information Fig. S6A). SRI-42127 also suppressed cytoplasmic HuR under hypoxic conditions by ~50%, although the degree of HuR translocation was less prominent than with PMG (Supporting Information Fig. S6B). We next looked at the effect of SRI-42127 on cytokine mRNA expression with LPS stimulation of astrocytes. There was a marked induction of IL1β, IL-6, IL-10, CXCL1, CCL2 and CCL3 (Supporting Information Fig. S7). CXCL1 was induced by more than 30,000-fold. SRI-42127 suppressed these inductions in a dose dependent pattern by as much as 3-fold (IL-6) at 1 μM. TNF-α and TGF-β1 mRNAs were only minimally suppressed, and at the highest dose TNF-α actually increased over vehicle control. IL-10 mRNA was induced at higher doses of SRI-42127 (3-fold at 1 μM). Analysis of culture media by ELISA indicated that LPS stimulation increased the production of key cytokine and chemokines, including IL-1β, IL-6, IL-10, TNF-α, CXCL1, CCL2 and CCL3, by as much as a 1100-fold (CXCL1) (Fig. 4B). IL-1β and IL-10 were undetected in unstimulated cells and increased to low but detectable levels. SRI-42127 treatment suppressed all inflammatory cytokines and chemokines by as much as 8-fold (IL-6). IL-10 was induced at lower doses of drug and only minimally suppressed at 1 μM. TGF-β1 levels did not change. There were several notable differences between PMG and astrocyte responses to LPS stimulation (based on the testing of similar numbers of cells). IL-1β, IL-6, TNF-α and IL-10 protein levels were higher in PMG versus astrocytes (590-, 5-, 13- and 147-fold respectively). CXCL1, on the other hand, was 5-fold higher with astrocyte stimulation. CCL2 and TGF-β1 were similar between the two cell types. Taken together, SRI-42127 suppressed microglial and astroglial production of inflammatory cytokines but had little effect on anti-inflammatory factors.

Figure 4: SRI-42127 suppresses HuR translocation in astrocytes and the production of inflammatory cytokines and chemokines.

(A) Primary astrocytes were stimulated with vehicle, LPS (1 μg/ml) + vehicle, or LPS + SRI-42127, and then fixed and immunostained with HuR and GFAP antibodies. HuR translocation was quantified based on a merged signal with GFAP (yellow) using methods described for PMG (Supporting Information Fig. S2). A HuR N/C ratio was calculated the mean ± SEM ratio for 12 astrocytes per condition is shown. (B) Cytokines and chemokines were measured by ELISA in conditioned media from cultured astrocytes. Vehicle alone was added to all samples that were not treated with SRI-42127. Each data point is the mean ± SD of three independent culture samples. Scale bar, 10 μm. P values: *< 0.05, ** < 0.01, ***< 0.001, ****< 0.0001.

3.3. SRI-42127 blocks neuroinflammatory responses in the brain

As our prior work indicates that SRI-42127 has good penetration into CNS tissues with concentrations reaching approximately 1.3 μM at 0.5 h after i.p. injection (Filippova et al., 2021), we next assessed the in vivo effect of SRI-42127 on inflammatory cytokine production in the brain. Systemic injection of LPS produces an acute neuroinflammatory response in the CNS including activation of microglia with production of inflammatory cytokines and chemokines (Cazareth, Guyon, Heurteaux, Chabry, & Petit-Paitel, 2014; Dutta, Zhang, & Liu, 2008). We used this model to test the effect of SRI-42127 on neuroinflammatory responses in the CNS. C57BL/6J mice were injected with LPS and one dose of vehicle or SRI-42127 i.p. At 2 h, AB, MB, and PB brain regions were excised for qPCR analysis of cytokine expression (Fig. 5A). Compared to non-LPS controls, there was a marked induction (35 to 2500-fold) of pro-inflammatory cytokines and chemokines (IL1β, TNF-α, CXCL1 and CCL2) and the anti-inflammatory cytokine IL-10 in all three brain regions (Fig. 5B). Arg1 and iNOS showed minimal induction, with the exception of AB which showed a decrease for Arg1. TGF-β1 showed essentially no induction. With SRI-42127 treatment, there was significant attenuation of all inflammatory cytokine mRNAs by as much as 2 to 3-fold in some brain regions (e.g. IL-1β, TNF-α, and CCL2). In contrast, Arg1 was induced in AB and MB regions and IL-10 was induced in all three regions by up to 2-fold (MB). TGF-β1 was minimally induced in AB and MB. Taken together, these data indicate that peripherally administered SRI-42127 attenuates the induction of inflammatory mediators throughout the brain without a significant negative effect on anti-inflammatory factors. The data are consistent with the in vitro effect of SRI-42127 on LPS-stimulated glial cells.

Figure 5. SRI-42127 suppresses inflammatory cytokine mRNA induction in vivo.

(A) At 2 h post LPS injection and one dose of i.p. SRI-42127 (15 mg/kg), mouse brains were harvested and divided into three brain regions as shown: anterior (AB), middle (MB) and posterior (PB). Relevant anatomical loci are identified in these regions: Cb, cerebellum; Ctx, cortex; Hi, hippocampus; Hy, hypothalamus; Md, medulla; Mb, midbrain; Ob, olfactory bulb; Po, pons; Se, septum; Th, thalamus. (B) qPCR analysis of cytokine and chemokine mRNA expression in different brain regions. Values were expressed relative to unstimulated control mice (C) for that region which was set at 1. Data points represent the mean ± SEM of three mice per condition. P values: *< 0.05, ** < 0.01, ***< 0.001, comparing LPS v. LPS + SRI-42127, and ^# < 0.05 comparing DMSO to LPS groups when values were close.

3.4. SRI-42127 suppresses HuR translocation and the activation of microglia in vivo.

We next tested the effect of systemic SRI-42127 on HuR localization in microglia and the state of microglial activation. Mice were injected with LPS i.p. followed by SRI-42127 (or vehicle) i.p. after 30 min for a total of three doses in 24 h. We first assessed the impact of SRI-42127 on HuR localization. We focused on the hippocampus as it is a discrete and readily identifiable structure that allows for a consistent analysis and comparison between cohorts. Tissue sections were immunostained with HuR plus Iba1 or GFAP antibodies and intracellular localization of HuR was quantified. In vehicle-treated animals, there was translocation of HuR into the cytoplasm in microglia as indicated by a merged yellow signal whereas in SRI-42127 treated mice, there was little to no merged signal (Fig. 6A). Quantitative assessment of Iba1+ cells showed a ~2-fold increase in the N/C ratio in mice treated with SRI-42127 compared to vehicle consistent with inhibitor-induced nuclear retention of HuR. For astrocytes, we found no definite evidence of HuR translocation (Supporting Information Fig. S8). We next assessed the activation state of microglia by measuring Iba1 intensity. Sections from the hippocampal region were immunostained with Iba1 antibodies and fluorescence intensity was measured. We observed a significant attenuation of Iba1 intensity in SRI-42127 treated mice (Fig. 6B). In the same sections, the number of microglia were quantified and showed no differences between vehicle and SRI-42127-treated mice (Fig. 6C). In summary, SRI-42127 exerted nuclear retention effects on HuR and suppressed microglial activation.

Figure 6. SRI-42127 blocks HuR translocation in microglia in vivo and suppresses microglial activation.

Mice were injected with LPS i.p. followed by SRI-42127 (15 mg/kg) or vehicle for a total of 3 doses over a 24 h period of time. At 24 h, HuR nuclear and cytoplasmic localization was quantified in microglia in the hippocampal region with Fiji software using the strategy outlined in Methods and Supporting Information Fig. S2. (A) Representative photomicrographs of dual immunofluorescent staining with HuR and Iba1 antibodies. A nuclear/cytoplasmic ratio was calculated from hippocampal sections in three mice per group as described in Materials and Methods. (B) In the hippocampus, overall microglial activation was determined by measuring Iba1 relative fluorescence intensity (RFI). Representative micrographs are shown above the graph. (C) In the same sections, microglial counts were determined based on Iba1+ cells. ***, P < 0.001. Scale bars, 15 μm.

3.5. SRI-42127 suppresses glial-mediated chemoattraction of monocytes and neutrophils

Since we observed a striking suppression of chemokine secretion by SRI-42127 for activated PMG and astroglia, including CXCL1 and CCL2, we assessed the effect on chemoattraction of monocytes and neutrophils using a dual chamber transmigration assay. Monocytes and neutrophils were harvested from murine bone marrow and plated in the upper chamber with conditioned media from LPS-activated glia treated with SRI-42127 (at varying doses) or vehicle in the lower chamber. We observed a significant attenuation of monocyte and neutrophil migration for both PMG and astrocyte conditioned media which was dose-dependent (Fig. 7). Since systemic LPS induces neutrophil and monocyte chemotaxis into the CNS (Giles et al., 2018; Thomson, McColl, Graham, & Cavanagh, 2020), likely the result of chemokine induction, we reasoned that SRI-42127 would inhibit this infiltration. After LPS stimulation and treatment with SRI-42127, we assessed the three brain regions (AB, MB, and PB) for monocytes and neutrophils using flow cytometry (Fig. 8). CD11b+ cells were gated with Ly6c and Ly6g markers to quantify monocytes (Cd11b+/Ly6c^hi/ ly6g−) and neutrophils (Cd11b+/Ly6c^lo/Ly6g+). A representative gating for vehicle and SRI-42127-treated mice is shown in Fig. 8A. With drug treatment, there was significant suppression of neutrophils in all brain regions by more than 2-fold. Monocytes were also significantly reduced by 1.5 to 2-fold. To determine the contribution of microglial HuR, we performed the same experiment on mice in which HuR is selectively knocked out in microglia using CX3CR1-driven Cre recombinase (Wang, Leavenworth, et al., 2019). Compared to littermate controls, HuR^−/− mice showed reduced monocytes in all three brain regions, similar to SRI-42127, whereas neutrophils were not significantly changed. In summary, HuR inhibition with SRI-42127 led to the suppression of immune cells infiltrating the CNS, and deletion of microglial HuR could not fully recapitulate this effect. Taken together, these results indicate the HuR inhibition by SRI-42127 blocks the chemotaxis/migration of neutrophils and monocytes in response to neuroinflammatory signals.

Figure 7. SRI-42127 blocks glial-produced chemoattraction/migration signals for neutrophils and monocytes.

Cultured PMG and astrocytes were stimulated with LPS and treated with vehicle or SRI-42127 for 1.5 h. The conditioned media was then placed in the bottom chamber of a transwell culture plate and peripherally-isolated neutrophils or monocytes cells were plated in the upper chamber. Migrated cells were quantified by filter staining. Representative micrographs of migrated cells are shown above each graph. Data points are the mean ± SEM of 20 random 10x fields in five (microglia) or three (astrocytes) independent cell culture samples per condition. P values: *< 0.05, ** < 0.01. Scale bars: 25 μm for monocytes and 20 μm for neutrophils.

Figure 8. SRI-42127 suppresses neutrophil and monocyte infiltration into brain tissue.

Wild-type mice were administered LPS (1 mg/kg) and given 3 equally spaced doses of SRI-42127 (15 mg/kg) over a 24 h time period. Single cell suspensions from the three regions (see schematic in Fig. 5) and the whole brain (WB) were assessed by flow cytometry to quantify monocytes and neutrophils. (A) gating strategy for monocytes (Ly6c⁺, Ly6g⁻) and neutrophils (Ly6c low, Ly6g⁺) as a percent of total CD11b⁺ cells. (B) Comparison between vehicle- and SRI-42127-treated mice. (C) Comparison between mice in which HuR is deleted from microglia (KO) and wild-type (WT) littermate controls. Data points represent the mean ± SEM of 6 mice per group. ** P < 0.01.

3.6. SRI-42127 exerts mRNA target-dependent effects on RNA stabilization and promoter activity of key pro-inflammatory targets

Since one of the functions of HuR is to stabilize mRNA targets, we assessed the impact of SRI-42127 on the RNA kinetics of key inflammatory mediators in microglial-like BV2 cells. Although these cells are transformed, our prior work using HuR siRNA showed similar patterns of cytokine suppression to PMG (Matsye et al., 2017). Here, we observed similar dose-dependent suppression of IL-1β, TNF-α, iNOS, and IL-6 mRNA induction with SRI-42127 treatment (Fig. 9). Similar to PMG, TGF-β1 mRNA was actually induced with higher doses. TNF-α, on the other hand, was suppressed by more than 60-fold at 1 μM (versus 1.6-fold in PMG). To assess RNA kinetics, cells were treated with either vehicle alone, LPS plus vehicle, or LPS plus SRI-42127 at 1.0 μM for 24 h followed by an actinomycin D pulse for different time intervals. RNA levels were assessed by qPCR to estimate the mRNA half-life (T_1/2). LPS stimulation induced mRNA stabilization, as estimated by an increase in T_1/2, for IL-1β (~ 1.8 h to > 4.0 h) and iNOS (~ 2.3 h to > 4.0 h). With those targets, concomitant treatment with SRI-42127 did not alter the T_1/2 up to 4 h, although degradation curves began to show some divergence at the 4 h time point. There was a destabilizing effect of SRI-42127 for IL-6 (T_1/2 of 2.2 h versus > 4.0 h for LPS plus vehicle). For TNF-α and TGF-β1, there was no reduction in half-life with SRI-42127 treatment. TNF-α showed the typical short T_1/2 of ~ 0.4 h for all conditions. These findings raised the possibility that the suppression of some target mRNAs could be related to a transcriptional effect as we have previously observed with HuR silencing or inhibition (Kwan, Floyd, Kim, et al., 2017; Matsye et al., 2017; Wang, Hjelmeland, Nabors, & King, 2019). To address the possibility of a transcriptional effect, we assessed promoter activity of IL-1β, TNF-α and three control promoters (SV40, the neuronal-specific promoter, Hel-N1 (King, 1996), and TREM-1 (Hosoda et al., 2011; Li et al., 2012) using luciferase reporters (Fig. 10A). With LPS treatment, there was a 2 to 3-fold increase in luciferase activity for TNF-α and IL-1β, but no induction with the control promoters (Fig. 10B). Treatment with SRI-42127 suppressed this activity back to unstimulated states for both promoters, but did not have any effect on the control promoters. Taken together, SRI-42127 had a mixed effect on transcriptional and posttranscriptional levels of gene regulation for different cytokine targets.

Figure 9. SRI-42127 significantly attenuates inflammatory cytokine mRNAs in BV2 microglial cells and destabilizes select cytokines.

BV2 microglial cells were used to assess the kinetics of cytokine mRNAs after treatment with SRI-42127. Cells were stimulated with LPS (1 mg/kg) and treated with varying doses of SRI-42127 for 24 h. Cytokine mRNA levels were measured by qPCR and showed a significant dose-dependent attenuation of pro-inflammatory cytokine mRNAs and an induction of the anti-inflammatory cytokine, TGF-β1 (shown in the left graph of each panel). For RNA kinetics, actinomycin D (ActD) was added at 24 h to LPS-stimulated cells treated with SRI-42127 (1.0 μM) or vehicle, and to unstimulated control cells treated with vehicle alone. RNA was collected at 0, 0.5, 1.0, 2.0 and 4.0 h following the ActD pulse. Cytokine mRNA levels were measured by qPCR and expressed as a % of RNA remaining compared to levels before ActD was added. Results are shown in the right graph of each panel. The estimated half-lives are shown in parentheses in the figure legends. P values: ** < 0.01, ***< 0.001, ****< 0.0001.

Figure 10. SRI-42127 suppresses promoter activity of select cytokine genes in BV2 microglial cells.

(A) Schematic diagram of luciferase reporter constructs with promoters for TNF-α, IL-1β, TREM1 and Hel-N1assessed in BV2 microglial cells. Positions relative to transcription initiation site are shown. (B) Luciferase activity was measured after cells were treated with vehicle, LPS + vehicle, or LPS and SRI-42127 (1.0 μM). Activity was adjusted to an internal transfection control and expressed as a fold-change over vehicle. control. The data points represent the mean ± SEM of three independent transfections. ***, P < 0.001, ****< 0.0001.

4. DISCUSSION

Our prior work identified ARE-directed posttranscriptional regulation as a major control point for glial production of key neuroinflammatory cytokines (e.g. IL-1β, TNF-α, and IL-6) and chemokines (CXCL1–3 and CCL2), (Kwan, Floyd, Kim, et al., 2017; Li et al., 2012; Matsye et al., 2017; Wang, Leavenworth, et al., 2019). In these reports, different models for glial stimulation were used including, LPS, traumatic stretch injury, and glioma conditioned media, supporting the role of HuR and posttranscriptional regulation as an essential downstream node for glial activation independent of the triggering event. In this report, we have used LPS-induced glial activation to show that SRI-42127 blocks HuR nucleocytoplasmic translocation in glial cells, attenuates production of key inflammatory cytokines and chemokines, dampens glial activation and the infiltration of neutrophils and monocytes into the CNS. This work builds on our prior reports underscoring the importance of posttranscriptional regulation in the molecular cascade of neuroinflammation and the opportunities for novel therapeutic intervention.

A major hallmark of HuR activation is its translocation to the cytoplasm, and we have previously shown this occurs in acute and chronic CNS diseases including SCI (astroglia), stroke (astroglia), and ALS (microglia) (Ardelt et al., 2018; Kwan, Floyd, Kim, et al., 2017; Kwan, Floyd, Patel, et al., 2017; Lu et al., 2007; Matsye et al., 2017). HuR is predominantly a positive regulator of mRNAs with AREs and its cytoplasmic translocation is necessary for mRNA transport, stabilization, and enhancement of mRNA translational efficiency (Barreau, Paillard, & Osborne, 2006; Gorospe, Tominaga, Wu, Fahling, & Ivan, 2011). SRI-42127 blocks HuR homodimerization which inhibits its cytoplasmic translocation and these downstream functions (Filippova et al., 2021; Meisner et al., 2007). The mechanism by which inhibition of dimerization leads to nuclear retention of HuR is not known, but it might disrupt the interaction with nuclear export proteins including transportins, pp32, APRIL or CRM1, that participate in its translocation (Gallouzi, Brennan, & Steitz, 2001; Rebane, Aab, & Steitz, 2004).

Loss of cytoplasmic HuR by inhibiting dimerization can push the equilibrium toward RNA degradation and dissociation from polysomes as negative ARE-RNA regulators such as KSRP or Tristetraprolin (TTP) gain access to the mRNA (Briata, Chen, Ramos, & Gherzi, 2013; Dhamija et al., 2011; Fu & Blackshear, 2017; Galban et al., 2008; Linker et al., 2005). The ~40% reduction in half-life of IL-6 mRNA after SRI-42127 treatment is consistent with this mechanism and could explain the decrease in IL-6 mRNA levels. The reduction in HuR expression with SRI-42127 treatment, which may be related to loss of autoregulation (Al-Ahmadi, Al-Ghamdi, Al-Haj, Al-Saif, & Khabar, 2009), would add to this imbalance. KSRP, the RNA destabilizer, however, is also positively regulated by HuR (Pullmann et al., 2007) and decreased with SRI-42127 treatment. Other mRNA targets like TNF-α and iNOS showed no definite change in RNA half-life, suggesting that their reduction may be related to a transcriptional effect. This was borne out by reporter assays where SRI-42127 selectively suppressed TNF-α and IL-β promoter activity, a finding similar to what we previously observed with HuR knockdown in BV2 microglial cells and astroglia (Kwan, Floyd, Kim, et al., 2017; Matsye et al., 2017). This effect may result indirectly from suppression of key transcription factor/regulators which are regulated posttranscriptionally by HuR or via changes in HuR-regulated lncRNAs or other non-coding RNAs that modulate transcription (Matsye et al., 2017; Shen et al., 2019; Yang et al., 2020; Zhou et al., 2018). It is also possible that increased nuclear HuR could affect splicing or directly interfere with transcriptional regulators or other post-transcriptional regulators.

Our data suggest that for some mRNA targets, HuR inhibition with SRI-42127 reduced translational efficiency, a process that is regulated by cytoplasmic HuR through its association with polysomes (Barreau et al., 2006; Gorospe et al., 2011). We suspect this possibility when suppression of protein levels is disproportionate to that of the mRNA. In PMG, for example, TNF-α and CCL2 had modest decreases in mRNA levels (~ 20% of control at 1.0 μM) in contrast to a 70–80% decrease in protein levels. In astrocytes, a similar dissociation was observed with TNF-α and CCL2. Polysome profiling, however, would be necessary to address this possibility.

Interestingly, TGF-β1 mRNA, which has AREs in the 3’ UTR and is a binding target of HuR (Nabors, Gillespie, Harkins, & King, 2001), showed no decrease in protein or mRNA expression with SRI-42127 treatment. This is consistent with our previous RNA sequencing analysis of LPS-stimulated PMG with HuR knockdown (Matsye et al., 2017), but in contrast to an attenuating effect when HuR^−/− PMG were treated with glioma conditioned medium (Wang, Leavenworth, et al., 2019). In other cells systems, such as myocytes and glioma cells, chemical inhibition of HuR by KH-3 and MS-444 respectively attenuated TGF-β1 expression (Green et al., 2019; Wang, Hjelmeland, et al., 2019). These variable responses might reflect differences in the mechanism of HuR inhibition, potential off-target effects, or a changing role of HuR depending on cell type and stimulatory conditions. In fact, HuR drives RNA destabilization and/or reduced protein expression of certain mRNA targets in myocytes, macrophages and fibroblasts (Cammas et al., 2014; Chang et al., 2010; Katsanou et al., 2005; Meng et al., 2005; Sanchez et al., 2019).

The overall effect of SRI-42127 treatment was similar between PMG and astrocytes, predominantly attenuating pro-inflammatory mediators associated with M1- and A1-like polarization (Acioglu, Li, & Elkabes, 2021; Lyu et al., 2021). The effect on markers of M2-like (anti-inflammatory) polarization was mixed with IL-10 decreasing in PMG but increasing in astrocytes at lower doses in vitro. IL-10 was induced in CNS tissue by SRI-42127 in LPS-stimulated mice. TGF-β1, YM1 or Arg1 levels (in PMG for the latter 2 targets) were not suppressed. This pattern suggests that SRI-42127 may have therapeutic application as pro-inflammatory mediators produced by activated microglia and astroglia, including IL-1β, IL-6,TNF-α, CXCL1, CCL2 and iNOS, drive tissue-damaging effects in many acute and chronic diseases (Brown & Vilalta, 2015). These include acute SCI or brain injury (David & Kroner, 2011; Orr & Gensel, 2018; Simon et al., 2017; Wofford, Loane, & Cullen, 2019); stroke (Garcia-Culebras et al., 2018), and neurodegenerative diseases such as ALS, Parkinson’s and Alzheimer’s (Acioglu et al., 2021; Sheeler et al., 2020; Song & Colonna, 2018). In our prior report of HuR and SCI, for example, we showed that stretch injury to astrocytes (mimicking the viscoelastic forces of CNS trauma) triggered HuR translocation to the cytoplasm and a significant induction of pro-inflammatory cytokines (Kwan, Floyd, Kim, et al., 2017). This induction was muted either with siRNA-mediated HuR silencing or inhibition with MS-444. In an ischemic stroke model, we showed that transgenic HuR in astrocytes worsens vasogenic edema and functional recovery (Ardelt et al., 2018). It is well recognized that activated glia in the earliest stages of stroke can exacerbate tissue damage, blood brain barrier permeability, edema, and infiltration of peripheral immune cells through release of inflammatory mediators regulated by HuR (Garcia-Culebras et al., 2018; Patabendige, Singh, Jenkins, Sen, & Chen, 2021). Our findings that SRI-42127 suppressed HuR translocation in glial cells under hypoxic conditions suggest that it might have therapeutic benefit in the acute phase of stroke.

Another important effect of HuR inhibition in the current study was the reduced recruitment of peripheral neutrophils and monocytes. Our in vitro migration assays suggest that this effect is related to an altered secretome in glial cells treated with SRI-42127 and is consistent with our previous observations of migration/invasion defects with HuR silencing, genetic deletion, or chemical inhibition (Kwan, Floyd, Kim, et al., 2017; Matsye et al., 2017; Wang, Leavenworth, et al., 2019). In the current study, microglial HuR^−/− mice did not show a reduction in CNS-infiltrating neutrophils versus SRI-42127 treatment, suggesting that astroglial HuR (or another cell type) may be responsible for neutrophil chemotaxis in this model. While many inflammatory cytokines were induced to a much greater degree in PMG with LPS stimulation, astrocytes produced 4-fold higher levels of CXCL1, the major chemokine for neutrophil recruitment (Kolaczkowska & Kubes, 2013). Although we did not observe definite LPS-induced translocation of HuR in astrocytes in vivo as we did in vitro, we only assessed these cells at 24 h following LPS administration so it is possible that translocation occurred earlier. We have previously shown marked HuR translocation in astrocytes following SCI, but this condition has sustained neuroinflammatory signaling (Kwan, Floyd, Kim, et al., 2017; Kwan, Floyd, Patel, et al., 2017). Because of the overlapping action of many chemokines suppressed by SRI-42127, such as CCL2, 3 and 4, it would be difficult to link a specific recruitment defect to any one of them (Griffith, Sokol, & Luster, 2014; Savarin-Vuaillat & Ransohoff, 2007). Other CNS cells such as neurons and oligodendrocytes, also express these factors and may be subject to the same inhibitory effects of the drug (de Haas, van Weering, de Jong, Boddeke, & Biber, 2007; Jiang, Liu, & Gao, 2020; Savarin-Vuaillat & Ransohoff, 2007). Lastly, it is possible that HuR-regulated chemokine receptors, such as CCR2, 3 and 5 (Matsye et al., 2017), may have been attenuated, thus contributing to decreased immune cell chemotaxis.

Therapeutic targeting of glia in CNS disease is a balancing act since these cells also exert neuroprotective and neuroplastic effects depending on the phase of recovery from CNS injury or stage of neurodegenerative disease. In the initial phases after SCI, TBI or stroke, the pro-inflammatory activation of glia worsens secondary tissue injury and triggers pathways of chronic neuropathic pain in contrast to the more chronic phases where glia become protective (Lyu et al., 2021; Ma, Wang, Wang, & Yang, 2017; Orr & Gensel, 2018; Simon et al., 2017). In neurodegenerative processes like ALS and Alzheimer’s, glia also play changing roles during the course of the disease (Acioglu et al., 2021; Kwon & Koh, 2020; Puentes, Malaspina, van Noort, & Amor, 2016). Several findings from our work suggest that SRI-42127 would be beneficial in acute CNS injury. First, through suppression of multiple inflammatory mediators, HuR modulates a diversity of pro-inflammatory pathways in glia. This would have a distinct advantage over targeting individual cytokines. For example, treatment blocking TNF-α would not necessarily prevent tissue damage or chronic pain induced by IL-6, IL-1β or other inflammatory mediators. Also, iNOS/NO toxicity and chemokine-induced infiltration of neutrophils and monocytes may not be affected. The failure of numerous therapeutic trials targeting specific inflammatory mediators in SCI underscores the merits of a multi-pronged therapeutic approach (David, López-Vales, & Wee Yong, 2012; Priestley, Michael-Titus, & Tetzlaff, 2012). Second, there was no overt cell toxicity for glial or neuronal cells (Fig. 1 and (Filippova et al., 2021)). Third, peak CNS drug concentrations achieved by systemic administration of SRI-42127 (Filippova et al., 2021) exceeded 1.0 μM which was the concentration that maximally suppressed glial production of pro-inflammatory cytokines in cultured glial cells. Importantly, cytokine mRNA induction in the brain was blocked by SRI-42127 and anti-inflammatory cytokine mRNAs like IL-10, TGF-β1, and Arg-1 were actually increased.

For more chronic neurodegenerative diseases, benefits may be offset by the potential impact of long-term HuR inhibition in the CNS and peripherally. Activation of myeloid cells (including microglia) and the production of inflammatory cytokines is a necessary defense against foreign pathogens. Chemokines such as CCL2, which were significantly suppressed by SRI-42127, also play a neurotrophic role and function as neuromodulators of synaptic transmission (Savarin-Vuaillat & Ransohoff, 2007; Semple, Kossmann, & Morganti-Kossmann, 2010). HuR positively regulates other trophic factors produced by glia such as vascular endothelial growth factor (VEGF) and leukemic inhibitory factor (LIF) (Kwan, Floyd, Kim, et al., 2017; Lin, Niimi, Clausi, Kanal, & Levison, 2020; Matsye et al., 2017; Sun et al., 2018; Wang, Leavenworth, et al., 2019), and anti-apoptotic factors such as the Bcl-2 family (Filippova et al., 2021; Filippova et al., 2011; Ishimaru et al., 2009; Wang, Hjelmeland, et al., 2019). Knockout of HuR in motor neurons leads to an ALS-like phenotype with loss of motor neurons due to apoptosis (Sun et al., 2018).

In conclusion, we show that inhibition of HuR with the small molecule inhibitor SRI-42127 potently suppresses cytokine production in activated glia and may represent a promising new approach for treatment of neurological diseases driven by neuroinflammation.

Main Points.

• HuR promotes expression of inflammatory cytokines in LPS-activated glial cells.

• SRI-42127 inhibits HuR multimerization and blocks downstream HuR functions including shuttling, mRNA stability, translational efficiency, and gene promoter regulation.

• SRI-42127 suppresses pro-inflammatory cytokines in LPS-activated glial cells.