LPA3 agonist-producing Bacillus velezensis ADS024 is efficacious in multiple neuroinflammatory disease models

Neuroinflammation is a common component of neurological disorders. In the gut-brain-immune axis, bacteria and their metabolites are now thought to play a role in the modulation of the nervous and immune systems which may impact neuroinflammation. In this respect, commensal bacteria of humans have recently been shown to produce metabolites that mimic endogenous G-protein coupled receptor (GPCR) ligands. To date, it has not been established whether plant commensal bacteria, which may be ingested by animals including humans, can impact the gut-brain-immune axis via GPCR agonism. We screened an isopropanol (IPA) extract of the plant commensal Bacillus velezensis ADS024, a non-engrafting live biotherapeutic product (LBP) with anti-inflammatory properties isolated from human feces, against a panel of 168 GPCRs and identified strong agonism of the lysophosphatidic acid (LPA) receptor LPA3. The ADS024 IPA extracted material (ADS024-IPA) did not agonize LPA2, and only very weakly agonized LPA1. The agonism of LPA3 was inhibited by the reversible LPA1/3 antagonist Ki16425. ADS024-IPA signaled downstream of LPA3 through G-protein-induced calcium release, recruitment of β -arrestin, and recruitment of the neurodegeneration-associated proteins 14 – 3-3 γ , ε and ζ but did not recruit the β isoform. Since LPA3 agonism was previously indirectly implicated in the reduction of pathology in models of Parkinson ’ s disease (PD) and multiple sclerosis (MS) by use of the nonselective antagonist Ki16425, and since we identified an LPA3-specific agonist within ADS024, we sought to examine whether LPA3 might indeed be part of a broad underlying mechanism to control neuroinflammation. We tested oral treatment of ADS024 in multiple models of neuroinflammatory diseases using three models of PD, two models of MS, and a model each of amyotrophic lateral sclerosis (ALS), Huntington ’ s disease (HD), and chemo-induced peripheral neuropathy (CIPN). ADS024 treatment improved model-specific functional effects including improvements in motor movement, breathing and swallowing, and allodynia suggesting that ADS024 treatment impacted a universal underlying neuro-inflammatory mechanism regardless of the initiating cause of disease. We used the MOG-EAE mouse model to examine early events after disease initiation and found that ADS024 attenuated the increase in circulating lymphocytes and changes in neutrophil subtypes, and ADS024 attenuated the early loss of cell-surface LPA3 receptor expression on circulating white blood cells. ADS024 efficacy was partially inhibited by Ki16425 in vivo suggesting LPA3 may be part of its mechanism. Altogether, these data suggest that ADS024 and its LPA3 agonism activity should be investigated further as a possible treatment for diseases with a neuroinflammatory component.


Introduction
Neuroinflammation has now been identified as a common component of neurological disorders (De Marchi et al., 2023;Gilhus and Deuschl, 2019;Gorji, 2022;Guzman-Martinez et al., 2019).Most often, neurodegenerative diseases are studied in isolation and seen as distinct from one another, possibly due to the wide variation in their initiating causes.Efforts have generally focused on developing therapies aimed at those initiating causes while less focus has been on developing therapies for the ensuing damage caused by the response of the immune system to the initiating stimulus.However, the role of the gut-brain-immune axis is gaining attention, since bacteria that interact with and modify an organism's immune system are increasingly found to be positively or negatively associated with neuroinflammatory diseases (Park and Kim, 2021;Tran et al., 2022).Bacteria within the gastrointestinal (GI) tract can also play a significant role in neural development and behavior via the gut-brain-immune axis (Cryan and Dinan, 2012;Jacobson et al., 2021;Morais et al., 2021;Sharon et al., 2016).Many GI bacteria directly or indirectly alter neurotransmission through interaction with the neuroendocrine system, immune system, circulatory system, or the vagus nerve (Chu et al., 2019;Liu et al., 2022).This relationship between microbes in the GI tract and the brain developed through shared evolutionary history (Suzuki et al., 2022) as even simple organisms, such as Caenorhabditis elegans, have a gut-brain axis (Ortiz De Ora and Bess, 2021).Some bacteria have adapted to life within animal hosts (Suzuki et al., 2022), while others may temporarily pass through the GI tract (Leeming et al., 2019), allowing for the possibility of either consistent or intermittent interaction with the gut-brain-immune axis.Pathogenic bacteria can also affect this axis, but in a detrimental way, contributing to various neuroinflammatory diseases (Jameson et al., 2020;Lotz et al., 2021).
While advances have been made, it is still unclear how some bacteria can have such a strong influence on development, behavior, and other aspects of neurological function.One possibility is the production of unique metabolites given that millions of years of co-evolution have resulted in bacteria that produce potent compounds which allow the microorganisms to increase their odds of survival in their environment (Stein, 2005) and their hosts may have evolved to use these pre-existing microbial molecules for other purposes.Examples of host-modulating molecules include short-chain fatty acids, aryl hydrocarbon receptor agonists, and neurotransmitters, among others (Chen et al., 2021).Recent studies have shown that commensal bacteria can also make molecules that mimic G-protein coupled receptor (GPCR) ligands (Chen et al., 2023;Cohen et al., 2017Cohen et al., , 2015)).GPCRs constitute a very large family of receptors that have enormous communication and signaling capabilities.These signaling receptors represent targets of approximately 35% of all approved drugs (Sriram and Insel, 2018) and constitute some of the current targets for neurological indications such as schizophrenia, Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), multiple sclerosis (MS), anxiety, depression and pain (Azam et al., 2020).Given the capacity of some bacteria to produce GPCR-modulating molecules, there may be an opportunity to harness the properties of certain bacteria to positively influence the host via modulating GPCRs involved in the gut-brain-immune axis.
Like host-derived agonists, bacterial compounds may signal through many pathways available to a GPCR such as those involving G proteins, β-arrestins, and the more recently identified 14-3-3 proteins (Kongsamut and Eishingdrelo, 2023).14-3-3 proteins are intracellular dimers that preferentially bind phosphorylated proteins to regulate their functions by modifying protein-protein interactions, trafficking, signaling, metabolism, translation and other functions (Kleppe et al., 2011;Yang et al., 2006).14-3-3 proteins are exclusively eukaryotic and have been identified in animal, plant, and fungal cells and may represent one of the earliest regulators of protein signaling within eukaryotic cells.The 14-3-3 proteins are highly expressed in the vertebrate brain constituting around 1% of total soluble proteins, and all seven of the known mammalian 14-3-3 isoforms have been associated with neurodegenerative diseases (Cornell and Toyo-oka, 2017;Fan et al., 2019).Studies have demonstrated that GPCRs can modulate 14-3-3 signal transduction, and conversely, 14-3-3 proteins can modulate GPCR activity (Eishingdrelo et al., 2022;Kongsamut and Eishingdrelo, 2023;Li et al., 2016;Yuan et al., 2019).Some, but not all GPCRs, have been tested for 14-3-3 binding.Given the early evolutionarily development of 14-3-3 proteins, their prevalence in the brain, and bioinformatic analysis which predicts that 90% of GPCRs contain at least one putative 14-3-3 binding motif (Yuan et al., 2019), the possibility that bacterial compounds could interact with this pathway is also relevant for potential drug discovery efforts for neurological disorders.
We have isolated and identified a bacterium, ADS024, from human fecal matter with the ability to produce and secrete bioactive molecules (O'Donnell et al., 2022).The live biotherapeutic product (LBP) Bacillus velezensis ADS024 is a member of the operational group Bacillus amyloliquefaciens, which consists of the soil-borne B. amyloliquefaciens and the plant-associated Bacillus siamensis and B. velezensis.B. velezensis is known primarily as a commensal of plants and provides its host with both nutrients and protection from pathogens and stress (Fan et al., 2018;Rabbee et al., 2019).The life cycle of B. velezensis also includes ingestion and potential dispersion by animals (Kang et al., 2022).We have shown that dosing of ADS024 in humans and animals does not lead to engraftment in the GI tract (Allegretti et al., 2023;Murphy et al., 2023), and in animal models, ADS024 reduced damage and inflammation induced by toxigenic Clostridioides difficile as well as by dextran sodium sulfate (DSS), an agent used to induce a condition resembling ulcerative colitis (UC) (Irwin et al., 2024;Murphy et al., 2023;Xie et al., 2022).To begin to understand how this bacterium may influence its host during transit and reduce inflammation while still being able to protect against pathogenic bacteria, we sought to determine if ADS024 was able to produce metabolites that altered the activity of any of a large panel of GPCRs.We found that an ADS024 extract selectively agonized the lysophosphatidic acid receptor 3 (LPA3), a member of the EDG family of GPCR receptors.This family appears to be involved in the initiation and resolution of inflammation and includes the S1P receptors which are current drug targets for ulcerative colitis and multiple sclerosis.Given previous indirect associations of LPA3 with protection in PD and MS models that were identified using a nonselective antagonist, we sought to determine if the LPA3-specific agonism associated with ADS024 had the potential for an impact on neuroinflammation by investigating its efficacy in eight models of five neuroinflammatory diseases.

Bacterial strains, fermentations, and isopropanol extraction of lyophilized bacteria
100 ml overnight cultures of ADS024 or ADS012, two strains of B. velezensis, were used to inoculate at 1% (v/v) either baffled shake flasks containing 350-500 ml SY medium or 10 L fermenters.50 ml overnight cultures of Escherichia coli Nissle 1917 were used to inoculate 500 ml of LB medium at 1% (v/v).Flask cultures were shaken at 200 rpm and 37 • C for 28-54 hrs, and then centrifuged at 4,000 rpm for 30 min.10 L fermentations were harvested at 9-28 hrs depending on growth conditions.Following growth, the cultures were centrifuged, cell pellets were washed in PBS and re-centrifuged.The pellets were resuspended in cryoprotection buffer and aliquoted into Lyoguard trays and freeze dried, and resulting dried cells were stored in vacuum-sealed bags at 4 • C. Bacteria were dosed as live bacteria in colony forming units (CFU) or live plus dead bacteria as total cell counts (TCC) depending on the study.For extraction, freeze-dried cells (2 g) were rehydrated in maximum recovery diluent (MRD), centrifuged, and the resulting cell pellet was extracted by vortexing for 10 min in 20 ml 70% isopropanol (IPA) as previously described (O'Donnell et al., 2022).After centrifugation, the resulting liquid IPA extract, which was enriched in membrane-bound and internal bacterial metabolites, was lyophilized.Dried material was resuspended in DMSO for use in downstream assays.

LPA3 sequence binding site prediction for 14-3-3 proteins and binding assay
Identification of potential binding sites in the cytoplasmic domain of LPA3 was performed using three methods: the artificial neural network (ANN), position-specific scoring matrix (PSSM), and support vector machines (SVM) as described (Madeira et al., 2015;Yuan et al., 2019).

LPA3 and 14-3-3 interaction LinkLight assay
The LPA3-TEV expression plasmid was constructed by fusing a Tobacco Etch Virus (TEV) protease to the C-terminus of human LPA3 cDNA and was transiently expressed in LinkLight 14-3-3z permuted luciferase reporter cells (14-3-3z-pLuc) (Li et al., 2016) using PEI transfection reagent (Longo et al., 2013).24 hrs after transfection, cells were resuspended in 2% FBS DMEM culture medium and seeded in a 384-well cell white culture plate with 20,000 cells/20 μL/well.Cells were cultured overnight.The next day, 5 μL of serial 1:3 dilutions of LPA3 agonist oleoyl-LPA (a positive control) or ASD024-IPA were added to cells and incubated in a 37 • C cell culture incubator for 180 min.Following incubation, the cell culture plate was equalized to room temperature, and 10 μL of luciferase detection reagent Luci-Glo was added to each well.Relative light unit (RLU) is recorded by using a PerkinElmer EnspireTM instrument.All data points were in triplicate, and assays were repeated.Concentration-response curves were analyzed using Prism software (GraphPad Software, Inc.San Diego, CA).All values are expressed as the mean ± SD (n = 3).The Linklight assays were performed using technical triplicates and the data was graphed in GraphPad Prism using log(agonist) vs. response, least squares fit model.Error bars represent standard deviation of the triplicates and at some concentrations the replicates were so similar that GraphPad prism did not draw error bars where error bars would have been smaller than the symbol size.All samples were examined simultaneously for comparison purposes and the study was repeated with new preparations to confirm replication of the binding curves and a representative study is presented.The goodness of fit degrees of freedom was 33 for all curves.The R squared of curves for oleoyl-LPA and LPA3/14-3-3γ, ζ, and ε were 0.91, 0.80, 0.92, respectively and for ADS024-IPA and LPA3/14-3-3γ, ζ, and ε were 0.84, 0.78 and 0.97, respectively.

Animal models of neuroinflammation
Zebrafish lines with random mutations were created by a forward genetic method (Driever et al., 1996; see Supplemental Information Table S2).Phenotypic screening of motor movement deficits followed by fin clip DNA sequencing identified founders in genes of interest: pink1, htt, and tdp-43.Neuroinflammation was initiated in other models using chemical or peptide induction as indicated below.All animal studies were carried out under the guidance of the appropriate institutional IACUC committee oversight and followed ARRIVE guidelines 2.0, the NIH Guide for the Care and Use of Laboratory Animals and PHS Policy on Humane Care and Use of Laboratory Animals.Both sexes were used in zebrafish studies.In mice, to reduce the number of animals needed, only one sex was used per study, however, both sexes were used overall.Unless otherwise noted, data was analyzed in GraphPad Prism.Infrequently, outliers were identified and removed using ROUT (Q=1%) as indicated, and unless otherwise noted data was analyzed by one-way ANOVA followed by a Sidak's multiple comparisons test.Comparisons between the untreated model versus each group were reported; additional comparisons were evaluated for specific purposes defined in advance and are described within the Material and Methods section below for each specific model.Unless indicated otherwise, all data are displayed graphically as the mean and standard deviation of means with adjusted p-values, where significance was defined at p(adj) < 0.05(*), p (adj) < 0.01 (**), p(adj) < 0.001 (***) and p(adj) < 0.0001(****).

MPTP zebrafish model
Zebrafish larvae (96 hrs post fertilization (hpf)) were randomly sorted into zebrafish embryo water (E3) that was either sham treated (DMSO only; n = 35), MPTP treated (250 µM; n = 30) or MPTP+bacteria (ADS024 or ADS012) treated (4x10 6 colony forming units (CFU)/ml; n = 42).Following 24 hrs of treatment, movement was evaluated with Noldus Ethovision TM during 50 min of testing consisting of 10-min periods of alternating darkness/bright light (McKinley et al., 2005).ADS024 efficacy was observed in four independent studies and a representative study is shown.Data were analyzed in GraphPad prism using one-way ANOVA followed by a Sidak's multiple comparison test of vehicle vs. other groups.Error bars represent standard deviation.Statistical parameters for Fig. 4B are: F(3, 145) = 22.41, p < 0.0001 where F(DFn, Dfd), and abbreviations are Fstatistic (F), degrees of freedom between groups (DFn), degrees of freedom residual (DFd).MPTP zebrafish studies were run at ZeClinics in Barcelona, Spain.

pink1 -/-α-synuclein zebrafish model
A pink1 -/-zebrafish line with a null mutation due to deletion starting from 114 bps upstream of pink1 start codon until exon 1 was subjected to α-synuclein injection to exacerbate the phenotype (see Supplemental Information Fig. S3-1).Dosing of test agents was performed on 6-, 7-and 8-days post fertilization (dpf) via water (embryo medium) dissolution and daily replacement.Levodopa (Sigma CAS: 59-92-7) and Carbidopa (Sigma CAS: 38821-49-7) were dosed at a 1:4 wt ratio (10 pg and 40 pg mix per 50 ml, respectively) and ADS024 was dosed at 4x10 6 CFU/ml.Motor coordination was assayed on Day 9 by swim tunnel (n = 24/grp) (see Supplemental Information Fig. S3-2).Measurement of latency was stopped if zebrafish larvae were still swimming at 60 s.Data were analyzed in GraphPad prism using one-way ANOVA followed by a Sidak's multiple comparison test of vehicle vs. other groups.Error bars represent standard deviation.Statistical parameters for Fig. 4E are F (4,115) = 331.3p< 0.0001.Studies were run at Pentagrit, Chennai India.

Myelin oligodendrocyte glycoprotein experimental autoimmune encephalomyelitis (MOG-EAE) zebrafish model
EAE was induced in larvae (5 dpf) by immunization with human MOG peptide 35-55 (1 nL of 0.6 mg/ml) in Complete Freund's Adjuvant (CFA) (Sigma-Aldrich, 9007-81-2) under anesthesia with 0.03% Tricaine (Sigma-Aldrich) solution for 2-3 min as described with few modifications (Kulkarni et al., 2017).A volume of 1 nL of MOG peptide was delivered intracranially into the ventral part of the brain along the precaudal vertebrae.Phenol red tracer was used for injection validation.Replacement of the water after 24 hrs was precisely followed to improve survival odds.Animals were randomly sorted into study groups (n = 24/ group, in 50 ml embryo medium) and dosed by water dissolution on 9, 10 and 11 dpf with replacement each 24 hrs.Groups included naive (not treated with MOG), and MOG model groups: vehicle, Tecfidera-2 µg/g body weight, ADS024 (1x10 7 TCC/ml), ADS024 (1x10 7 TCC/ml) + Ki16425 (30 µM), and Ki16425 (30 µM).Assays were performed on 12 dpf.Raw tracks from video files were processed using Image J software and the data extracted in CSV format was converted to XLS format and were transferred to Microsoft Excel and Prism software.The total swim distance was derived as described in Supplementary information for mhtt zebrafish (see Supplementary information S4) using the same data acquisition, processing, and analysis procedures.Two outliers in the naïve group were identified and removed using ROUT (Q=1%) analysis in GraphPad Prism.Data were analyzed using one-way ANOVA followed by a Sidak's multiple comparison test of vehicle vs. all other groups and comparison of ADS024 to ADS024 + Ki16425.Error bars represent standard deviation.Statistical parameters for Fig. 5A F(5, 136) = 63.80,P<0.0001.Studies were conducted at Pentagrit, Chennai, India.

MOG-EAE mouse model
EAE was initiated on Day 0 in C57BL/6J (Jackson Labs) mice (age 11 wk females) by immunization with an emulsion of MOG 35-55 /CFA followed by injection of pertussis toxin on Days 0 and 1 in all groups except the Naïve group which were untreated (n = 12).A fresh solution of MOG was prepared prior to inoculation by dissolving the RP-HPLC purified lyophilized powder in phosphate-buffered saline (PBS) for a solution at a final injected concentration of 2 mg/ml (200 μg MOG in 100 μl PBS).
CFA containing heat-killed mycobacterium tuberculosis H37 Ra (3 mg/ ml) was used as supplied.Prior to inoculation on Study Day 0, equal volumes of MOG and CFA were mixed, at a ratio of 1:1 using two syringes connected by a Luer fitting until a stable emulsion was achieved.
A total dose volume of 200 μl was administered subcutaneously (SC) to each animal.MOG solution was dosed as 2 × 100 μl SC bilateral injections over the paralumbar regions.Prior to immunostimulant injection on Study Day 0, the pertussis toxin (PT) stock solution was freshly at a stock concentration of 100 μg/ml in distilled water.Prior to injection on Study Days 0 and 2, the PT stock solution (100 μg/ml) was diluted in PBS and vortexed vigorously for a final injected concentration of 2 μg/ ml.Each mouse was injected with 0.15 ml PT via IP (approximately 300 ng/mouse).Undiluted stock solution (100 μg/ml) was kept refrigerated at 2-8 • C and was reused for the second PT injection on Study Day 2 and discarded after 48 h.Thorough vortexing was required just prior to the PT injection session.Fingolimod (FTY720) was prepared in distilled water at a concentration of 0.6 mg/ml.Aliquots of 1.8 ml were stored at − 20 • C. Fingolimod treatments (0.6 mg/ml, 3 mg/kg, 5 ml/kg, per os (PO, QD), n = 8) were administered starting on Day − 3. Before each dosing, an aliquot of FTY720 was thawed and animals were held firmly in one hand and then, an appropriate size gavage needle was inserted gently through the mouth cavity.Once the gavage needle was sufficiently inserted through the throat and into the stomach, the agent was slowly injected.After the injection was performed, the needle was slowly removed in a manner that would not harm the animal.For ADS024 dosing, animals were administered ADS024 twice daily (BID) (PO, 1x10 9 CFU, 5 ml/kg, 100 μl, n = 12).Animals in the vehicle group were administered PBS (BID, PO, n = 12/grp) similarly to the dosing regimen for ADS024.The clinical score was defined as 0 = no clinical evidence of disease, 1 = limp tail / weakness of tail / paralysis of tail, 2 = abnormal gait, 3 = severe hind-limb weakness / partial hind-limb paralysis, and 4 = complete hind limb paralysis.Clinical scores were analyzed by one-way ANOVA followed by comparison of each treatment group to the Vehicle group using Dunnett's multiple comparisons test.Statistical parameters for Fig. 6A are F(3,64) = 25.41,P<0.0001.A repeat study of shorter duration also demonstrated ADS024-induced delay and reduction in clinical scores.The studies were run at MD Biosciences Innovalora Ltd (Israel).

Huntington's disease mutant mhtt zebrafish model
Zebrafish from a homozygous mhtt line with chorea and motor movement deficits due to a K32Q missense mutation in the htt gene (see Supplemental Information Fig. S4-1) were randomly selected and assigned to study groups (n = 24/group in 50 ml embryo medium) dosed on 6, 7, and 8 dpf with 24 hr replacement in fresh water.Tetrabenazine was used at 28 pg per 50 ml and bacteria were used at 4x10 6 CFU/ml.
Chorea and total swim distance were measured on Day 9 as described (Supplemental information Fig. S4-2).Data were analyzed in GraphPad prism using one-way ANOVA followed by a Sidak's multiple comparison test of vehicle vs. all other groups and the ADS024 group vs. E. coli group.Error bars represent standard deviation.One-way ANOVA parameters were: Panel 8A: F (4, 115) = 101.0,p < 0.0001, Panel 8B: F(7, 115) = 1468, p < 0.0001.The findings were repeated in a separate study.Studies were conducted at Pentagrit, Chennai, India.

Chemotherapy-induced peripheral neuropathy cisplatin mouse model
Neuropathic pain was induced in female mice using cisplatin (2.3 mg/kg), administered IP, for 5 consecutive days (0-4), followed by 5 days off (5-9), and then for an additional 5 consecutive days (10-14).All animals in the groups were injected with cisplatin (n = 43) except for Naive controls which were injected with saline (n = 12).Cisplatintreated animals were dosed PO with ADS024 at 1x10 9 CFU BID (Day -3 to Day 18; n = 11) or with vehicle (PBS; n = 12); the positive control consisted of IP 150 mg/kg gabapentin one hour before the assay (n = 7).The development of neuropathic pain was determined by measuring mechanical allodynia (Von Frey test) on Day -1 (baseline) and on Days 15 and 18 at 2 hrs post dosing of test agents.Allodynic response to tactile stimulation is assessed using the Von Frey apparatus (Touch Test®).Briefly, the mouse is placed on a metal mesh surface with free movement in an enclosure covered with red cellophane to diminish environmental disturbances.After cessation of exploratory behavior, the test is initiated.Starting with the least force fiber, the tip is pressed against the plantar skin surface of the foot at right angles and the force of application increases until the fiber bends which is at the maximum force attained.Forces (g) generated: 0.008, 0.02, 0.04, 0.07, 0.16, 0.40, 060, 1.00, 1.40, 1.7, 2.0.Mice indicate sensations by paw withdrawal and the minimal force needed to elevate the withdrawal reflex is recorded.Each Von Frey test is performed identically starting at the lowest force and proceeding to larger forces, and forces required for left and right paw withdrawal reflex values are averaged.Decreases in force needed to induce withdrawal are indicative of allodynia, as the force applied is a non-painful stimulus under normal conditions.Von Frey test was measured on Day -1 (baseline) and study Days 15 and 18.Data were analyzed in GraphPad prism using one-way ANOVA followed by a Sidak's multiple comparison test of vehicle vs. all other groups within one time point.Error bars represent standard deviation.One-way ANOVA parameters were F (11, 114) = 79.88,p < 0.0001.The study was conducted twice, once with taxol and once with cisplatin, both demonstrating efficacy.Peripheral neuropathy studies were run at MD Biosciences Innovalora Ltd (Israel).

ADS024 produces an agonist of the GPCR lysophosphatidic acid receptor 3 (LPA3)
To determine if B. velezensis ADS024, a strain that previously displayed anti-inflammatory activity in a UC model (Irwin et al., 2024), could produce metabolites that were capable of agonizing GPCRs, we tested ADS024-IPA, a mixture of compounds isolated by isopropyl alcohol extraction (IPA) of lyophilized ADS024 bacterial cells, on a PathHunter panel of 168 GPCRs using a β-arrestin format (Wang et al., 2004).The mechanism of signal generation in this system is specific to an individual stably transfected GPCR; each GPCR is tagged with a partial enzyme that produces a downstream signal when it binds a β-arrestin molecule tagged with a complementary enzyme fragment.Because ADS024-IPA is a complex mixture of extracted material resuspended in DMSO near the mixture solubility limit, only potent molecules or molecules that are present at very high percentages in the complex mixture would provide a strong signal in the screen.The initial screen yielded five agonist hits with Lysophosphatidic Acid Receptor 3 (LPA3, also known as LPAR3 or EDG7) the only hit that reached 100% of the positive control signal.
To confirm the agonism of LPA3 detected in the screen, we compared dose-response curves of oleoyl-LPA, the positive control agonist, with ADS024-IPA in the LPA3 β-arrestin assay (Fig. 1A).Oleoyl-LPA is one of many LPA molecules locally produced naturally by cells and which can activate all nearby LPA receptors.In humans, there are at least six known receptor types: LPA1, LPA2, LPA3, LPA4, LPA5, and LPA6.Oleoyl-LPA is a prototypical LPA with one double bond in its 18-carbon fatty acid chain and comparative analyses show that its potency is about 10-fold greater for LPA1, LPA2, and LPA4 than for LPA3, LPA5, and LPA6 (Kano et al., 2019).Oleoyl-LPA is useful for in vitro studies of LPA receptors where relative receptor levels can be controlled but is less useful for in vivo studies since it activates all 6 LPA receptors and is insoluble in aqueous solutions.Oleoyl-LPA demonstrated a sigmoidal curve with an effective concentration at 50% maximum (EC50) of 0.38 µg/ml (0.87 µM).ADS024-IPA agonism displayed a similar but slightly steeper sigmoidal curve with a mean EC50 of 44 µg/ml (range of 11 -106 µg/ml) for several independent ADS024-IPA preparations.While the curves are displayed in µg/ml for ease, we cannot directly compare the potency of oleoyl-LPA and the active compound in ADS024-IPA until the active compound is purified and the molecular weight is known so that a molar comparison can be performed.Because different ADS024-IPA preparations yielded slightly different EC50s, this indicates that different preparations may contain slightly different percentages of the LPA3 agonist.To determine the specificity of the signal, we evaluated LPA3 agonism by an IPA extract of B. velezensis ADS012, another strain also isolated from human feces.ADS012-IPA displayed an EC50 of greater than 270 µg/ml suggesting it may either contain a reduced level of the agonist or a different agonist.An IPA extract of E. coli Nissle 1917, an unrelated bacterium with anti-inflammatory properties, did not show activity in the LPA3 assay (Fig. 1A).In addition to β-arrestin signaling, LPA3 is known to cause calcium release via signaling through Gq.To determine if agonism by ADS024-IPA could be detected by changes in calcium levels, we ran a calcium-based assay using the same LPA3transfected cells.In this calcium assay, oleoyl-LPA displayed an EC50 of 0.008 µM (Fig. 1B) which is approximately 44-fold lower than that determined in the β-arrestin assay.This confirms that the LPA3 receptor couples efficiently with Gq and the amplified calcium signaling provides a more sensitive readout than β-arrestin coupling for oleoyl-LPA (Kajitani et al., 2023).While the parent cells do not express high levels of endogenous LPA receptors, caution must be used in interpreting calcium signaling since it is not directly coupled to the transfected GPCR.In the case of ADS024-IPA agonism of LPA3, calcium signaling occurred with an EC50 of 0.21 µg/ml (Fig. 1B) which is approximately 53-fold lower than the β-arrestin EC50 seen for this extract (β-arrestin EC50 for this preparation was 11 µg/ml).Since the obtained ratio of G-protein EC50/ β-arrestin EC50 for ADS024-IPA (53-fold) was very similar to that measured for the endogenous ligand oleoyl-LPA (44-fold), it suggests that LPA and ADS024-IPA behave similarly in initiating calcium and β-arrestin pathways.
To confirm and expand on the specificity of ADS024 agonism of LPA3 identified in the original screen, we ran dose-response curves of ADS024-IPA on LPA1 and LPA2, GPCRs which are also agonized by oleoyl-LPA.LPA2 was run in β-arrestin mode, the preferred mode for initial screening, and LPA1 was run in calcium mode since a β-arrestin assay was unavailable for LPA1.Like LPA3, both LPA1 and LPA2 are receptors for several LPA molecules that differ in chain length or saturation and displayed agonism by oleoyl-LPA (Fig. 1C-D).However, ADS024-IPA only very weakly agonized LPA1 in the calcium assay since the signal reached only 20% efficacy at 100 µg/ml while agonism by oleoyl-LPA had an EC50 of 0.0017 µg/ml (0.0038 µM) and reached a maximum signal at < 0.1 µg/ml (Fig. 1C).These results established that the parent CHO-K1 cells used in these assays do not contain a significant background signal for ADS024-IPA activity in a calcium-based assay, confirming the strength and specificity of the ADS024-IPA LPA3 calcium signal.Furthermore, ADS024-IPA showed no agonism of LPA2 (Fig. 1D).Given that oleoyl-LPA activates LPA1, LPA2 and LPA3 while ADS024-IPA is selective for LPA3, these results indicate that the active agent in ADS024 is not oleoyl-LPA.Furthermore, the active agent is not likely a common 1-acyl-LPA found in human plasma, since the prevalent derivatives (LPAs 16:0, 18:0, 18:1, 18:2, 18:3, 20:0, 20:4) all agonize LPA1 more efficiently than LPA3 (Fujiwara et al., 2005).In our quest to determine specificity, we also ran agonist assays of other EDG family receptors, the sphingosine-1-phosphate receptors S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (see Supplemental Information Table S1), as well as other LPA receptors LPA4 and LPA5.No other EDG receptors demonstrated strong agonism by ADS024-IPA under the conditions tested except for S1PR4 which displayed an EC50 for ADS024-IPA in β-arrestin mode that was > 10-fold higher (>500 µg/ml) than that for LPA3, indicating that ADS024-IPA was highly selective for LPA3.

LPA3 agonist activity of ADS024-IPA is blocked by the LPA1/3 antagonist Ki16425
To determine if the agonism of ADS024-IPA might be due to a compound(s) that binds to the same active site as oleoyl-LPA, we attempted to block the receptor activity with Ki16425.Ki16425 is a reversible competitive inhibitor of LPA binding and was shown to inhibit inositol phosphate production in RH7777 cells with Ki values of 0.34, 6.5, and 0.93 µM for human LPA1, LPA2, and LPA3 receptors, respectively (Ohta et al., 2003).Since it is more effective at inhibiting LPA1 and LPA3 than LPA2, it is often referred to as an LPA1/3 inhibitor.We used a dose of 10 µM of Ki16425 since this dose was previously shown to be sufficient to block calcium influx and downstream effects of both LPA1 and LPA3.As expected, 10 µM Ki16425 fully inhibited the agonism of LPA3 by oleoyl-LPA at low LPA doses but was less effective at higher LPA doses (Fig. 2A).Ki16425 was similarly effective at inhibiting the agonism by ADS024-IPA (Fig. 2B).Ki16425 also inhibited β-arrestin binding to LPA3 initiated by both oleoyl-LPA and ADS024-IPA (data not shown).

Efficacy of ADS024 in neuroinflammatory disease models
LPA3 is in the EDG family of receptors which includes S1P receptors, which are known to mediate and regulate inflammatory processes in diverse diseases such as UC and MS which suggests a common underlying inflammatory pathology.Since LPA3 had been indirectly implicated in protection against neuroinflammation in both a PD and MS mouse model, we hypothesized that LPA3 may be involved in the control of a common element in multiple neuroinflammatory diseases and that ADS024 treatment, which has selective LPA3 agonism, might be efficacious in models of these diseases.ADS024 can be dosed orally both in mice and zebrafish and our reasoning behind species model choice was as follows: 1) mouse models were generally preferred over zebrafish due to higher similarity to humans, 2) zebrafish models were chosen when a potential mouse model was not available to us or was not practical due to required long periods of twice a day oral gavage dosing which would stress fragile mice excessively, and 3) zebrafish models were chosen when shorter mouse models that were available did not show the outcome of interest.Since evidence of impact in models of multiple neuroinflammatory diseases in two species with multiple outcomes would further suggest a common pathology rather than a pathway unique to an outcome, disease, or species, we chose an array models and outcomes as described below.

Efficacy of ADS024 in Parkinson's disease models
A possible role for LPA1 and/or LPA3 in neuroprotection in a Parkinson's disease model was recently identified (Choi et al., 2018).Thus, we tested ADS024 in two neurotoxin (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)) models: a zebrafish model (Razali et al., 2021) and a mouse sub-chronic model (Machado et al., 2016).MPTP is routinely used for its known selective impact on dopaminergic neurons and short model duration.In zebrafish, MPTP dosed into the water has been shown to cause loss of dopaminergic neurons and reduction of locomotor activity (Vaz et al., 2018).Since zebrafish express LPA3, we tested the ability of ADS024 to protect the zebrafish from MPTP-induced motor movement deficits.As indicated above, we were unable to use oleoyl-LPA as a benchmark in this and other animal studies because it has several drawbacks: 1) it agonizes at least six LPA receptors and its potency for LPA1, LPA2, and LPA4 is approximately 10-fold greater than for LPA3, LPA5, and LPA6 (Kano et al., 2019) which may lead to opposing effects depending on timing of LPA receptor agonism (Ho et al., 2015), 2) it has in vivo proinflammatory effects likely due to the interaction with multiple LPA receptors (Chiang et al., 2021;Zhao and Natarajan, 2013), and 3) it is highly hydrophobic making it difficult to dose.Zebrafish larvae were exposed to MPTP from 96 to 120 hpf and assayed for distance moved over time in an alternating light-dark cycle.As expected, naive zebrafish moved during the dark cycle but had limited movement in the light cycle (Fig. 4A).MPTP caused a reduction in the dark cycle movement which was attenuated by the concurrent addition of lyophilized ADS024 in the water (Fig. 4A, B).ADS012 at the same dose had no effect in this study; however, further studies suggested some protective activity was occasionally present in different ADS012 fermentation lots (data not shown) suggesting that it may have the capability to produce active agent at lower levels than ADS024.This is consistent with the LPA3 agonism data.Studies of naïve wildtype

AA Position
Peptide zebrafish demonstrated that ADS024 had no effect on total distance moved or latency to fall in a swim tunnel assay indicating that the effect of ADS024 on swimming movement is specific to the induction of neurological damage (data not shown).We then tested ADS024 in the mouse sub-chronic mouse model in which mice were treated with MPTP at 20 mg/kg IP QD for 4 days with sacrifice on Day 7.This model does not display overt motor deficits but displays some aspects of neuroinflammation along with reduced dopamine and dopamine metabolite levels (Machado et al., 2016;Mat Taib and Mustapha, 2020).As expected, MPTP significantly reduced the levels of dopamine and DOPAC in the striatum as measured by quantitative HPLC indicating potential damage to the dopaminergic neurons (Fig. 4C, D).Prophylactic ADS024 treatment (5x10 8 CFU BID starting on Day -3) mildly but significantly increased the striatal dopamine and DOPAC levels (28% and 48% increase relative to MPTP only treatment, respectively, p < 0.05), whereas the equivalent dose of cryoprotectant in which the ADS024 is lyophilized, showed no significant effect.We then used a third PD model to determine if the protection was more generally applicable beyond neurotoxin-induced loss of dopaminergic neurons.We tested the ability of ADS024 to protect against a Parkinsonian phenotype in a mutant pink1 -/-zebrafish model injected with α-synuclein.pink1 -/-is a null mutation due to a deletion of 114 bps starting upstream of exon 1 and terminating in exon 1 which led to the loss of the start codon (see Supplemental Information Fig. S3-1).Disease progression is further exacerbated in the model with intracranial microinjection of α-synuclein into 5-day-old larvae.Agent treatment was performed on days 6, 7, and 8 and, on day 9, we measured latency to fall in a swim tunnel assay.The model had significantly reduced latency to fall relative to wildtype naïve zebrafish (mean of 23.6 s vs. 58.7 s, respectively; p < 0.0001) indicating a deficit in motor movement.Treatment with the positive control combination Carbidopa:Levodopa demonstrated a 43% increase in latency to fall relative to the vehicletreated model (Fig. 4E, p < 0.0001) and ADS024 treatment resulted in a 38% increase (p < 0.0001).In contrast to the improvement in swimming ability by ADS024, ADS012 led to a 30% reduction, further demonstrating that not all B. velezensis strains dosed similarly may result in the same outcomes.Together, these results suggest that ADS024 can protect dopaminergic neurons from neurotoxin and genetic insults and thereby improve neurochemical and motor outcomes.

Efficacy of ADS024 in multiple sclerosis models
A recent study in a mouse experimental autoimmune encephalomyelitis (EAE) model demonstrated that oleoyl-LPA dosed intraperitoneally reduced clinical symptoms while inhibition of LPA1/3 by the inhibitor Ki16425 worsened outcomes (Choi et al., 2021a).Thus, to extend our findings beyond PD, we assessed the effect of ADS024 in zebrafish and mouse EAE models of MS induced by MOG peptide (Constantinescu et al., 2011;Kulkarni et al., 2017).In the zebrafish MOG-EAE model, total swim distance was reduced relative to naive animals and was slightly improved by Tecfidera, a drug used clinically for MS (25% increase over Vehicle (the untreated model), p < 0.05) (Fig. 5A).ADS024 greatly increased the total swim distance (110% increase, p < 0.0001) bringing it into the normal range in this study.The large window of efficacy and small variance for swim distance provided an opportunity to evaluate the ability of Ki16425 to inhibit ADS024 efficacy in vivo.Ki16425 has been shown to selectively block zebrafish LPA1, LPA2a, LPA2b and LPA3 receptors but not LPA4, LPA6a, or LPA6b and Ki16425 at a dose of 30 µM had previously been shown to significantly impact hematopoietic differentiation in 2-6 dpf larvae (Li et al., 2014).Ki16425 (30 µM) dosed concurrently with ADS024 on 9-11 dpf partially blocked the efficacy of ADS024 (approximately 50%) and Ki16425 on its own very slightly increased swim distance relative to the untreated model, possibly due to its ability to also block LPA1 or LPA2.We were unable to use higher doses of Ki16425 due to the complication of decreased zebrafish survival in this model.Tracing of video recordings of movement and well exploration from a separate study demonstrated that the MOG Vehicle group exhibited atypical movement, contrasting sharply with the typical exploration observed in the Naïve group which swim primarily at the exterior of the wells in a circular movement pattern (Fig. 5B).In contrast to the MOG Vehicle group, the study groups treated with Tecfidera and ADS024 display some rescue in normal movement patterns where more larvae comprehensively explored well exterior walls as exemplified by circular tracings, though not all zebrafish recovered normal movement in this study.
In the mouse MOG-EAE model, prophylactic treatment with ADS024 delayed onset and reduced clinical scores (Fig. 6A,B).Fingolimod, a clinically used treatment, reduced clinical scores to 0 in this study indicating it was maximally effective at the dose used.An attempt to use Ki16425 dosed intraperitoneally to block the efficacy of ADS024 resulted in the early death of mice.It is unclear if this was due to a worsening of the disease or possible unrelated toxicity.Therefore, the role of LPA3 in the efficacy of ADS024 in a MOG-EAE model was determined using the zebrafish model where we could dose Ki16425 in the water as described previously (Fig. 5A).Because of the evidence of an early ADS024-induced delay in the appearance of clinical scores in the mouse model, we chose to examine circulating blood immune cells by flow cytometry on Day 7 after induction.We hypothesized that ADS024 might act prophylactically to reduce the overall autoimmune response to the MOG-EAE protocol.Results indicated that, as expected, circulating neutrophils, monocytes and lymphocyte numbers were greatly increased in the MOG-EAE model (Vehicle) relative to Naïve mice (Fig. 6C-F).In healthy mice, lymphocytes are the dominant circulating white blood cell (Ivetic et al., 2019), whereas neutrophils had become dominant in the model (Vehicle group) on Day 7.These changes Fig. 3. 14-3-3 protein recruitment to LPA3 by oleoyl-LPA and ADS024-IPA.Interactions between LPA3 and 14-3-3 proteins 14-3-3ε (A), (14-3-3γ (B), and 14-3-3ζ (C) in transiently transfected HEK293 cells were measured in the presence of increasing concentrations of oleoyl-LPA or ADS024-IPA using a Linklight reporter system as indicated in Materials and Methods.Each concentration was run in triplicate and error bars represent standard deviation.Where no error bar is evident, the deviation was smaller than the size of the plotted superimposed points.This study was repeated with similar results.RLU: relative light units.
indicated that the immune response to the induction was evident at this time point.Treatment with ADS024 partially attenuated the increase in lymphocytes and CD4+ T cell numbers but did not significantly alter numbers of neutrophils and monocytes.The ratio of neutrophils to lymphocytes in the model was increased slightly by ADS024 from 3.6 to 4.8 (p < 0.0001) suggesting the observed changes were not due to changes in blood volume.Because neutrophils are known to be a first responder to bacteria and yet absolute neutrophil numbers were not significantly changed by ADS024 in this model, we examined two markers on neutrophils, CD62L and CXCR4, to evaluate a potential impact on neutrophil subtypes.CD62L (L-selectin) is a surface adhesion protein involved in immune cell extravasation from the blood into tissues and is shed after neutrophil priming during inflammation (Ivetic et al., 2019;Snyder et al., 2021).CXCR4 is a chemokine receptor that is increased on neutrophils during the aging process and assists in homing aged neutrophils to the bone marrow for clearance.We noted a large 150 µM MPTP (Vehicle, n = 30) and MPTP with lyophilized ADS024 (4x10 6 CFU/ml, n = 42) or MPTP with ADS012 (4x10 6 CFU/ml, n = 42) in the water.Following 24 hrs of treatment the zebrafish were assayed for swim distance movement in a light-dark alternating assay.Multiple repeat studies with different preparations showed similar results.In C, D: dopaminergic damage in mice was induced (n = 10/group) by dosing with MPTP or saline for four consecutive days (Days 0-3, 20 mg/kg, QD, IP).Oral treatment with ADS024 (5x10 8 CFU), saline, or the cryoprotectant buffer (CryoP) (dose matched to ADS024) was performed from Day -3 to Day 7. On Day 7, mice were sacrificed, and the striatum was dissected from the brain, homogenized and dopamine and DOPAC were measured by reverse phase HPLC-EDC.A repeat study demonstrated attenuation of the reduction of the dopamine metabolite homovanillic acid.In E,F: pink1 -/-zebrafish were injected with α-synuclein and on 5 dpf were treated for 3 days with agents in the water (D6-8; n = 24/group, ADS024 at 4x10 6 CFU/ml, Carbidopa:Levidopa at a 1:4 ratio as a mix of 10 pg and 40 pg per 50 ml, respectively).On D9 zebrafish were assayed for motor coordination in a swim tunnel assay where latency to fall was measured.Each point represents one zebrafish.A repeat study showed similar results.Data were analyzed by one-way ANOVA followed by a Sidak's multiple comparisons test between the vehicle-treated model versus all other groups as described in Materials and Methods.One data point for dopamine in the vehicle group was identified as an outlier using ROUT (Q=1%) and was removed.Data are displayed as mean and error bars indicate standard deviation.Significance is determined by adjusted p-values defined as p(adj) < 0.05(*), p(adj) < 0.01 (**), p(adj) < 0.001 (***) and p(adj) < 0.0001(****) and ns indicates a comparison was performed but was not significant.increase in the percentage of neutrophils that had shed their CD62L (CD62L-) in the model (89% CD62L-vs.8% CD62L-for Vehicle vs. Naïve, p < 0.0001) (Fig. 6G) which was partially attenuated by ADS024 (89% vs. 75% for CD62L-neutrophils in Vehicle vs. ADS024-treated, p < 0.0001).However, the percentage of CXCR4+ neutrophils, which was low in naïve mice (approximately 1%), was unchanged at Day 7 in the model or by ADS024 treatment of the model (Fig. 6H).We also examined naïve (CD62L+CXCR4-) and aged (CD62L-CXCR4+) (Casanova-Acebes et al., 2013) neutrophils (Fig. 6I-J) and found that ADS024 partially attenuated the model-induced reduction in the percentage of naïve neutrophils but had no effect on the small increase in aged neutrophils at this time point.Together, these data suggests that ADS024 influences circulating lymphocyte numbers and some neutrophil subtypes in this model which are both consistent with an anti-inflammatory phenotype.
Because ADS024 contains an agonist of LPA3 and was effective in this model, we evaluated LPA3 levels on the circulating immune cells on Day 7 which was prior to evidence of clinical observations.We observed Fig. 5. Efficacy of ADS024 in MOG-EAE zebrafish MS disease model.Total swim distance per minute was measured in a MOG-EAE zebrafish model (A) and in a separate study well exploration was evaluated (B).All groups (n = 24/grp) were MOG-EAE treated on 5 dpf except Naive which was mock treated.Treatments were dosed in the water on 9, 10, and 11 dpf and included Tecfidera (2 µg/g), ADS024 (1x10 7 TCC/ml), ADS024 (1x10 7 TCC/ml) + Ki16425 (30 µM), and Ki16425 alone (30 µM).Assays were performed on 12 dpf.Data were analyzed by one-way ANOVA followed by a Sidak's multiple comparisons test between the vehicle-treated model versus the other groups and between ADS024 and ADS024 + Ki16425 groups as described in Materials and Methods.Data are displayed as mean ± standard deviation.Significance was determined by adjusted p-values defined as p(adj) < 0.05(*), p(adj) < 0.01 (**), p(adj) < 0.001 (***) and p(adj) < 0.0001(****).Studies were repeated with similar results.(B) In a separate study, unique 1-minute trajectories of the zebrafish larvae in the wells (n = 24/grp, 1 larvae/well) were video recorded and the summary movement evaluated by tracing the movement of larvae within its well.a uniform reduction of LPA3 on circulating neutrophils, monocytes, and lymphocytes in the model (Vehicle group) relative to Naïve mice (Fig. 6K-N); in CD4+ T cells, LPA3 cell-surface expression was reduced to near zero (Fig. 6N).The reduction of LPA3 cell-surface expression was attenuated by ADS024 on neutrophils, lymphocytes and CD4+ lymphocytes while the effect on monocytes was variable between experiments (Fig. 6L and data not shown).Taken together, these results suggest that there is a change in LPA3 cell-surface expression on circulating immune cells in the model and that ADS024 treatment, which contains an LPA3 agonist, may cause LPA3 cell-surface stabilization or increased receptor expression under these conditions.CXCR2, another GPCR on circulating neutrophils, was upregulated in the MOG-EAE model on Day 7, and its expression was not modified by ADS024 treatment, while CXCR1 was downregulated in the model and slightly increased by ADS024, and CCR5 was unchanged under all conditions suggesting some specificity of the ADS024 effect on immune cells (see Supplemental Information Fig. S6).A preliminary study showed increased LPA3 expression on neutrophils in spinal cord on Day 16 in the model relative to naïve mice and this increase was attenuated by ADS024 treatment, an effect opposite that on circulating blood immune cells on Day 7 (data not shown).Thus, regulation of LPA3 expression may be dependent on timing and/or location in the model.Further studies of dosing optimization for maximum clinical response are warranted prior to a detailed examination of the time course of changes in immune cell numbers and inflammatory response both in the periphery and the CNS.The efficacy of ADS024 in the mouse and zebrafish MS models along with the alteration of LPA3 expression and the attenuation of ADS024 protection by Ki16425 together suggest that ADS024 may provide some protection against MOG-EAE induced pathology, at least partially through modulation of LPA3.Fig. 7. Efficacy of ADS024 in a tdp-43 -/-Amyotrophic Lateral Sclerosis zebrafish model.Operculum movement (A) and Buccal movement (B) were measured in wildtype and tdp-43 -/-zebrafish.Fish (n = 24/group) were treated with Riluzole (10 µM), ADS024 (1x10 6 total cell counts (TCC)/ml)), E.coli Nissle (1x10 6 TCC/ml), or Ki16425 (30 µM) on Days 6, 7, and 8 with replacement before assaying on Day 9.Each larva was transferred to a capillary tube with embryo medium and movement was recorded for 3 min under a stereo microscope on a backlight stage.For operculum movement, video was manually analyzed for number of beats per minute (bpm) by counting complete cycles of opening and closing of the operculum, a thin bony flap covering the gill complex.Each complete cycle was considered one beat.Buccal movement, the opening of the buccal cavity and the closing of the operculum followed by suction feeding, was evaluated using video recorded for 1 min under feeding conditions.Each complete cycle of these movements was counted as one buccal movement cycle in bpm.A repeated study yielded similar results.Data were analyzed by one-way ANOVA followed by a Sidak's multiple comparisons test between the groups as indicated by bars above and as described in Materials and Methods.Data are displayed as mean and error bars indicate standard deviation.Significance is determined by adjusted p-values defined as p(adj) < 0.05(*), p (adj) < 0.01 (**), p(adj) < 0.001 (***) and p(adj) < 0.0001(****).The designation ns indicates that the comparison was performed but was not statistically significant.

Efficacy of ADS024 in an amyotrophic lateral sclerosis disease model
LPA1 and LPA2 signaling have been implicated in disease progression of amyotrophic lateral sclerosis (ALS) (Gento-Caro et al., 2022;Puigdomenech-Poch et al., 2021), though less is known about the role of LPA3.Thus, we examined the effect of ADS024 in an ALS zebrafish model containing a mutation in the tdp-43 gene (see Supplemental information for model generation and characteristics Figs.S5-1 and S5-2).This mutation constitutes a complete null genotype due to a 322-bps deletion which includes the start codon of the gene.Operculum movement, which represents breathing movement in zebrafish, was significantly reduced in the tdp-43 -/-mutant model (p < 0.0001) and was improved by Riluzole (18% increase over vehicle treated, p < 0.0001) which blocks the release of glutamate and is used in ALS patients to extend survival or time to tracheostomy.Though ADS024 slightly reduced operculum movement in wildtype zebrafish, ADS024 increased it significantly in the tdp-43 -/-ALS model to near wildtype levels (58% increase over vehicle, p < 0.0001) (Fig. 7A) suggesting that the increase in operculum movement is contingent on the presence of neuropathology in the model.Ki16425 significantly attenuated the ADS024 improvement in operculum movement (74% inhibition of the ADS024induced improvement, p < 0.0001) though Ki16425 treatment alone slightly increased operculum movement suggesting the possibility that LPA1 or LPA2 inhibition may be beneficial in the model.E. coli Nissle did not alter operculum movement.Buccal movement, which represents feeding movement in zebrafish, was also significantly reduced in the model relative to wildtype zebrafish (p < 0.0001) and was greatly increased by Riluzole (73% improvement, p < 0.0001) (Fig. 7B).ADS024 alone slightly reduced buccal movement in wildtype zebrafish (16% reduction relative to untreated, p < 0.001), however it significantly improved buccal movement in the ALS model (94% increase over vehicle, p < 0.0001).Unlike the results for operculum movement, Ki16425 had no effect on the ADS024-induced improvement in buccal movement.Anti-inflammatory E. coli Nissle increased buccal movement (63% relative to untreated, p < 0.0001).Together this suggests that ADS024 and its component LPA3 agonist may impact operculum and buccal movement, but the mechanism of ADS024 improvement for each movement may be different.Thus, ADS024, which potentially has multiple active agents, could potentially be beneficial for the treatment of breathing and swallowing issues in ALS.

Efficacy of ADS024 in a Huntington's disease model
Similar to other neurodegenerative diseases, Huntington's disease (HD) has an inflammatory component (De Marchi et al., 2023;Guzman-Martinez et al., 2019).To further investigate the efficacy of ADS024 in neuroinflammatory disease outcomes, we tested the impact of ADS024 in an mhtt HD zebrafish model in which the htt gene was mutated leading to chorea (jerky movements) and a reduced ability to swim.Previous studies had shown that inhibition of the LPA1/3 receptors in two HD models reduced the motor improvement due to treatment with gintoninderived LPA (Jang et al., 2019).In those studies, gintonin was shown to have antioxidant and anti-inflammatory activities through nuclear factor erythroid 2-related factor (Nrf2) signaling, which has been shown to be downstream of LPA3 signaling (Chen et al., 2020).In the case of human HD, the genetic cause is an amplification of glutamine residues in the Htt protein, however, there is still some debate over whether the phenotype is primarily caused by a loss of function, a gain of function, or both (Schulte and Littleton, 2011).In the zebrafish line used here, the phenotype is caused by a single lysine to glutamine conversion (K32Q), and yet the phenotype is pronounced in the mhtt larvae where they display chorea, which is one of the most common symptoms in human HD, as well as wall butting behavior (see Supplemental Information S4 for model description).We examined the effect of tetrabenazine, a clinically used drug for chorea, in the mhtt model.Tetrabenazine reduced the number of twitching events per minute (40% reduction vs. vehicle, p < 0.0001) (Fig. 8A).ADS024 and the unrelated antiinflammatory bacterium E. coli Nissle also both reduced chorea in the model (53% and 60% reduction vs. vehicle, respectively, p < 0.0001).In addition, we examined total swim distance, which represents productive movement, which was greatly reduced in the model relative to control zebrafish (75% reduction vs. wildtype, p < 0.0001) (Fig. 8B).Not surprisingly, tetrabenazine further reduced total swim distance per minute (49% reduction vs. vehicle, p < 0.0001) since its mechanism leads to depletion of monoamines in neurons; a side effect of tetrabenazine in humans includes drowsiness and sedation (Jankovic and Beach, 1997).However, in contrast to tetrabenazine, ADS024 improved swim distance (52% increase over mhtt, p < 0.0001).We were unable to use Ki16425 to determine if it blocked ADS024 improvement due to severe hyperactivity induced by its Ki16425 in the mhtt model characterized by highvelocity rapid turning and darting-like erratic movements within a single behavioral bout (data not shown).The reasoning behind this is unclear but warrants further study.The anti-inflammatory bacterium E. coli Nissle improved swim distance (26% increase), though ADS024 was superior to E. coli Nissle (p < 0.0001).Altogether, these data suggest that some bacteria may affect HD symptoms and that ADS024 may be a useful agent for individuals suffering from HD. Fig. 8. Efficacy of ADS024 in an mhtt Huntington's Disease zebrafish model.Chorea (A) and Total Swim Distance per minute (B) were measured in wildtype and mhtt zebrafish.Fish (n = 24/group) were treated with Tetrabenazine (28 pg), ADS024 (4x10 6 CFU/ml), or E. coli Nissle 1917 (4x10 6 CFU/ml) on Days 6, 7, and 8 with replacement before assaying on Day 9. To study chorea (jerky movements per minute), 9 dpf larvae were acclimatized to a study well for 10 min and the well plate was placed under stereomicroscope where larvae were observed for phenotypic signs of chorea and jerky movements were recorded manually.Total swim distance was derived from video recordings as described in Supplemental Information (Section S4).The study was repeated with similar results.Data were analyzed by oneway ANOVA followed by a Sidak's multiple comparisons test between the vehicle-treated model versus the other groups and between the ADS024 group and the E. coli Nissle group as described in Materials and Methods.Data are displayed as mean and error bars indicate standard deviation.Significance is determined by adjusted p-values where p(adj) < 0.0001(****) and ns indicates the comparison was performed but was not significant.

Efficacy of ADS024 in a peripheral neuropathy model
To determine if the effect of ADS024 on neuroprotection of the central nervous system extended to the peripheral nervous system, we examined the effect of prophylactic ADS024 treatment in a mouse chemotherapy-induced peripheral neuropathy (CIPN) model.Paclitaxelinduced neuropathy was previously found to be absent in LPA3 receptordeficient mice (Uchida et al., 2014), thus it was not clear a priori if ADS024 would increase or decrease pain sensation in a cisplatin model.Cisplatin was dosed in two rounds of 5 days each on Days 0-4 and Days 10-14.Response to Von-Frey fiber stimulation was measured on Days 15 and 18, where lower scores in the cisplatin-treated (Vehicle) mice indicated higher sensitivity to an uncomfortable stimulus.As expected, short-term gabapentin treatment diminished the sensitivity that had developed, yielding a higher score than in the Vehicle mice (Fig. 9).On study Day 15, the withdrawal force for the ADS024-treated group was 0.83 ± 0.11 g, a significantly higher force than needed for paw withdrawal for the Vehicle-treated animals (0.18 ± 0.01 g; p < 0.0001).Similar results were seen on study Day 18.Thus, ADS024 given prophylactically significantly reduced the sensitivity to cisplatin neuropathy on both Days 15 and 18.These results suggest that, in addition to the possible use of ADS024 in central nervous system neuroinflammatory diseases, ADS024 may also be beneficial in prevention of peripheral neuropathy.

Discussion
Neurological disorders have increasingly been found to involve neuroinflammation which may be impacted by the gut-brain axis (Agirman et al., 2021;De Marchi et al., 2023;Gilhus and Deuschl, 2019;Gorji, 2022;Guzman-Martinez et al., 2019).This complex communication system has recently been referred to by other terms such as the gut microbiome-immune system-brain axis (Foster et al., 2021) due to the accumulation of data indicating the importance of both bacteria and the immune system within the axis (Yuan et al., 2023).GI contents (e.g.food, bacteria, and metabolites) have been identified as potential modulators of the immune and nervous systems and some agents have been associated with diseases such as MS, HD, Alzheimer's, ALS and many others (Altieri et al., 2023;Chen et al., 2022;Wasser et al., 2020;Wright et al., 2018).Bacterial metabolites, such as short chain fatty acids, serotonin, indole derivatives and bile metabolites (Park and Kim, 2021) have been linked to many effects on the gut-brain-immune axis and some bacterial metabolites can mimic GPCR ligands (Chen et al., 2023;Cohen et al., 2017Cohen et al., , 2015)).We, therefore, evaluated the ability of an extract of ADS024, an anti-inflammatory Bacillus velezensis strain isolated from human feces (O'Donnell et al., 2022), to signal through human GPCRs.From a screen of 168 GPCRs, we identified a strong signal for agonism of the lysophosphatidic acid receptor, LPA3.A previous study screening a simplified human microbiome for bacterial metabolite agonists of GPCRs demonstrated no agonism of LPA3 (EDG7) (Colosimo et al., 2019) indicating that LPA3 is not a common target of the microbiota.The responsible agonist in ADS024-IPA was highly selective for LPA3 over LPA1, LPA2, LPA4, and LPA5 indicating that it was not oleoyl-LPA nor likely to be another common LPA molecule based on LPA specificity studies for the different LPA receptors (Fujiwara et al., 2005).This suggests that the active moiety may constitute a very different chemical structure than oleoyl-LPA.ADS024-IPA agonism was inhibited with a competitive antagonist of LPA1/3, Ki16425, suggesting that the activity is blocked by reversible binding of Ki16425 to the LPA binding site.LPA signaling through its receptors has been associated with synaptic transmission, neural progenitor cell function, myelination, inflammation, and brain immune responses, among others (Knowlden and Georas, 2014;Yung et al., 2015) and LPA levels are dysregulated in neuroinflammatory conditions such as MS (Jiang et al., 2018;Schmitz et al., 2017).What is less clear is the specific role each LPA receptor plays in neuroinflammation.
ADS024-IPA was more selective for LPA3 agonism than typical LPA molecules, however, it did signal similarly through the pathways downstream of LPA3, namely calcium signaling, β-arrestin binding, and the newly identified recruitment of 14-3-3 proteins This is the first report of a direct binding between LPA3 and members of the 14-3-3 family of regulatory molecules.Recruitment of 14-3-3 proteins to LPA3 is of particular interest since 14-3-3 proteins are highly expressed in the brain and have been associated with various neurological diseases (Foote and Zhou, 2012;Kaplan et al., 2017;Pair and Yacoubian, 2021).Among the isotypes, 14-3-3γ is mainly expressed in neurons and has been implicated in neurodegenerative diseases as well as neurodevelopmental and neuropsychiatric disorders (Cho and Park, 2020).14-3-3γ plays a protective role in EAE pathology since its absence by genetic knockout in mice leads to increased pathology (Lee et al., 2015).LPA is also protective in the mouse EAE model through the LPA1/3 receptors (Choi et al., 2021a), therefore it is possible that ADS024 is efficacious in this model and other neuroinflammatory models by initiating recruitment of 14-3-3γ to LPA3.14-3-3γ and 14-3-3ε proteins were found to be reduced in an α-synuclein transgenic PD mouse model and overexpression led to resistance to toxin-induced pathology (Yacoubian et al., 2010), thus it is possible that the protective activity of ADS024 in PD is due in part to its initiation of 14-3-3 protein recruitment to LPA3.Much more research is needed to understand the complex biology between LPA3 and the 14-3-3 proteins since 14-3-3 proteins heterodimerize and bind many other proteins while modulating signaling pathways.
Ki16425 only partially inhibited the efficacy of ADS024 in vivo.This suggests that either the distribution and quantity of Ki16425 in vivo may have been insufficient to reach its target to fully block the activity of ADS024, or that the ability of Ki16425 to also block LPA1 and LPA2 may have led to additional effects that complicate interpretation, or alternatively, there may be other mechanisms also involved in the ability of ADS024 to reduce neuroinflammation.Indeed, a potential benefit of a live biotherapeutic product (LBP) for neuroinflammatory diseases is the possibility of a multi-modal mechanism within one agent.For example, ADS024, like other B. velezensis strains, has the capacity to take up and convert parent isoflavones from soy cultures into more active antiinflammatory derivatives (Kondo et al., 2023;Dias et al., 2018;Razeghi Jahromi et al., 2014;Spagnuolo et al., 2018;data not shown).Another important benefit of LBPs is the ability to dose them orally to effectively deliver agents that may be otherwise challenging such as highly hydrophobic compounds like LPA-type molecules or the isoflavones.Furthermore, unlike most other anti-inflammatory agents that reduce immune responses to pathogens, some B. velezensis strains, including ADS024, have been shown to enhance anti-infection capabilities (Kang et al., 2022;Murphy et al., 2023;Yun et al., 2023).
In the mouse MOG-EAE model, ADS024 attenuated the MOGinduced increase in circulating lymphocytes and altered neutrophil subtype distribution.In addition, ADS024 protected against the loss of LPA3 on the major circulating immune cells, together suggesting that impact on the immune system may play a role in its efficacy.Indeed, ADS024 was effective in the eight models representing five neuroinflammatory diseases reported here regardless of the target and cause of neuronal damage.This supports a role for ADS024 in modifying the ensuing immune response to the insult, rather than impacting the cause of the injury itself.For example, ADS024 ameliorated symptoms that were induced either chemically (e.g. by cisplatin or MPTP) or genetically (e.g. by mutations in the models mhtt, tdp-43 -/-or in a pink1 -/- mutant treated with exogenous α-synuclein).Given the broad efficacy of ADS024 and the common feature of neuroinflammation in all the neurodegenerative diseases represented by these models, targeting neuroinflammation may be an approach that could be broadly protective (De Marchi et al., 2023;Guzman-Martinez et al., 2019).
The immune system has evolved to protect against pathogens and damage by initiating, regulating, and then terminating an inflammatory response.If regulation and resolution of inflammation do not occur, ongoing inflammation can lead to excessive damage.One hypothesis is that LPA3 may be part of an off switch for inflammation since it has been shown to be important for increased expression of Nrf2, a protein involved in reduction of inflammation and oxidative damage (Chen et al., 2020).In the EAE model, the efficacy of an LPA-containing extract of ginseng was determined to be due to stabilization of Nrf2 signaling through increased expression and activity of LPA1 and/or LPA3 receptors (Choi et al., 2021b).Thus, LPA3 may be part of an inflammation resolution pathway and inadequate activation may lead to excessive ongoing inflammation which could result in neuronal and glial damage and loss.If so, agonism and/or modulation of LPA3 expression on immune cells by ADS024 may play a role in reducing excessive inflammation leading to the functional benefits observed in the animal models reported here.
In addition to the role of LPA3 in impacting the immune system, it is possible that LPA3 agonism also plays a more direct role on cells of the nervous system.Under normal conditions, high LPA3 expression has been identified in astrocytes, with low or no expression in microglia or neurons (Fujita et al., 2008;Spohr et al., 2008;Uhlen et al., 2010).However, LPA3 is upregulated on neurons after injury and its expression in the brain is increased by the inflammatory stimulus lipopolysaccharide LPS (Goldshmit et al., 2010;Plastira et al., 2020).These results support a role for increased LPA3 activity in the nervous system in response to neuroinflammatory stimuli.The connection between LPA3, inflammation and the nervous system is further supported by recent studies in autism spectrum disorder (ASD).In addition to other behavioral changes, LPA3 knockout zebrafish display reduced social interaction (Chen et al., 2020;Lin et al., 2020), and in humans ASD individuals show evidence of peripheral and central neuroinflammation (Depino, 2013), have altered calcium signaling in their astrocytes, and have impaired Nrf2 signaling (Nadeem et al., 2022;Schrier et al., 2022;Yang et al., 2020).When transferred to mice, human ASD astrocytes derived from stem cells can induce repetitive behavior and impair memory (Allen et al., 2022).Because of the apparent connection between LPA3 and its expression in astrocytes, stimulation of Nrf2, effect on neuroinflammatory diseases, and impact on social behavior, we ran a preliminary study examining the effect of ADS024 on the mouse genetic autism model, BTBR.ADS024 treatment of BTBR mice for only 6.5 days showed a small but significant increase in sociability (data not shown), suggesting that an increase in LPA3 signaling may be able to correct a neuroinflammatory issue in ASD.Again, this further suggests that the efficacy of ADS024 through LPA3 signaling is agnostic of the cause of neuroinflammation, supporting its potential use in multiple neuroinflammatory diseases.
One of the limitations of this study is the use of Ki16425 which is not a selective LPA3 antagonist since it displays strong antagonist activity for both LPA1 and LPA3 and weaker activity for LPA2 in mammals, and strong antagonism for LPA1, LPA2a, LPA2b and LPA3 in zebrafish.Thus, any dose of Ki16425 used will also impact LPA1 in addition to LPA3 since the compound has similar IC50s for both receptors and we cannot rule out an impact on LPA2 at high concentrations (Ohta et al., 2003).We observed side effects of the use of Ki16425 such as death in MOG-EAE mice and hyperactivity in diseased HD zebrafish.This limited the use of Ki16425 in some studies.Also, Ki16425 was only used to block efficacy of ADS024 when a large effect window was evident so that the study could be powered to see a 50% inhibition, if present.In addition, the pharmacokinetic properties of Ki16425 are not well established.One study showed poor brain penetration (Sánchez-Marín et al., 2018), therefore it is possible that we have underestimated the role of LPA3 agonism in ADS024 activity using Ki16425 to block the receptor.Furthermore, we cannot fully rule out the possible involvement of LPA1 or S1PR4 in some of the observed activity of ADS024, though ADS024-IPA contains a much stronger agonist of LPA3 than either S1PR4 or LPA1; currently, there is no available S1PR4-selective antagonist that is known to be active in vivo.The development of in vivo active LPA3specific inhibitors, LPA1-specific inhibitors, and S1PR4-selective inhibitors with demonstrated abilities to cross the blood-brain barrier, which are not available now, would alleviate these concerns in the future.
Another study limitation is that we did not perform all possible outcomes in all models.Choice of outcomes was determined by relevance to human disease (e.g.movement in Huntington's and breathing in ALS), outcome availability depending on species type (e.g.pain perception in mice, swimming in α-synuclein treated pink1 -/-zebrafish), and preference for functional over non-functional outcomes.However, non-functional outcomes were sometimes added to gain particular mechanistic information.Deep analysis of mechanism requiring extensive work per model will be the basis for future studies.Furthermore, benchmark molecules were chosen as follows: clinically used compounds were used as positive controls when available and useful in the model, oleoyl-LPA was not used due to its promiscuity for all LPA receptors and due to its hydrophobicity, and additional species of bacteria were sometimes used as a comparator for ADS024.These included ADS012, an independently isolated B. velezensis strain, and the unrelated anti-inflammatory E. coli Nissle bacteria.Due to limits of study sizes, not all studies used all possible groups.Another limitation is that we cannot yet conclude if increased LPA3 activity through agonism by ADS024 is critical for efficacy, or if instead, it is the subsequent downregulation of the receptor that is important, as has been shown with fingolimod, another agonist of some members of the EDG family of receptors.Fingolimod agonizes the S1P receptors S1PR1, S1PR3, S1PR4 and S1PR5 but has been shown to work by downregulating the receptors, and thus fingolimod is often called a modulator rather than an agonist.Future studies concerning the changes in cell-surface expression of LPA3 on immune and non-immune cells are needed to better understand the exact mechanism by which ADS024 impacts LPA3.Furthermore, while the endogenous agonists collectively called LPA are known players in inflammation, we have only touched upon the impact of ADS024 agonism of LPA3 on many inflammatory processes.ADS024 was previously shown to reduce IL-6 and TNF-α levels in a GI inflammatory model (Chesnel and Acton, 2022;Irwin et al., 2024) and here we have shown impact on immune cells in the MOG-EAE model, but future studies are needed to evaluate the impact of ADS024 on downstream effects of LPA3 agonism such as activation of the Nrf2 pathway and its role in regulation of cytokine production and oxidative stress control enzymes.Finally, work is underway to isolate and identify the active LPA3 agonist to determine if, when given alone, it can produce similar results in the animal models seen here and thereby potentially constitute a therapy for human neuroinflammatory diseases.

Conclusions
Bacillus velezensis ADS024 produces at least one potent agonist of LPA3 and is efficacious in a diverse spectrum of neuroinflammatory diseases when given orally, suggesting it targets a common underlying pathology.An extract of ADS024, ADS024-IPA, was shown to promote signaling of LPA3 through G-proteins, β-arrestin and 14-3-3 proteins.The LPA3 agonist in ADS024-IPA was found to be highly specific for LPA3 versus other LPA receptors suggesting it may have a different chemical structure than known LPA molecules.When given orally to animal models of diverse neuroinflammatory diseases including PD, HD, MS, ALS and CIPN, ADS024 improved outcomes regardless of the target and cause of neuronal damage which supports a role for ADS024 in modifying the response to the insult, rather than the cause of the injury itself and this appeared to be at least partially due to LPA3 agonist activity.Furthermore, ADS024 altered LPA3 expression on circulating immune cells suggesting an in vivo impact on the receptor beyond the GI tract.This data supports a link between the ability of a bacterium to produce a GPCR agonist and an effect on the immune system and nervous system of an animal via its GI tract.Altogether, these results suggest that ADS024 may be beneficial as a potential prophylaxis or treatment for a range of neuroinflammatory diseases.

Funding sources
This work was funded by Adiso Therapeutics, Concord, MA USA.

Fig. 2 .
Fig. 2. Effect of LPA1/3 inhibitor Ki16425 on LPA3 agonism by oleoyl-LPA and ADS024-IPA.Calcium signaling was measured in LPA3 stably transfected CHO-K1 cells using the PathHunter system (see Materials and Methods).Oleoyl-LPA dose-response curves (A) and ADS024-IPA dose-response curves (B) were run in the absence or presence of 10 µM Ki16425.Efficacy is defined as % Activity test sample = 100% x (mean RFU of test sample − mean RFU of DMSO) / (mean MAX RFU oleoyl LPA − mean RFU of DMSO).All doses were run in duplicate and where it appears there may be only one replicate, there are two superimposed duplicates.The study was repeated in β-arrestin mode with similar results.LPA: Lysophosphatidic Acid; IPA: isopropyl alcohol extract; RFU: relative fluorescence units.

Fig. 4 .
Fig. 4. Impact of ADS024 on outcomes in Parkinson's models.The effects of ADS024 on PD outcomes were measured in an MPTP zebrafish model (A,B), an MPTP mouse model (C,D) and a pink1 -/-α-synuclein model (E).In A, B: 4-day-old zebrafish larvae were treated for 24 hrs simultaneously with no additive (Naïve, n = 35),

Fig. 6 .
Fig. 6.Efficacy of ADS024 in MOG-EAE mouse MS disease model and impact on immune cells.Measurement of clinical scores (A,B), immune cell types (C-F), neutrophil subgroups (G-J), and LPA3 protein expression (K-N).All groups were stimulated with MOG except for the Naïve group which was mock treated.Treatments included Fingolimod (D0-D23, 0.6 mg/ml, 3 mg/kg, 5 ml/kg, PO QD, n = 8, black circles − all clinical scores were 0 in this study), ADS024 (Day 0 to Day 23, 1x10 9 CFU PO BID, 5 ml/kg, n = 12, aqua squares), and PBS (vehicle, Day 0 to Day 23, PO BID, 5 ml/kg, n = 12, brown circles).A: Clinical score: 0 = no clinical evidence of disease, 1 = limp tail/weakness of tail/paralysis of tail, 2 = abnormal gait, 3 = severe hind-limb weakness/partial hind-limb paralysis, and 4 = complete hind limb paralysis.Data was analyzed using one-way ANOVA followed by Dunnett's test and plotted as mean + S.E.M, *p < 0.05.B) Cumulative clinical scores starting at first evidence of disease (from Day 12 onward) were plotted as mean ± standard deviation where each point corresponds to an individual animal.A shorter repeat study confirmed the early attenuation by ADS024 at days 11-13.C-N) In additional studies, flow cytometry was used to evaluate circulating immune cells in blood on Day 7 after disease initiation.LPA3 surface expression was measured using an antibody to mouse LPA3.Neutrophils were defined as CD45+Ly6G+ within the granulocyte population as indicated in Materials and Methods.Percentages of naïve neutrophils (CD62L+CXCR4-) and aged neutrophils (CD62L-CXCR4+) were determined within the total neutrophil population.Immune cell data were analyzed by one-way ANOVA followed by a Sidak's multiple comparisons test between the vehicle-treated model versus the other groups as indicated in Materials and Methods.Data are displayed as mean and error bars indicate standard deviation.Significance is determined by adjusted p-values defined as p(adj) < 0.05(*), p(adj) < 0.01 (**), p(adj) < 0.001 (***) and p(adj) < 0.0001 (****) and ns indicates a comparison was performed but was not significant.The study was repeated with similar results except LPA3 expression on monocytes was not significant (p = 0.41) in the repeated study but trended in the same direction.

Fig. 9 .
Fig. 9. Efficacy of ADS024 in a cisplatin-induced peripheral neuroinflammatory model.Mechanical allodynia (representing neuropathic pain) was induced by two rounds of cisplatin treatment (2.3 mg/kg IP on Days 0-4 and Days 10-14) in mice.All animals in the groups were injected with Cisplatin (n = 43) except for Naive controls which were injected with saline (n = 12).Cisplatin-treated animals were dosed PO with ADS024 at 1x10 9 CFU BID (D-3 to D18; n = 11) or with vehicle (PBS; n = 12); the positive control consisted of IP 150 mg/kg Gabapentin one hour before the assay (n = 7).The development of neuropathic pain was confirmed by measuring mechanical allodynia (Von Frey test) on day − 1 (baseline) and on Days 15 and 18 at 2 hrs after oral dosing of test agents.Von Frey Score was recorded as the largest fiber required to initiate paw withdrawal.Higher scores indicate a lower touch sensitivity to a negative stimulus.For a positive control agent, mice were dosed acutely with Gabapentin (150 mg/kg, IP) one hour before the assay.Data were analyzed by one-way ANOVA followed by a Sidak's multiple comparisons test between all groups.Data are displayed as mean and error bars indicate standard deviation.Significance is determined by adjusted p-values defined as p(adj) < 0.05(*), p (adj) < 0.01 (**), p(adj) < 0.001 (***) and p(adj) < 0.0001(****), ns indicates the comparison was performed but was not significant.A similar study using taxol as the chemotherapy confirmed efficacy.