First Trimester Human Umbilical Cord Perivascular cells (HUCPVC) Modulate the Kynurenine Pathway and Glutamate Neurotransmission in an LPS-induced Mouse Model of Neuroinflammation

First Trimester Human Umbilical Cord Perivascular cells (HUCPVC) Modulate the Kynurenine Pathway and Glutamate Neurotransmission in an LPS-induced Mouse Model of Neuroinflammation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First Trimester Human Umbilical Cord Perivascular cells (HUCPVC) Modulate the Kynurenine Pathway and Glutamate Neurotransmission in an LPS-induced Mouse Model of Neuroinflammation Fyyaz Siddiqui, Denis Gallagher, Hannah Shuster-Hyman, Lianet Lopez, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2238679/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The Kynurenine Pathway (KP) of tryptophan degradation and glutamate toxicity is implicated in several neurological disorders, including depression. Although mesenchymal stromal cells (MSC)-mediated immunomodulation and neuroprotection have been studied in many of these disorders, their potential to influence KP and the glutamatergic system has not yet been investigated. Hence, this study sought to investigate the effect of HUCPVC, a rich and potent source of MSC, on Lipopolysaccharide (LPS)-activated KP metabolites, KP enzymes, and key components of glutamate neurotransmission. Methods The immunomodulatory effect of peripherally administered HUCPVC on the expression profile of kynurenine pathway enzymes and metabolites was assessed in the plasma and brain of mice treated with LPS. An assessment of the glutamatergic system, including selected receptors, transporters and proteins was also conducted. Results HUCPVC were found to modulate LPS-induced activation of KP enzymes and metabolites in the brain associated with neurotoxicity. Moreover, the reduced expression of the glutamatergic components due to LPS was also found to be significantly improved by HUCPVC. Conclusions The immunomodulatory properties of HUCPVC appear to confer neuroprotection, at least in part, through their ability to modulate the KP in the brain. This KP modulation enhances neuroprotective regulators and downregulates neurotoxic consequences, including glutamate neurotoxicity, which is associated with neuroinflammation and depressive behavior. Kynurenine pathway neuroinflammation mesenchymal stromal cells HUCPVC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Mounting evidence has supported a direct and positive relationship between immune-activated proinflammatory cytokines and psychiatric disorders [ 1 – 3 ]. Pro-inflammatory cytokines elicited during systemic infection, cancer, and autoimmune diseases have been shown to play a significant role in the manifestations and propagation of a broad spectrum of depressive symptoms [ 4 , 5 ]. Signaling of these peripherally originated proinflammatory cytokines to the brain through multiple pathways is implicated in neuroinflammation, perturbation of neurotransmitters’ metabolism, transport and function, and induction of depressive symptoms [ 6 – 9 ]. One such pathway that is activated by the immune response is the kynurenine pathway, which has been shown to play a key role in the activation of the central nervous system (CNS) and the pathogenesis of neuroinflammation and depression [ 10 , 11 ]. The kynurenine pathway (KP) (Fig. 1 A) claims the major share of tryptophan metabolism for the production of kynurenine and subsequent metabolites, while a meager amount of ingested tryptophan is converted to serotonin by the methoxyindole pathway [ 12 , 13 ]. When induced by inflammatory signals, the extrahepatically expressed enzyme, indoleamine 2,3- dioxygenase (IDO), triggers activation of the kynurenine pathway by oxidatively breaking down tryptophan to kynurenine. IDO expressed in the brain contributes to centrally produced kynurenine which is further augmented by peripheral kynurenine crossing the blood-brain barrier [ 14 ]. Downstream catabolism of kynurenine takes two distinct routes leading to the production of excitatory and anti-excitatory metabolites- notably, quinolinic acid (QUIN) and Kynurenic acid (KYNA) which are a functional agonist and antagonist to the glutamate receptor- N-methyl-D-aspartate (NMDAR), respectively [ 15 – 17 ]. The association of these compounds with the glutamate receptor and extracellular glutamate availability, renders neuroprotective or neurotoxic character to these distinct routes of KP and their constitutive metabolites. Dysregulation of KP resulting in a perturbation in the synthesis of neuroactive metabolites, notably KYNA and QUIN, have been associated with a plethora of neurodegenerative diseases and psychiatric disorders, including depressive illness [ 18 ]. Pharmacological interventions include the development of analogs of KYNA and inhibitors of neurotoxic enzymes that lead to the production of QUIN [ 18 – 20 ]. Though Mesenchymal Stromal Cells (MSC), owing to their capabilities to regulate the immune and inflammatory response, have been an attractive therapeutic candidate for treating various diseases with inflammatory components [ 21 ], their therapeutic potential has so far not been tested for modulation of the immune-activated kynurenine pathway. We have previously demonstrated the efficacy of MSC in mitigating neuroinflammation and depressive behavior in both stress-induced, as well as LPS-induced preclinical models of depression and, have also outlined a phagocytosis-driven immunomodulation mechanism [ 22 , 23 ]. In the current study, we aim to investigate the immunomodulatory and neuroprotective capabilities of a young source of MSC, HUCPVC’s, through a potential impact on kynurenine pathway modulation in an LPS-based mouse model of neuroinflammation; which to our knowledge has never been explored. Glutamate excitotoxicity is implicated in many neurodegenerative diseases and psychiatric disorders [ 24 , 25 ]. By their direct action on the glutamate receptors, QUIN and KYNA not only influence excitatory neurotransmission but also play an active role in the uptake and release of glutamate [ 26 , 27 ]. Moreover, glutamate-induced excitotoxicity is directly related to a compromised glutamate transport system, that is associated with neuropathological conditions, including depression [ 28 ]. Thus, this study further investigates the possible role of MSC in the modulation of key glutamatergic neurotransmission components, including expression profiles of glutamate receptors (NMDA), glutamate transporters, and synaptosomal proteins. Materials And Methods Animal Treatment & Cells Previously established and characterized, pathogen-free lines of first trimester HUCPVC were used for this study, with ethics approval by the University of Toronto REB (#28889) and by an independent accredited ethics board (VERITAS, #2576) [ 22 ]. 7–8-week-old male C57BL/6J mice were obtained from Charles River (Laval, Quebec). After arrival, mice were group-housed in standard shoebox cages, habituated for a week, and allowed ad libitum food and water access. General health was monitored daily by veterinary technicians or research staff. MSC lines from human first-trimester umbilical cords were established and maintained as described previously [ 29 ]. As a control, human fibroblasts (HS-68 foreskin-derived) were purchased from ATCC (Manassas, VA). All cells were grown using minimum essential media with alpha modification, 10% fetal bovine serum, and 1% penicillin/streptomycin purchased from Gibco (Gaithersburg, MD). A fresh Solution of LPS (serotype O111:B4, New England Biolabs (Whitby, ON) was prepared on the day of injections by dissolving the compound in a sterile endotoxin-free isotonic saline. LPS (0.83mg/kg) was administered intraperitoneally (i.p) in the LPS group. This dose is known to induce a full spectrum of the acute sickness response [ 30 ] and robustly increase IDO activity in the brain – one of the mechanisms associated with LPS-induced depressive behavior in mice [ 31 ]. 1 x 10 6 HUCPVC or HS68 cells resuspended in 200µl of saline were injected intravenously (i.v) simultaneously with LPS in the LPS + HUCPVC or LPS_HS68 groups, respectively. Untreated animals were included as a control group. RNA extraction, Reverse Transcription & Real-time qPCR Total RNA from whole-brain samples was extracted using Qiagen’s RNeasy Mini Kit (catalog# 74104), according to the manufacturer’s protocol. The eluted RNA was quantified using Nanodrop (NanoVue, GE). 1µg of total RNA was used for reverse transcriptase reactions that were carried out in a Verity 96-well Thermal cycler (Applied Biosystems, model# 9902), using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, catalog# 4368814), according to the manufacturer’s protocol. Real-time qPCR was performed for 40 cycles on a QuantStudio5 thermocycler (Applied Biosystems). Primers used were Gapdh (NM_008084), Ido1 (NM_008324), Kat2 (NM_011834), Kmo (NM_133809), Haao (NM_025325), Qprt (NM_133686.1), NR1 (NM_008169.3). NR2A (NM_008170.4), NR2B (NM_008171.4). GAPDH was used as an endogenous reference gene for the normalization of mRNA levels and relative quantification of gene expression. Fold change from the qPCR data was measured by the delta-delta Ct method. Immunohistochemistry, Image analysis & Quantification Mice were euthanized by cervical dislocation and trans-cardially perfused with cold PBS followed by 4% paraformaldehyde (PFA). Brains were harvested and fixed in 4% PFA for 24 h at 4°C. Cryopreservation was done by immersing the harvested brains in 30% sucrose for 1–2 days, followed by snap-freezing in optimum cutting temperature (OCT) formulation (Electron Microscopy Sciences). Serial coronal tissue sections were obtained at a 16–18µm thickness on a cryostat at TCP (the Centre for Phenogenomics, Toronto). For each animal (n = 3 animals per group) 6–8 sections were processed for immunohistochemistry. After briefly rinsing with PBS, the sections were fixed in 4% PFA for 15 min and blocked with 5% Normal Goat or Donkey serum (Sigma) supplemented with 1% BSA (Sigma), and 0.4% Triton-X-100 (Fisher) in PBS, for 1–2 h at room temperature. Tissue sections were then incubated overnight at 4°C with the primary antibodies in 50% diluted blocking solution, followed by 1 h incubation with suitable fluorescently labeled secondary antibodies (Alexa Fluor1:1000) at room temperature. For immunostaining of nuclei, the sections were incubated with DAPI for 5 min before mounting the slides with coverslips using mounting media (Abcam). Primary antibodies used were: Mouse monoclonal anti-GFAP antibody [2A5] (1:200, Abcam, catalog# ab4648); rabbit monoclonal EAAT2 [E3P5K] (1:50, Cell Signalling Technology, catalog# 20848); anti-IBA1 (1:1000, Wako Chemicals, Richmond, VA). Confocal microscopy was performed using a Leica TCS SP8 confocal microscope at the advanced optical microscopy facility (AOMF), University Health Network, Toronto. The confocal microscope was equipped with fully spectral 400 to 700nm filters and HyD high-sensitivity detectors. Images were processed using Leica LAS-X software. 6–9 representative images were taken at x20 magnification spanning the cerebral cortex and hippocampus. Image thresholds and tissue surface area marked by positive staining were analyzed using ImageJ software. For quantification of EAAT2 and GFAP expression, the mean percent area of EAAT2 positive staining was normalized with that of GFAP for a given field of view for all images across different experimental groups. Western Blotting Whole-brain homogenization Brains were harvested and immediately snap-frozen in liquid nitrogen and stored at -80°C. Brain tissues were later placed in RIPA buffer (Sigma, catalog # 0278) in 2ml silica tubes prefilled with 3mm zirconium beads and subjected to high-velocity impact homogenization using the BeadBug 6, Six Position Homogenizer (Benchmark Scientific, catalog # D1036). Homogenization was done at 3000rpm for 30 seconds followed by 30 seconds on ice-, repeating the cycle 3 times. The homogenate was then centrifuged at 12000rpm for 15 min at 4°C and the supernatant was collected in the pre-chilled tubes. Synaptosome protein fractionation Synaptic protein extraction was performed following the protocol from Thermo Scientific. Briefly, freshly harvested whole brains, excluding the cerebellum (200-400mg), were homogenized in 10 volumes of the Syn-PER synaptic protein extraction reagent (Thermo Fisher; catalog# 87793) using a Dounce tissue grinder, performing 10–12 up-and-down strokes. The homogenate was then centrifuged at 1200 g for 10 minutes to remove cell debris, and the supernatant was centrifuged at 15,000 g for 20 minutes. The pellets, containing synaptosomes, were gently resuspended in 1–2 ml of the Syn-PER reagent. The resulting whole-brain homogenate/synaptosomal protein lysate was assayed for protein concentration using a BCA protein assay kit (Thermo Scientific, catalog# 23225), and stored at − 80°C. These protein samples were placed in a reducing buffer containing β-mercaptoethanol and heated at 90°C for 10 min. Samples were then subjected to SDS–polyacrylamide gel electrophoresis in 4–12% Tris-Glycine 1mm precast gels (Invitrogen, catalog# XP04120BOX), and then transferred to polyvinylidene fluoride membranes using a semi-wet Mini Blot Module transfer unit (Life Technologies, catalog# B1000). The membranes were blocked in LiCor blocking buffer for 1 h at room temperature and probed with the primary antibodies in 0.1% Tween LiCor blocking buffer overnight at 4°C. The next day, the membranes were washed 3 times for 10 min each in 0.01% tween phosphate buffer solution before probing with an appropriate mix of IR-Dye-labeled Licor secondary antibodies in 0.1% Tween, 0.01% SDS LiCor blocking buffer for 1 h at room temperature. Washes, as described above, were repeated after incubation with secondary antibodies. Western blot images were obtained on a Licor Odyssey Imaging System. Relative quantification of the protein bands was assessed by densitometry analysis using ImageJ software. Primary antibodies used were: rabbit monoclonal EAAT2 [E3P5K] (1:1000, Cell Signalling Technology, catalog# 20848); rabbit monoclonal anti PSD95 antibody [EPR23124-118] (1:2000, Abcam, catalog# ab238135); mouse monoclonal anti NMDAR2B/NR2B (1:500, Invitrogen, catalog# MA-1-2014), rabbit monoclonal anti Drebrin antibody [EPR12634] (1:10,000, Abcam, catalog# ab178408) and rabbit monoclonal anti Synaptophysin antibody [YE269] (1:10,000, Abcam, catalog# ab32127). Extraction and Quantification of Brain and Plasma Samples - LCMS Analysis Sample extraction & preparation, LC-MS/MS analysis, and quantification were performed by the Analytical Facility for Bioactive Molecules (AFBM), Hospital for Sick Children, Toronto, Canada. All LC-MS/MS grade solvents were purchased from Caledon Laboratories Ltd (Georgetown, ON). Autosampler vials/glass inserts used in the sample extraction were purchased from Chromatographic Specialties Ltd (Brockville, ON). 150–180 mg of frozen brain tissues were weighed and transferred into Precellys homogenization tubes containing ceramic beads (Bertin Technologies, Rockville, Washington DC). The entire brain was processed for this assay which required two tubes per sample depending on the brain weight. Tissue samples were kept overnight in the − 80°C freezer until extraction. The following day, extraction solvent was added to each Precellys tube to achieve a target concentration of 150 mg/mL and homogenized using a Precellys 24 high-throughput homogenizer (Bertin Technologies). Plasma (100 µL) and brain samples (67 µL of the homogenized suspensions (corresponding to 10 mg tissues) were transferred into Eppendorf tubes containing 1 ml 90:10 acetonitrile (ACN):methanol (MeOH) alongside standards, quality control standards, and deuterated internal standards. Tubes were vortexed and then centrifuged at 20,000 g. Supernatants were transferred to a conical tube and taken to dryness under a gentle stream of nitrogen. Samples were reconstituted in 90/10 H 2 O/ACN + 0.1% formic acid and analyzed by LC/MS/MS. An Agilent 1200 UPLC system (Agilent Technologies, Santa Clara, CA, USA) fitted with a Sciex Q-Trap 5500 mass spectrometer (AB Sciex, Framingham, MA, USA) was used in Electron Spray Ionization (ESI) mode. Two methods were employed. Except for a few analytes, most analytes were quantified using a Kinetex PFP column (2.6 µm, 100Å, 50 x 3.0 mm; Phenomenex, Torrence, CA). A gradient mobile phase of 10 minutes at a flow rate of 0.4 ml/min was used for the elution of the biogenic amines with mobile phase A (MPA): 1% acetic acid in water and mobile phase B (MPB): 1% acetic acid in 1:1 MeOH:ACN. For the remainder of the analytes, an EZ Fast 4uaaa-MS column was used. A gradient mobile phase of 10 minutes at a flow rate of 0.4 ml/min was used with MPA (0.1% formic acid, 0.1% heptafluorobutyric acid in water) and MPB (0.1% formic acid in MeOH). Quantification was performed with Analyst 1.6.1 software (ABSciex: Framingham, Massachusetts, USA) by plotting the sample peak area ratios (Analyte peak area/Internal Standard peak area) of the biogenic amine standards against a standard curve generated from various standard concentrations from 0.05 ng to 100 ng, spiked with the same amount of internal standard used for the samples and extracted using the same conditions. Statistical Analysis All data were represented as mean ± SEM and analyzed using Ordinary one-way analysis of variance followed by post hoc pairwise Tukey’s multiple comparisons tests comparing all the experimental groups using GraphPad Prism (GraphPad Software, San Diego, CA). Results Peripheral HUCPVC infusion modulates LPS-induced activation of kynurenine pathway enzymes in the brain Proinflammatory cytokines mediate the induction of IDO which serves as a molecular switch for triggering the initiation of the KP [ 32 ]. Enzymatic activity and expression of IDO, which is also present in brain endothelial cells, perivascular macrophages, astrocytes, and microglia [ 33 ], have a direct influence on brain tryptophan metabolism [ 34 ]. Our previous study demonstrated significant mitigation of LPS-induced proinflammatory cytokines in the brain by peripherally administered HUCPVC in mice [ 23 ]. Thus, we sought to investigate the impact of the immunomodulatory potential of HUCPVC on the gene expression profile of Ido1 and the subsequent metabolic enzymes of the pathway in the LPS-activated CNS (Fig. 1 A). Our results indicate significant induction of Ido1 in the brain by LPS (p < 0.0001) and its equally significant downregulation back to control levels as a result of HUCPVC treatment (p < 0.0001) (Fig. 1 B). Ido -mediated synthesis of kynurenine is centrally placed in the KP as it is further favorably catabolized by the enzymes Kmo and Kat towards putative neurotoxic or neuroprotective branches, respectively [ 19 ]. In the LPS group, we recorded no significant changes in mRNA levels of Kat2 , when compared to control (p = 0.2949) or LPS + HUCPVC (p = 0.9846) groups (Fig. 1 C). Conversely, a significant LPS-induced upregulation of Kmo (p = 0.0020) was seen to be modulated back to near control levels by HUCPVC (p = 0.0176) (Fig. 1 D). The excitatory properties associated with the ability of quinolinic acid to stimulate NMDA receptors directly and selectively are well known [ 17 ]. This neurotoxic compound is synthesized by the enzymatic activity of Haao. We observed that LPS significantly increased the expression of Haao (p = 0.0002) and that the treatment of LPS combined with HUCPVC helped downregulate its level significantly (p = 0.0001) (Fig. 1 E). Accumulation of toxic concentrations of quinolinic acid also depends on the rate of metabolism of quinolinic acid to NAD + by the enzyme Qprt. Our results indicate that the immune response to LPS resulted in a significant lowering of Qprt expression (p = 0.0033) when compared to the control group and LPS + HUCPVC effected a significant upregulation of the enzyme transcript (p < 0.0001) (Fig. 1 F). This data suggests that the influence of HUCPVC treatment on Qprt expression may be instrumental in regulating the synthesis of KP metabolites. Influence of HUCPVC on LPS-activated kynurenine pathway metabolites and serotonin in the brain and plasma: The induction of KP metabolites following 24h of LPS treatment and intervention by HUCPVC were assessed by LC-MS/MS. The standard curves were linear over the concentration ranges. The calibration curves were as follows: y = 0.166 x + 0.166, R 2 = 0.9922 for tryptophan; y = 0.349x + 0.0344, R 2 = 0.9860 for kynurenine; y = 0.0943x + 0.000927, R 2 = 0.9934 for kynurenic acid; y = 0.00715x + 0.0175, R 2 = 0.9857 for quinolinic acid; y = 0.196x + 0.00148, R 2 = 0.9930 for serotonin; y = 1.12e-005x + -5.35e-005, R 2 = 0.9989 for glutamine and y = 3.67e-005x + -0.00039, R 2 = 0.9973 for glutamate. The representative chromatograms of these analytes are shown in Fig. 2 A-G. As detailed in Table 1 , LPS significantly upregulated brain levels of tryptophan (p = 0.0197), kynurenine (p < 0.0001), kynurenine:tryptophan (p < 0.0001) and quinolinic acid (p = 0.0003), when compared to control. No significant difference was found in the levels of kynurenic acid (p = 0.9503) or serotonin (p = 1417). However, the neurotoxic index, quinolinic acid:kynurenic acid was found to be significantly increased by LPS (p = 0.0117), when compared to control. The combination of LPS with HUCPVC significantly modulated the brain levels of tryptophan (p = 0.0217), kynurenine (p = 0.0390), kynurenine:tryptophan (p = 0.0401) and quinolinic acid (p = 0.0042) when compared to LPS alone. No significant difference was found in the levels of kynurenic acid (p = 0.9598) or serotonin (p = 0.7892). However, quinolinic acid:kynurenic acid was found to be significantly increased in this condition (p = 0.0149). Since brain kynurenine levels are directly impacted by peripheral circulation [ 35 ], plasma tryptophan metabolism was also assessed (Table 1 ). No significant effect of LPS was found in the plasma levels of tryptophan (p > 0.9999), kynurenine (p = 0.2420), quinolinic acid (p = 0.9409), kynurenic acid (p = 0.6114), quinolinic acid:kynurenic acid (p = 0.7065) or serotonin (p = 0.9999) when compared to control. However, the IDO activation index, Kynurenin:tryptophan was found to be significantly increased by LPS (p = 0.0320) when compared to control. No significant modulation was noted by HUCPVC on plasma levels of tryptophan (p = 0.9930), kynurenine (p > 0.9999), kynurenine:tryptophan (p = 0.8341), kynurenic acid (p > 0.9999), quinolinic acid (p = 0.4393), quinolinic acid:kynurenic acid (p = 0.7698) or serotonin (p = 0.9978) when compared to LPS-treated animals. The immunomodulatory effect of a control non-MSC cell type was tested by using human foreskin-derived fibroblasts (HS68) in lieu of HUCPVC, and their influence on LPS-activated KP metabolites was assessed. There was no significant effect on brain levels of tryptophan (p = 0.2513), kynurenine (p = 0.2901), kynurenic acid (p > 0.9999), quinolinic acid (p = 0.9980), quinolinic acid:kynurenic acid (p = 0.9998) or serotonin (p = 0.9984) when compared to LPS-treated animals. However, a significant effect was recorded for kynurenine:tryptophan (p = 0.0035). Moreover, HS68 was found to have no significant effect on the plasma levels of tryptophan (p = 0.9991), kynurenine (p = 0.7195), kynurenine:tryptophan (p = 0.4029), kynurenic acid (p = 0.9485), quinolinic acid (p = 0.9752), quinolinic acid:kynurenic acid (p = 0.8721) or serotonin (p = 0.8125). These experimental read-outs support the possibility that peripherally infused HUCPVC have an influence on the KP metabolism in the brain. Table 1 TRY, tryptophan; KYN, kynurenine; KYNA, kynurenic acid; QUIN, quinolinic acid; 5-HT, serotonin; Glu, glutamate; Gln, glutamine, LPS, lipopolysaccharide; HUCPVC, human umbilical cord perivascular cells; HS68, human foreskin fibroblast cells. Data represents mean (± SEM). n = 5–8 mice per group, (except HS68, n = 3). a Significant effect of LPS (versus Control), b significant effect of HUCPVC (versus LPS), c significant effect of HS68 (versus LPS), Ordinary One-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. Metabolite Control LPS LPS + HUCPVC LPS + HS68 Brain TRY (nM) 172 (8.09) 209 (7.28) a* 170 (7.16) b* 236 (17.1) KYN (nM) 12.8 (0.96) 61.1 (3.46) a**** 43 (5.97) b* 47.9 (9.93) KYN:TRY 0.077 (0.009) 0.338 (0.01) a**** 0.238 (0.025) b* 0.173 (0.079) c** KYNA (nM) 1.257 (0.155) 1.388 (0.060) 1.513 (0.237) 1.39 (0.206) QUIN (nM) 3044 (604) 7892 (362) a*** 4086 (651) b** 8090 (1311) QUIN:KYNA 3081 (511) 5717 (330) a* 3080 (676) b* 5790 (229) 5-HT (nM) 385 (40.9) 520 (50.6) 464 (42.6) 507 (3.33) Glu (µM) 909 (66) 893 (36) 893 (43) 1103 (281) Gln (µM) 1853 (179.5) 2025 (162.1) 2012 (138.4) 2374 (583) Plasma TRY (µM) 0.758 (0.030) 0.756 (0.033) 0.741 (0.046) 0.746 (0.103) KYN (µM) 0.824 (0.061) 1.155 (0.168) 1.155 (0.133) 0.915 (0.040) KYN:TRY 1.106 (0.103) 1.71 (0.212) a* 1.54 (0.104) 1.28 (0.208) KYNA (µM) 0.159 (0.005) 0.14 (0.014) 0.141 (0.010) 0.151 (0.003) QUIN (µM) 4.389 (0.392) 4.751 (0.507) 3.808 (0.465) 4.403 (0.336) QUIN:KYNA 27.9 (3.16) 44.3 (17.7) 30.1 (4.38) 29.2 (2.75) 5-HT (µM) 3.19 (0.411) 3.24 (0.525) 3.12 (0.494) 2.47 (0.070) Glu (µM) 11.1 (1.82) 12 (2.19) 9.31 (1.88) 10.1 (3.12) Gln (µM) 170.1 (26.6) 169.5 (24.4) 135.1 (18.1) 176.5 (20.5) Assessment of Kynurenine pathway metabolites, serotonin, glutamate and glutamine levels by LC-MS after 24h LPS treatment along with either HUCPVCs or HS68. Impact of peripherally administered HUCPVC on LPS-induced neuroinflammation and glutamatergic neurotransmission Microglial activation by LPS and modulation by HUCPVC Corroborating our previous study where we reported significant mitigation of LPS-induced proinflammatory cytokines in the brain by peripherally administered HUCPVC [ 23 ], in this study we demonstrate the potential of HUCPVC potential to modulate neuroinflammation by measuring LPS-induced microglial activation in the hippocampus (Fig. 3 A,B,C ) and cortex (Fig. 3 D,E,F), LPS significantly increased activation of microglia as assessed by increased positive staining for ionized calcium-binding adapter molecule 1 (IBA1) in the dentate gyrus region of the hippocampus (p = 0.0028) (Fig. 3 G) as well as the cortex (p = 0.0003) (Fig. 3 H). Significant modulation of this induction by HUCPVC was seen both in the dentate gyrus (p = 0.0018) (Fig. 3 G) as well as the cortex (p = 0.0003) (Fig. 3 H). The regulation of glutamate transporters & receptors by HUCPVC infusion Glutamate-mediated excitotoxicity is linked to various neurodegenerative diseases and psychiatric disorders [ 36 , 37 ]. Since the synaptic levels of glutamate are predominantly maintained by excitatory amino acid transporters (EAATs), we tested the expression of EAAT2 in the brain in response to peripheral immune activation and HUCPVC infusion. In this experiment, EAAT2 was co-stained with glial fibrillary acidic protein (GFAP), a marker for astrocytes, where it is primarily localized [ 38 ]. Overall EAAT2 expression was assessed by normalizing its positive signal with that of GFAP in the cerebral cortex and dentate gyrus (DG) region of the hippocampus. The results revealed that LPS significantly diminished EAAT2 expression levels in the cerebral cortex (p = 0.0319, Fig. 4 B, G) and hippocampus (p = 0.0182, Fig. 4 E, H). Conversely, LPS in combination with HUCPVC significantly reinstated the transporter’s expression level both in the cerebral cortex (p = 0.0102, Fig. 4 C, G) and hippocampus (p = 0.0180, Fig. 4 F, H). This differential EAAT2 protein expression in control, LPS and LPS + HUCPVC-treated animals was also demonstrated by Western blot. We observed an obvious loss of EAAT2 immunoreactivity due to LPS, which was regained close to the control level in the HUCPVC-treated group (Fig. 4 I). The densitometric data analysis (Fig. 4 J) confirmed significant downregulation of EAAT2 expression by LPS (p = 0.0010) and significant rescue by HUCPVC treatment (p = 0.0034). NMDAR is one of the prominent glutamate receptors that mediate the activity of glutamate and 2 main subunits, NR1 and NR2A/B, are obligatory for the receptor’s activity and signaling [ 39 ]. We tested the transcript levels of these NMDAR subunits in the mRNA isolated from whole brains of the control, LPS, and LPS + HUCPVC groups (Fig. 5 A-C). LPS was found to significantly upregulate the transcript expression of NR2A (p < 0.0001) and NR2B (P < 0.0001). Conversely, HUCPVC significantly downregulated NR2A (P = 0.0006) and NR2B (p < 0.0001) transcripts. However, no significant change was observed in the NR1 gene expression in both LPS (p = 0.4128) and LPS + HUCPVC (p = 0.3258) groups, when compared to control. Extended role of HUCPVC in regulating synaptosomal proteins linked to glutamate trafficking & receptor activity The synaptosomal fraction is enriched with several proteins that impact glutamate receptor (NMDAR) activity and signaling. By Western blot, we tested some of these proteins such as NMDAR subunit- NR2B, postsynaptic density protein (PSD95), synaptophysin, and drebrin which are associated with glutamate trafficking and NMDAR function [ 40 , 41 , 42 ]. A significant increase in NR2B expression by LPS (p = 0.0386) was observed which was equally counteracted by HUCPVC intervention (p = 0.0299) (Fig. 6 A, B). The expression level of drebrin was found to be significantly downregulated by LPS (p = 0.0013) and rescued by HUCPVC (p = 0.0386) (Fig. 6 C, D). However, no change in the expressions of either PSD95 (Fig. 6 C, D) or synaptophysin ( Fig. 6 E, F ) was found to be induced by LPS alone or in combination with HUCPVC. Discussion Exploiting the well-known immunomodulatory potential of MSC, this study is the first, to our knowledge, to examine the influence of intravenously infused MSC on the activated kynurenine pathway (KP) and glutamate neurotransmission. Here, we report that peripherally administered HUCPVC regulate KP enzymes and metabolites in the LPS-activated CNS. Furthermore, these MSC were also found to exert a modulatory effect on the expression profile of glutamate receptor subunits and glutamate transporters. Thus, this study lends a novel approach to target aberrant signaling of KP and the subsequent glutamate excitotoxicity that are known to have diverse neuropathological consequences. Inflammation-associated upregulation and activation of IDO (product of ido1 gene) in the brain is a critical step in the break down of tryptophan to kynurenine and initiation of the KP (Fig. 1 A) [ 11 , 43 , 44 ]. Moreover, IDO activation is shown to be crucial for depressive-like behavior in mice treated with LPS for 24h [ 11 , 32 ]. We have previously reported that HUCPVC modulate neuroinflammation and depressive behavior after 24h of LPS injection in mice [ 23 ]. In this study, we report that HUCPVC regulate IDO at the transcriptional level and the enzyme’s activation in the brain, as assessed by kynurenine:tryptophan ratio. This finding informs us of the possibility that the modulation of inflammation-associated depressive behavior by HUCPVC could be due to their ability to influence the catalytic function of IDO. Kynurenine synthesized by IDO can be a favorable substrate for either astrocytic enzyme KAT leading to the production of neuroprotective KYNA or microglial enzymes KMO and HAAO leading to the production of neurotoxic QUIN. Consistent with other studies [ 45 , 46 ], we recorded an LPS-induced aberration in the brain mRNA levels of Kmo, Haao and Kat2 , which is a predominant isomer of the KAT enzyme responsible for KYNA production [ 47 ]. Since the inadequate metabolism of QUIN by the enzyme QPRT contributes to increased neurotoxicity [ 48 ], we also tested the brain mRNA level of the enzyme, which was sharply downregulated by LPS. Thus, the recovery of an LPS-activated imbalance of the KP enzymes by HUCPVC treatment indicates the capability of MSC to influence the KP enzymatic machinery of glia governing the central production of KP metabolites. KP metabolite levels in the brain have been intensely interrogated owing to their strong affiliation with many CNS disorders [ 1 , 49 ]. In the current study, we evaluate the downstream immunomodulatory effect of intravenously administered HUCPVC on the neurotoxicity index, as assessed by the brain QUIN/KYNA ratio. Since the majority of brain kynurenine is peripherally derived during inflammation [ 13 ], plasma levels of the KP metabolites were also evaluated. Furthermore, to delineate the potential MSC-associated immunomodulatory effect, human foreskin-derived fibroblast cells (HS68), which have a relatively low level of immunomodulatory potential compared to MSC [ 50 , 51 ], were independently injected into a group of animals. Our results indicate that compared to the brain, IDO activation in the plasma was not sufficient to increase the flux of circulating kynurenine. We hypothesize that this mild increase in kynurenine:tryptophan ratio, as shown in this study and by others [ 52 ], could merely be the statistical outcome of non-significant changes in tryptophan or kynurenine levels, which likely holds little metabolic or clinical significance. This lack of increase in plasma kynurenine may be due to its rapid clearance by the kidney and excretion of its metabolites in urine [ 53 , 54 ]. Sufficient IDO-induced tryptophan oxidation is required to exceed the effect of renal processing and result in appreciable levels of plasma kynurenine. Moreover, kynurenine being a substrate to KMO, the inflammation-induced expression and/or activity of KMO can counter the effect of IDO. In addition, since circulating proinflammatory cytokines are shown to peak within 1–6 hours of LPS treatment [ 42 , 55 ], arguably, these early timepoints could correspond to a relatively higher plasma IDO activity, as shown by Wirthgen et. al [ 56 ]. Thus, the low plasma levels of other KP metabolites in the current study could be the downstream effect of this lack of increase in kynurenine. Conversely, in the brain, we noted significant aberration of KP metabolite levels due to LPS. Significantly altered KP metabolites in the brain due to LPS were rescued back to basal levels by HUCPVC treatment; an effect not seen for the most part with fibroblast cells. These findings reveal significant and specific immunomodulatory effect of MSC on KP metabolism in response to LPS; thus maintaining homeostasis between two functionally contradictory branches of the pathway. Interestingly, and in accord with other studies [ 57 ], we found an increase in tryptophan by LPS. This increase, which could be due to LPS-induced lipolysis resulting in increased availability of albumin-free tryptophan to cross the blood-brain barrier (BBB) [ 58 ], was reversed by HUCPVC. Cytokine-stimulated IDO activation is also known to have a negative impact on serotonin (5HT) turnover [ 59 ]. Since the level of 5-hydroxy indole acetic acid (5HIAA), the metabolite of 5HT was not examined in this study, the unchanged 5HT level does not reflect its actual turnover and thus does not preclude the possibility of inflammation-afflicted modulation of serotonergic neurotransmission. The nexus between activated cerebral KP metabolism and NMDAR activation, leading to enhanced glutamate function, is associated with many neuropathological conditions [ 60 ]. The observed HUCPVC-induced modulation of KP metabolites in this study, notably that of QUIN, an endogenous NMDAR agonist, provoked further investigation into the expression profile of the obligatory subunits of NMDAR, that mediate the receptor’s activity, and whose upregulation is implicated in various brain pathologies, including inflammation-related depressive phenotype [ 61 ]. In this study, we report a significant increase in the transcript levels of the subunits, NR2A and NR2B , in response to LPS, which resonates with similar observations by others [ 62 , 63 ]. The demonstrated ability of HUCPVC to modulate these subunits may suggest a novel and relatively safer therapeutic alternative to the NMDAR subunit-targeting antidepressants that are shown to have psychoactive side effects and cardiovascular toxicity [ 64 ]. Glutamatergic circuitry is also negatively impacted by the perturbed transport mechanism responsible for the clearance of synaptic glutamate, leading to glutamate excitotoxicity [ 28 ]. Our study, for the first time, illustrates the potential of MSC, specifically HUCPVC, in regaining the LPS-induced decline in the astrocytic glutamate transporter EAAT2. Since the levels of glutamate (Glu) and glutamine (Gln) in the blood and brain also reflect glutamate excitotoxicity [ 65 ], we tested these metabolites in the plasma and whole-brain homogenate by LCMS. However, levels of Glu and Gln showed no significant changes between the control and LPS treatment groups. Since Glu and Gln cross BBB [ 66 ] and are expressed differentially in different brain regions [ 67 ], the lack of modulation in Glu and Gln levels reported in this study could be the consequence of the limitations of the methodology, which is unable to distinguish between the source of the metabolites in the plasma (central vs peripheral) or to delineate the region-specific expression of Glu and Gln in the brain. We further extended the scope of this study to investigate the immunomodulatory effect of systemically infused MSC on the neuroinflammation-associated synaptic imbalance implicated in neurodegenerative and psychiatric illnesses, including depression [ 68 ]. The effect of peripherally administered HUCPVC on LPS-induced dysregulation of synaptic markers such as drebrin, synaptophysin, PSD95, and NMDAR regulatory subunit NR2B, was tested in a synaptosomal isolate. Drebrin, an actin-binding protein in dendritic spines and one of the key players in the NMDAR-dependent synaptic neurotransmission, has been shown to be negatively regulated by the neuroinflammatory cascade associated with neurodegenerative diseases and psychiatric disorders [ 40 , 69 ]. The HUCPVC-mediated restoration of LPS-induced downregulation of drebrin expression suggests a novel potential application of MSC in restoration of synaptic loss. Furthermore, we report the upregulation of NR2B expression in response to LPS. This corroborates the previous findings that the proinflammatory cytokines in the brain facilitate the activation of the NMDAR subunit [ 70 ]. Pharmacological suppression of NR2B has been achieved using various selective antagonists of the NMDAR subunit in various brain pathologies [ 71 ]. However, these pharmacological agents cause undesirable side effects including neurotoxicity and hypertension [ 72 ]. Thus, our findings support the possibility that MSC may be a potentially safer and more efficient therapeutic alternative to target NR2B. There has been a considerable lack of understanding of the basis of systemically administered MSC’s ability to provide neuroprotection in many diseases and injury models. In our previous study using an LPS-induced mouse model of neuroinflammation and depression, we have demonstrated phagocytosis-driven immunomodulation by peripherally infused HUCPVCs [ 23 ]. The resulting systemic innate immune alteration from pro- to anti-inflammatory phenotype was plausibly correlated to the mitigation of LPS-induced neuroinflammatory response and depressive symptoms. In the current study, modulation of LPS-activated microglia via the kynurenine pathway in the CNS by HUCPVC may be another mechanism for peripheral immune modulation by the MSCs. Considering our previous findings that peripherally infused MSC fail to cross BBB [ 22 ], an alternate mechanism of MSC neuroprotective potential is its paracrine action, by which secreted factors are shown to mediate neuroprotection [ 73 – 75 ]. Contextually, studies showing the interplay between the kynurenine pathway and MSC support the involvement of IDO in the immunosuppressive effect of MSC [ 76 ]. Moreover, KYNA is also shown to regulate the expression of TNF-stimulated gene 6 (TSG-6), a paracrine factor, and promote TSG-6-mediated immunosuppressive and anti-inflammatory effects of MSC [ 77 ]. However, the modulation of a broad spectrum of activated kynurenine metabolites and the downstream signaling consequences vis-à-vis the glutamatergic system by MSC, as shown in this study, represents a novel finding. Conclusion This is the first study to examine and demonstrate the capability of intravenously injected MSC to regulate KP metabolites in a neuroinflammatory context, thus maintaining an optimal balance between ‘neuroprotective’ and ‘neurotoxic’ branches of the pathway. The novelty of this study is also marked by the demonstrated potential role of MSC in curbing glutamate excitotoxicity. In conclusion, our research findings further our knowledge to understand the mechanistic aspect of MSC neuroprotection, specifically through the KP. In addition, the specific anti-inflammatory properties we observed support the potential for use of MSC, particularly HUCPVC, as a therapeutic option for targeting inflammation-driven activated KP metabolites and glutamatergic systems linked to neurological diseases and affective disorders. Abbreviations ACN: Acetonitrile BBB: Blood-brain barrier CNS: Central nervous system DG: Dentate gyrus EAAT: Excitatory amino acid transporter GFAP: Glial fibrillary acidic protein Gln: Glutamine Glu: Glutamate 3HAA: 3-Hydroxy anthranilic acid 3HAAO: 3-Hydroxy anthranilic acid dioxygenase 5HIAA: 5-Hydroxy índole acetic acid 3HK: 3-Hydroxykynurenine HUCPVC: Human Umbilical Cord Perivascular cells IDO: Indoleamine 2,3- dioxygenase IP: Intraperitoneal IV: Intravenous KAT: Kynurenine aminotransferase KMO: Kynurenine monooxygenase KP: Kynurenine Pathway KYN: Kynurenine KYNA: Kynurenic acid LC-MS: Liquid Chromatography with tandem mass spectrometry LPS: Lipopolysaccharide MeOH: Methanol MSC: Mesenchymal stromal cell NAD: Nicotinamide adenine dinucleotide NMDAR: N-methyl-D-aspartate receptor OCT: Optimum cutting temperature PFA: Paraformaldehyde QPRT: Quinolinate phosphoribosyl transferase QUIN: Quinolinic acid 5HT: Serotonin TRY: Tryptophan TSG6: TNF-stimulated gene 6 Declarations Ethics approval All use and animal care were conducted and reported in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and approved by the Animal Care Committee of the University Health Network (AUP 5232.4, University of Toronto, Canada). Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interest The authors declare that they have no competing interests Funding This work was funded by the CReATe Fertility Centre Authors’ contributions Fyyaz Siddiqui: participated in study conception, animal handling, experimental design & execution, cell culture, imaging and data collection, data analysis, and manuscript preparation; Denis Gallagher: participated in study conception, supervision and manuscript preparation; Hannah Shuster-Hyman: participated in image analysis and reviewed the manuscript; Lianet Lopez: participated in cell culture and reviewed the manuscript; Andrée Gauthier-Fisher: participated in study design, data interpretation and preparation of the manuscript; Clifford Librach: participated in study design, data interpretation and preparation of the manuscript. Acknowledgments The authors wish to thank Fatima Sultani, Ashley St. Pierre, and the directors of the Analytical Facility for Bioactive Molecules, The Hospital for Sick Children, Toronto, Canada for assistance with the samples’ extraction/ preparation and LC-MS/MS analysis. The authors also acknowledge Andrea Archila, William Xiao and Roberto Lopez, Animal Resource Centre, University Health Network, Toronto, for their excellent technical support. References Haroon E, Raison CL, Miller AH. Psychoneuroimmunology meets neuropsychopharmacology: translational implications of the impact of inflammation on behavior. Neuropsychopharmacol. 2012; 37:137–162 Raison CL, Miller AH. Malaise, melancholia, and madness: the evolutionary legacy of an inflammatory bias. Brain Behav Immunol. 2013; 31:1–8 Raison CL, Capuron L, Miller AH. Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends Immunol . 2006; 27:24–31 Miller AH, Maletic V, Raison CL. 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Cell Death Differ. 2018; 25 (7):1209-1223. Additional Declarations No competing interests reported. Supplementary Files Westernblottinggels.tiff Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2238679","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":152228394,"identity":"97484669-d75f-4625-871a-f972d466713f","order_by":0,"name":"Fyyaz Siddiqui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBAC9gYQWQHlJTDIEdbCcwBEngERzCAtxkRqYWyDamEgSotEjuHHn/Ps8vnZzx988LDNgIG/vTuBkBZjCcltyZYze5KZDRKBWiTOnN2AV4u9RI6BhOG2AwYGB5LZJBLO/GEwkMjFrwVky4/EOQcM7M8/Zv+RcMaAKC1mEgcbgLZIJLMxJFQQo4XnWZllw7FkA4kbj40lgFp4CPqFhz15880fNXYG/P2JDz/+MDCQ42/vxa+FQSDDANUM/MpBgP/4A8KKRsEoGAWjYGQDAPr+QymwFR/cAAAAAElFTkSuQmCC","orcid":"","institution":"CReATe Fertility Centre","correspondingAuthor":true,"prefix":"","firstName":"Fyyaz","middleName":"","lastName":"Siddiqui","suffix":""},{"id":152228395,"identity":"e1d2c17f-77bf-4ad7-acca-0970534dc625","order_by":1,"name":"Denis Gallagher","email":"","orcid":"","institution":"CReATe Fertility Centre","correspondingAuthor":false,"prefix":"","firstName":"Denis","middleName":"","lastName":"Gallagher","suffix":""},{"id":152228396,"identity":"5a4cf7db-2a25-4fa4-9a56-ca588ba86227","order_by":2,"name":"Hannah Shuster-Hyman","email":"","orcid":"","institution":"CReATe Fertility Centre","correspondingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"","lastName":"Shuster-Hyman","suffix":""},{"id":152228397,"identity":"33d51bd9-d182-4d65-b2bb-4fa6eb9bb672","order_by":3,"name":"Lianet Lopez","email":"","orcid":"","institution":"CReATe Fertility Centre","correspondingAuthor":false,"prefix":"","firstName":"Lianet","middleName":"","lastName":"Lopez","suffix":""},{"id":152228398,"identity":"1a101595-b55a-4aeb-a29d-f5ef76e97212","order_by":4,"name":"Andrée Gauthier-Fisher","email":"","orcid":"","institution":"CReATe Fertility Centre","correspondingAuthor":false,"prefix":"","firstName":"Andrée","middleName":"","lastName":"Gauthier-Fisher","suffix":""},{"id":152228399,"identity":"799a43c4-d252-4075-8bdd-6454d1f022b9","order_by":5,"name":"Clifford L Librach","email":"","orcid":"","institution":"CReATe Fertility Centre","correspondingAuthor":false,"prefix":"","firstName":"Clifford","middleName":"L","lastName":"Librach","suffix":""}],"badges":[],"createdAt":"2022-11-04 15:14:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2238679/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2238679/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29257765,"identity":"fcd7fe31-56b3-45e3-85e0-e530c85bfd5d","added_by":"auto","created_at":"2022-11-18 20:22:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHUCPVC modulates LPS-induced activation of Kynurenine pathway enzymes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic diagram of the Kynurenine pathway (A). mRNA expression of Kynurenine pathway enzymes after 24h LPS and HUCPVC treatments (B-F). Total RNA from the brain was isolated from Control, LPS, and LPS+HUCPVC groups (n=3 per group) and subjected to qPCR. Individual measurements were normalized using GAPDH as a housekeeping gene and the Fold change relative to Control was calculated by the ΔΔCt method. *p\u0026lt;0.05, **p\u0026lt;0.005, ***p\u0026lt;0.0005, ****p\u0026lt;0.0001, \u003cem\u003ens \u003c/em\u003e= not significant, one-way ANOVA with Tukey’s multiple comparison tests. TRY, tryptophan; \u003cem\u003eido\u003c/em\u003e, indoleamine dioxygenase; KYN, kynurenine; \u003cem\u003ekat\u003c/em\u003e, kynurenine aminotransferase; \u003cem\u003ekmo\u003c/em\u003e, kynurenine monooxygenase; 3HK, 3-hydroxykynurenine; 3HAA, 3-hydroxyanthranilic acid; \u003cem\u003ehaao\u003c/em\u003e, 3-hydroxyanthranilic acid dioxygenase; \u003cem\u003eqprt\u003c/em\u003e, quinolinate phosphoribosyltransferase; NAD, Nicotinamide adenine dinucleotide.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2238679/v1/25a26b2049f463621ec76bee.png"},{"id":29257767,"identity":"83f43039-4a87-44f3-b589-61a4a1c16c3a","added_by":"auto","created_at":"2022-11-18 20:22:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":26079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatograms of kynurenine pathway metabolites.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Tryptophan, B) Kynurenine, C) Kynurenic acid, D) Quinolinic acid, E) Serotonin, F) Glutamine, G) Glutamate. Peak area ratios for the standards and the corresponding analytes are shown on the left and right sides respectively (Peak intensity on the y-axis and retention time on the x-axis of the chromatograms)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2238679/v1/57f05e461d1f2a21d6065ddb.png"},{"id":29257931,"identity":"424aacf0-147c-4f72-920c-a3f4d3614777","added_by":"auto","created_at":"2022-11-18 20:30:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":470708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHUCPVC modulate LPS-induced microglial activation. \u003c/strong\u003eRepresentative images showing positive immunostaining of microglial activation marker, ionized calcium-binding adapter molecule 1 IBA1 (green), in the hippocampus (A,B,C) and cortex (D,E,F) in the brain cryosections of control (A,D), LPS (B,E) and LPS+HUCPVC (C,F) groups. Nuclei counterstained with DAPI (blue). Scale bar = 50µm. The mean percent area of IBA1 positive signal in the hippocampus (G) and cortex (H) was assessed by Image J analysis, \u003cem\u003en\u003c/em\u003e=3 per group. *p\u0026lt;0.05, **p\u0026lt;0.005, ***p\u0026lt;0.0005, one-way ANOVA with Tukey’s multiple comparison tests.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2238679/v1/5aed65bfa44391cfbc095907.png"},{"id":29257770,"identity":"30b39751-9582-4288-b89b-bcbcb0746365","added_by":"auto","created_at":"2022-11-18 20:22:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":495202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHUCPVC rescues the LPS-induced decline of glutamate transporter in the brain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative images showing double staining of astrocyte marker glial fibrillary acidic protein (GFAP, red) and excitatory amino acid transporter 2 (EAAT2, green) in the cerebral cortex region (A,B,C) and dentate gyrus region of the hippocampus (D,E,F) in the brain cryosections of control (A,D), LPS (B,E), and LPS+HUCPVC (C,F) groups. Scale bar = 50µm. Immunohistochemistry data for cortex (G) and hippocampus (H) represented as the ratio of mean percent area of EAAT2 and the corresponding GFAP positive signals of a given field of view for each section was assessed by Image J analysis, \u003cem\u003en\u003c/em\u003e=3-5 per group. Representative Western blot of EAAT2 (I) expression in Control (1), LPS (2) \u0026amp; LPS+HUCPVC (3) groups. Densitometric analysis of EAAT2 protein expression (J). Data normalized to GAPDH, n=5-9 per group. *p\u0026lt;0.05, **p\u0026lt;0.005, ***p\u0026lt;0.0005, one-way ANOVA with Tukey’s multiple comparison tests.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2238679/v1/3ffb1cb39698b969b6bc8875.png"},{"id":29257768,"identity":"b308e56a-1b4f-49d5-8825-736102898d4b","added_by":"auto","created_at":"2022-11-18 20:22:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8012,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHUCPVC-mediated modulation of NMDAR in the brain. \u003c/strong\u003emRNA expression levels of glutamate receptor, NMDAR subunits, NR1 (A), NR2A (B) and NR2B (C) in Control, LPS and LPS+HUCPVC groups. Fold change calculated by ΔΔCt method. Data normalized to GAPDH, n=3 per group, *p\u0026lt;0.05, **p\u0026lt;0.005, ***p\u0026lt;0.0005, ****p\u0026lt;0.0001, \u003cem\u003ens\u003c/em\u003e= not significant, one-way ANOVA with Tukey’s multiple comparison tests.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2238679/v1/c1086577ba9d0317294591a9.png"},{"id":29257769,"identity":"6e43a94b-f7a2-488d-a7e9-8e89a2cd947d","added_by":"auto","created_at":"2022-11-18 20:22:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHUCPVC modulates the expression of synaptosomal proteins. \u003c/strong\u003eRepresentative Western blots and relative protein expression of NR2B (A, B), Drebrin (C, D), PSD95 (E, F), and Synaptophysin (G, H) in Control (1), LPS (2) \u0026amp; LPS+HUCPVC (3) groups. The data is normalized to GAPDH or b-actin as mentioned, n=6-9 per group, *p\u0026lt;0.05, ‘\u003cem\u003ens\u003c/em\u003e’, not significant, one-way ANOVA with Tukey’s multiple comparison tests.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2238679/v1/8683925bb45aefab7a6706cd.png"},{"id":32103750,"identity":"73362d95-bb7e-4019-8498-4a3ce936476d","added_by":"auto","created_at":"2023-01-27 05:59:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1887470,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2238679/v1/23ee95a5-91c0-4aa7-b670-efd1f3b6ca42.pdf"},{"id":29257932,"identity":"6b86bddb-c14e-4b84-af4b-74c518199458","added_by":"auto","created_at":"2022-11-18 20:30:17","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1759924,"visible":true,"origin":"","legend":"","description":"","filename":"Westernblottinggels.tiff","url":"https://assets-eu.researchsquare.com/files/rs-2238679/v1/ddd6fc4113ab3f4b1c6e55cf.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"First Trimester Human Umbilical Cord Perivascular cells (HUCPVC) Modulate the Kynurenine Pathway and Glutamate Neurotransmission in an LPS-induced Mouse Model of Neuroinflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMounting evidence has supported a direct and positive relationship between immune-activated proinflammatory cytokines and psychiatric disorders [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. Pro-inflammatory cytokines elicited during systemic infection, cancer, and autoimmune diseases have been shown to play a significant role in the manifestations and propagation of a broad spectrum of depressive symptoms [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. Signaling of these peripherally originated proinflammatory cytokines to the brain through multiple pathways is implicated in neuroinflammation, perturbation of neurotransmitters\u0026rsquo; metabolism, transport and function, and induction of depressive symptoms [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. One such pathway that is activated by the immune response is the kynurenine pathway, which has been shown to play a key role in the activation of the central nervous system (CNS) and the pathogenesis of neuroinflammation and depression [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eThe kynurenine pathway (KP) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) claims the major share of tryptophan metabolism for the production of kynurenine and subsequent metabolites, while a meager amount of ingested tryptophan is converted to serotonin by the methoxyindole pathway [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. When induced by inflammatory signals, the extrahepatically expressed enzyme, indoleamine 2,3- dioxygenase (IDO), triggers activation of the kynurenine pathway by oxidatively breaking down tryptophan to kynurenine. IDO expressed in the brain contributes to centrally produced kynurenine which is further augmented by peripheral kynurenine crossing the blood-brain barrier [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Downstream catabolism of kynurenine takes two distinct routes leading to the production of excitatory and anti-excitatory metabolites- notably, quinolinic acid (QUIN) and Kynurenic acid (KYNA) which are a functional agonist and antagonist to the glutamate receptor- N-methyl-D-aspartate (NMDAR), respectively [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. The association of these compounds with the glutamate receptor and extracellular glutamate availability, renders neuroprotective or neurotoxic character to these distinct routes of KP and their constitutive metabolites.\u003c/p\u003e\n\u003cp\u003eDysregulation of KP resulting in a perturbation in the synthesis of neuroactive metabolites, notably KYNA and QUIN, have been associated with a plethora of neurodegenerative diseases and psychiatric disorders, including depressive illness [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Pharmacological interventions include the development of analogs of KYNA and inhibitors of neurotoxic enzymes that lead to the production of QUIN [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Though Mesenchymal Stromal Cells (MSC), owing to their capabilities to regulate the immune and inflammatory response, have been an attractive therapeutic candidate for treating various diseases with inflammatory components [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e], their therapeutic potential has so far not been tested for modulation of the immune-activated kynurenine pathway.\u003c/p\u003e\n\u003cp\u003eWe have previously demonstrated the efficacy of MSC in mitigating neuroinflammation and depressive behavior in both stress-induced, as well as LPS-induced preclinical models of depression and, have also outlined a phagocytosis-driven immunomodulation mechanism [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the current study, we aim to investigate the immunomodulatory and neuroprotective capabilities of a young source of MSC, HUCPVC\u0026rsquo;s, through a potential impact on kynurenine pathway modulation in an LPS-based mouse model of neuroinflammation; which to our knowledge has never been explored.\u003c/p\u003e\n\u003cp\u003eGlutamate excitotoxicity is implicated in many neurodegenerative diseases and psychiatric disorders [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. By their direct action on the glutamate receptors, QUIN and KYNA not only influence excitatory neurotransmission but also play an active role in the uptake and release of glutamate [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, glutamate-induced excitotoxicity is directly related to a compromised glutamate transport system, that is associated with neuropathological conditions, including depression [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Thus, this study further investigates the possible role of MSC in the modulation of key glutamatergic neurotransmission components, including expression profiles of glutamate receptors (NMDA), glutamate transporters, and synaptosomal proteins.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eAnimal Treatment \u0026amp; Cells\u003c/h2\u003e\n \u003cp\u003ePreviously established and characterized, pathogen-free lines of first trimester HUCPVC were used for this study, with ethics approval by the University of Toronto REB (#28889) and by an independent accredited ethics board (VERITAS, #2576) [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. 7\u0026ndash;8-week-old male C57BL/6J mice were obtained from Charles River (Laval, Quebec). After arrival, mice were group-housed in standard shoebox cages, habituated for a week, and allowed ad libitum food and water access. General health was monitored daily by veterinary technicians or research staff.\u003c/p\u003e\n \u003cp\u003eMSC lines from human first-trimester umbilical cords were established and maintained as described previously [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. As a control, human fibroblasts (HS-68 foreskin-derived) were purchased from ATCC (Manassas, VA). All cells were grown using minimum essential media with alpha modification, 10% fetal bovine serum, and 1% penicillin/streptomycin purchased from Gibco (Gaithersburg, MD). A fresh Solution of LPS (serotype O111:B4, New England Biolabs (Whitby, ON) was prepared on the day of injections by dissolving the compound in a sterile endotoxin-free isotonic saline. LPS (0.83mg/kg) was administered intraperitoneally (i.p) in the LPS group. This dose is known to induce a full spectrum of the acute sickness response [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] and robustly increase IDO activity in the brain \u0026ndash; one of the mechanisms associated with LPS-induced depressive behavior in mice [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. 1 x 10\u003csup\u003e6\u003c/sup\u003e HUCPVC or HS68 cells resuspended in 200\u0026micro;l of saline were injected intravenously (i.v) simultaneously with LPS in the LPS\u0026thinsp;+\u0026thinsp;HUCPVC or LPS_HS68 groups, respectively. Untreated animals were included as a control group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003eRNA extraction, Reverse Transcription \u0026amp; Real-time qPCR\u003c/h2\u003e\n \u003cp\u003eTotal RNA from whole-brain samples was extracted using Qiagen\u0026rsquo;s RNeasy Mini Kit (catalog# 74104), according to the manufacturer\u0026rsquo;s protocol. The eluted RNA was quantified using Nanodrop (NanoVue, GE). 1\u0026micro;g of total RNA was used for reverse transcriptase reactions that were carried out in a Verity 96-well Thermal cycler (Applied Biosystems, model# 9902), using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, catalog# 4368814), according to the manufacturer\u0026rsquo;s protocol. Real-time qPCR was performed for 40 cycles on a QuantStudio5 thermocycler (Applied Biosystems). Primers used were \u003cem\u003eGapdh\u003c/em\u003e (NM_008084), \u003cem\u003eIdo1\u003c/em\u003e (NM_008324), \u003cem\u003eKat2\u003c/em\u003e (NM_011834), \u003cem\u003eKmo\u003c/em\u003e (NM_133809), \u003cem\u003eHaao\u003c/em\u003e (NM_025325), \u003cem\u003eQprt\u003c/em\u003e (NM_133686.1), \u003cem\u003eNR1\u003c/em\u003e (NM_008169.3). \u003cem\u003eNR2A\u003c/em\u003e (NM_008170.4), \u003cem\u003eNR2B\u003c/em\u003e (NM_008171.4). GAPDH was used as an endogenous reference gene for the normalization of mRNA levels and relative quantification of gene expression. Fold change from the qPCR data was measured by the delta-delta Ct method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eImmunohistochemistry, Image analysis \u0026amp; Quantification\u003c/h2\u003e\n \u003cp\u003eMice were euthanized by cervical dislocation and trans-cardially perfused with cold PBS followed by 4% paraformaldehyde (PFA). Brains were harvested and fixed in 4% PFA for 24 h at 4\u0026deg;C. Cryopreservation was done by immersing the harvested brains in 30% sucrose for 1\u0026ndash;2 days, followed by snap-freezing in optimum cutting temperature (OCT) formulation (Electron Microscopy Sciences). Serial coronal tissue sections were obtained at a 16\u0026ndash;18\u0026micro;m thickness on a cryostat at TCP (the Centre for Phenogenomics, Toronto). For each animal (n\u0026thinsp;=\u0026thinsp;3 animals per group) 6\u0026ndash;8 sections were processed for immunohistochemistry. After briefly rinsing with PBS, the sections were fixed in 4% PFA for 15 min and blocked with 5% Normal Goat or Donkey serum (Sigma) supplemented with 1% BSA (Sigma), and 0.4% Triton-X-100 (Fisher) in PBS, for 1\u0026ndash;2 h at room temperature. Tissue sections were then incubated overnight at 4\u0026deg;C with the primary antibodies in 50% diluted blocking solution, followed by 1 h incubation with suitable fluorescently labeled secondary antibodies (Alexa Fluor1:1000) at room temperature. For immunostaining of nuclei, the sections were incubated with DAPI for 5 min before mounting the slides with coverslips using mounting media (Abcam). Primary antibodies used were: Mouse monoclonal anti-GFAP antibody [2A5] (1:200, Abcam, catalog# ab4648); rabbit monoclonal EAAT2 [E3P5K] (1:50, Cell Signalling Technology, catalog# 20848); anti-IBA1 (1:1000, Wako Chemicals, Richmond, VA). Confocal microscopy was performed using a Leica TCS SP8 confocal microscope at the advanced optical microscopy facility (AOMF), University Health Network, Toronto. The confocal microscope was equipped with fully spectral 400 to 700nm filters and HyD high-sensitivity detectors. Images were processed using Leica LAS-X software. 6\u0026ndash;9 representative images were taken at x20 magnification spanning the cerebral cortex and hippocampus. Image thresholds and tissue surface area marked by positive staining were analyzed using ImageJ software. For quantification of EAAT2 and GFAP expression, the mean percent area of EAAT2 positive staining was normalized with that of GFAP for a given field of view for all images across different experimental groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003eWestern Blotting\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eWhole-brain homogenization\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBrains were harvested and immediately snap-frozen in liquid nitrogen and stored at -80\u0026deg;C. Brain tissues were later placed in RIPA buffer (Sigma, catalog # 0278) in 2ml silica tubes prefilled with 3mm zirconium beads and subjected to high-velocity impact homogenization using the BeadBug 6, Six Position Homogenizer (Benchmark Scientific, catalog # D1036). Homogenization was done at 3000rpm for 30 seconds followed by 30 seconds on ice-, repeating the cycle 3 times. The homogenate was then centrifuged at 12000rpm for 15 min at 4\u0026deg;C and the supernatant was collected in the pre-chilled tubes.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSynaptosome protein fractionation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eSynaptic protein extraction was performed following the protocol from Thermo Scientific. Briefly, freshly harvested whole brains, excluding the cerebellum (200-400mg), were homogenized in 10 volumes of the Syn-PER synaptic protein extraction reagent (Thermo Fisher; catalog# 87793) using a Dounce tissue grinder, performing 10\u0026ndash;12 up-and-down strokes. The homogenate was then centrifuged at 1200 g for 10 minutes to remove cell debris, and the supernatant was centrifuged at 15,000 g for 20 minutes. The pellets, containing synaptosomes, were gently resuspended in 1\u0026ndash;2 ml of the Syn-PER reagent.\u003c/p\u003e\n \u003cp\u003eThe resulting whole-brain homogenate/synaptosomal protein lysate was assayed for protein concentration using a BCA protein assay kit (Thermo Scientific, catalog# 23225), and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. These protein samples were placed in a reducing buffer containing \u0026beta;-mercaptoethanol and heated at 90\u0026deg;C for 10 min. Samples were then subjected to SDS\u0026ndash;polyacrylamide gel electrophoresis in 4\u0026ndash;12% Tris-Glycine 1mm precast gels (Invitrogen, catalog# XP04120BOX), and then transferred to polyvinylidene fluoride membranes using a semi-wet Mini Blot Module transfer unit (Life Technologies, catalog# B1000). The membranes were blocked in LiCor blocking buffer for 1 h at room temperature and probed with the primary antibodies in 0.1% Tween LiCor blocking buffer overnight at 4\u0026deg;C. The next day, the membranes were washed 3 times for 10 min each in 0.01% tween phosphate buffer solution before probing with an appropriate mix of IR-Dye-labeled Licor secondary antibodies in 0.1% Tween, 0.01% SDS LiCor blocking buffer for 1 h at room temperature. Washes, as described above, were repeated after incubation with secondary antibodies. Western blot images were obtained on a Licor Odyssey Imaging System. Relative quantification of the protein bands was assessed by densitometry analysis using ImageJ software. Primary antibodies used were: rabbit monoclonal EAAT2 [E3P5K] (1:1000, Cell Signalling Technology, catalog# 20848); rabbit monoclonal anti PSD95 antibody [EPR23124-118] (1:2000, Abcam, catalog# ab238135); mouse monoclonal anti NMDAR2B/NR2B (1:500, Invitrogen, catalog# MA-1-2014), rabbit monoclonal anti Drebrin antibody [EPR12634] (1:10,000, Abcam, catalog# ab178408) and rabbit monoclonal anti Synaptophysin antibody [YE269] (1:10,000, Abcam, catalog# ab32127).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003eExtraction and Quantification of Brain and Plasma Samples - LCMS Analysis\u003c/h2\u003e\n \u003cp\u003eSample extraction \u0026amp; preparation, LC-MS/MS analysis, and quantification were performed by the Analytical Facility for Bioactive Molecules (AFBM), Hospital for Sick Children, Toronto, Canada.\u003c/p\u003e\n \u003cp\u003eAll LC-MS/MS grade solvents were purchased from Caledon Laboratories Ltd (Georgetown, ON). Autosampler vials/glass inserts used in the sample extraction were purchased from Chromatographic Specialties Ltd (Brockville, ON).\u003c/p\u003e\n \u003cp\u003e150\u0026ndash;180 mg of frozen brain tissues were weighed and transferred into Precellys homogenization tubes containing ceramic beads (Bertin Technologies, Rockville, Washington DC). The entire brain was processed for this assay which required two tubes per sample depending on the brain weight. Tissue samples were kept overnight in the \u0026minus;\u0026thinsp;80\u0026deg;C freezer until extraction. The following day, extraction solvent was added to each Precellys tube to achieve a target concentration of 150 mg/mL and homogenized using a Precellys 24 high-throughput homogenizer (Bertin Technologies). Plasma (100 \u0026micro;L) and brain samples (67 \u0026micro;L of the homogenized suspensions (corresponding to 10 mg tissues) were transferred into Eppendorf tubes containing 1 ml 90:10 acetonitrile (ACN):methanol (MeOH) alongside standards, quality control standards, and deuterated internal standards. Tubes were vortexed and then centrifuged at 20,000 g. Supernatants were transferred to a conical tube and taken to dryness under a gentle stream of nitrogen. Samples were reconstituted in 90/10 H\u003csub\u003e2\u003c/sub\u003eO/ACN\u0026thinsp;+\u0026thinsp;0.1% formic acid and analyzed by LC/MS/MS.\u003c/p\u003e\n \u003cp\u003eAn Agilent 1200 UPLC system (Agilent Technologies, Santa Clara, CA, USA) fitted with a Sciex Q-Trap 5500 mass spectrometer (AB Sciex, Framingham, MA, USA) was used in Electron Spray Ionization (ESI) mode. Two methods were employed. Except for a few analytes, most analytes were quantified using a Kinetex PFP column (2.6 \u0026micro;m, 100\u0026Aring;, 50 x 3.0 mm; Phenomenex, Torrence, CA). A gradient mobile phase of 10 minutes at a flow rate of 0.4 ml/min was used for the elution of the biogenic amines with mobile phase A (MPA): 1% acetic acid in water and mobile phase B (MPB): 1% acetic acid in 1:1 MeOH:ACN. For the remainder of the analytes, an EZ Fast 4uaaa-MS column was used. A gradient mobile phase of 10 minutes at a flow rate of 0.4 ml/min was used with MPA (0.1% formic acid, 0.1% heptafluorobutyric acid in water) and MPB (0.1% formic acid in MeOH). Quantification was performed with Analyst 1.6.1 software (ABSciex: Framingham, Massachusetts, USA) by plotting the sample peak area ratios (Analyte peak area/Internal Standard peak area) of the biogenic amine standards against a standard curve generated from various standard concentrations from 0.05 ng to 100 ng, spiked with the same amount of internal standard used for the samples and extracted using the same conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eAll data were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and analyzed using Ordinary one-way analysis of variance followed by post hoc pairwise Tukey\u0026rsquo;s multiple comparisons tests comparing all the experimental groups using GraphPad Prism (GraphPad Software, San Diego, CA).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003ePeripheral HUCPVC infusion modulates LPS-induced activation of kynurenine pathway enzymes in the brain\u003c/h2\u003e\n \u003cp\u003eProinflammatory cytokines mediate the induction of IDO which serves as a molecular switch for triggering the initiation of the KP [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Enzymatic activity and expression of IDO, which is also present in brain endothelial cells, perivascular macrophages, astrocytes, and microglia [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e], have a direct influence on brain tryptophan metabolism [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our previous study demonstrated significant mitigation of LPS-induced proinflammatory cytokines in the brain by peripherally administered HUCPVC in mice [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Thus, we sought to investigate the impact of the immunomodulatory potential of HUCPVC on the gene expression profile of \u003cem\u003eIdo1\u003c/em\u003e and the subsequent metabolic enzymes of the pathway in the LPS-activated CNS (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Our results indicate significant induction of \u003cem\u003eIdo1\u003c/em\u003e in the brain by LPS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and its equally significant downregulation back to control levels as a result of HUCPVC treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). \u003cem\u003eIdo\u003c/em\u003e-mediated synthesis of kynurenine is centrally placed in the KP as it is further favorably catabolized by the enzymes \u003cem\u003eKmo\u003c/em\u003e and \u003cem\u003eKat\u003c/em\u003e towards putative neurotoxic or neuroprotective branches, respectively [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. In the LPS group, we recorded no significant changes in mRNA levels of \u003cem\u003eKat2\u003c/em\u003e, when compared to control (p\u0026thinsp;=\u0026thinsp;0.2949) or LPS\u0026thinsp;+\u0026thinsp;HUCPVC (p\u0026thinsp;=\u0026thinsp;0.9846) groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Conversely, a significant LPS-induced upregulation of \u003cem\u003eKmo\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0020) was seen to be modulated back to near control levels by HUCPVC (p\u0026thinsp;=\u0026thinsp;0.0176) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). The excitatory properties associated with the ability of quinolinic acid to stimulate NMDA receptors directly and selectively are well known [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. This neurotoxic compound is synthesized by the enzymatic activity of \u003cem\u003eHaao.\u003c/em\u003e We observed that LPS significantly increased the expression of \u003cem\u003eHaao\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0002) and that the treatment of LPS combined with HUCPVC helped downregulate its level significantly (p\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). Accumulation of toxic concentrations of quinolinic acid also depends on the rate of metabolism of quinolinic acid to NAD\u0026thinsp;+\u0026thinsp;by the enzyme \u003cem\u003eQprt.\u003c/em\u003e Our results indicate that the immune response to LPS resulted in a significant lowering of \u003cem\u003eQprt\u003c/em\u003e expression (p\u0026thinsp;=\u0026thinsp;0.0033) when compared to the control group and LPS\u0026thinsp;+\u0026thinsp;HUCPVC effected a significant upregulation of the enzyme transcript (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). This data suggests that the influence of HUCPVC treatment on \u003cem\u003eQprt\u003c/em\u003e expression may be instrumental in regulating the synthesis of KP metabolites.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003eInfluence of HUCPVC on LPS-activated kynurenine pathway metabolites and serotonin in the brain and plasma:\u003c/h2\u003e\n \u003cp\u003eThe induction of KP metabolites following 24h of LPS treatment and intervention by HUCPVC were assessed by LC-MS/MS. The standard curves were linear over the concentration ranges. The calibration curves were as follows: y\u0026thinsp;=\u0026thinsp;0.166 x\u0026thinsp;+\u0026thinsp;0.166, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9922 for tryptophan; y\u0026thinsp;=\u0026thinsp;0.349x\u0026thinsp;+\u0026thinsp;0.0344, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9860 for kynurenine; y\u0026thinsp;=\u0026thinsp;0.0943x\u0026thinsp;+\u0026thinsp;0.000927, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9934 for kynurenic acid; y\u0026thinsp;=\u0026thinsp;0.00715x\u0026thinsp;+\u0026thinsp;0.0175, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9857 for quinolinic acid; y\u0026thinsp;=\u0026thinsp;0.196x\u0026thinsp;+\u0026thinsp;0.00148, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9930 for serotonin; y\u0026thinsp;=\u0026thinsp;1.12e-005x + -5.35e-005, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9989 for glutamine and y\u0026thinsp;=\u0026thinsp;3.67e-005x + -0.00039, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9973 for glutamate. The representative chromatograms of these analytes are shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-G. As detailed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, LPS significantly upregulated brain levels of tryptophan (p\u0026thinsp;=\u0026thinsp;0.0197), kynurenine (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), kynurenine:tryptophan (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and quinolinic acid (p\u0026thinsp;=\u0026thinsp;0.0003), when compared to control. No significant difference was found in the levels of kynurenic acid (p\u0026thinsp;=\u0026thinsp;0.9503) or serotonin (p\u0026thinsp;=\u0026thinsp;1417). However, the neurotoxic index, quinolinic acid:kynurenic acid was found to be significantly increased by LPS (p\u0026thinsp;=\u0026thinsp;0.0117), when compared to control. The combination of LPS with HUCPVC significantly modulated the brain levels of tryptophan (p\u0026thinsp;=\u0026thinsp;0.0217), kynurenine (p\u0026thinsp;=\u0026thinsp;0.0390), kynurenine:tryptophan (p\u0026thinsp;=\u0026thinsp;0.0401) and quinolinic acid (p\u0026thinsp;=\u0026thinsp;0.0042) when compared to LPS alone. No significant difference was found in the levels of kynurenic acid (p\u0026thinsp;=\u0026thinsp;0.9598) or serotonin (p\u0026thinsp;=\u0026thinsp;0.7892). However, quinolinic acid:kynurenic acid was found to be significantly increased in this condition (p\u0026thinsp;=\u0026thinsp;0.0149). Since brain kynurenine levels are directly impacted by peripheral circulation [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e], plasma tryptophan metabolism was also assessed (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). No significant effect of LPS was found in the plasma levels of tryptophan (p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999), kynurenine (p\u0026thinsp;=\u0026thinsp;0.2420), quinolinic acid (p\u0026thinsp;=\u0026thinsp;0.9409), kynurenic acid (p\u0026thinsp;=\u0026thinsp;0.6114), quinolinic acid:kynurenic acid (p\u0026thinsp;=\u0026thinsp;0.7065) or serotonin (p\u0026thinsp;=\u0026thinsp;0.9999) when compared to control. However, the IDO activation index, Kynurenin:tryptophan was found to be significantly increased by LPS (p\u0026thinsp;=\u0026thinsp;0.0320) when compared to control. No significant modulation was noted by HUCPVC on plasma levels of tryptophan (p\u0026thinsp;=\u0026thinsp;0.9930), kynurenine (p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999), kynurenine:tryptophan (p\u0026thinsp;=\u0026thinsp;0.8341), kynurenic acid (p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999), quinolinic acid (p\u0026thinsp;=\u0026thinsp;0.4393), quinolinic acid:kynurenic acid (p\u0026thinsp;=\u0026thinsp;0.7698) or serotonin (p\u0026thinsp;=\u0026thinsp;0.9978) when compared to LPS-treated animals. The immunomodulatory effect of a control non-MSC cell type was tested by using human foreskin-derived fibroblasts (HS68) in lieu of HUCPVC, and their influence on LPS-activated KP metabolites was assessed. There was no significant effect on brain levels of tryptophan (p\u0026thinsp;=\u0026thinsp;0.2513), kynurenine (p\u0026thinsp;=\u0026thinsp;0.2901), kynurenic acid (p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999), quinolinic acid (p\u0026thinsp;=\u0026thinsp;0.9980), quinolinic acid:kynurenic acid (p\u0026thinsp;=\u0026thinsp;0.9998) or serotonin (p\u0026thinsp;=\u0026thinsp;0.9984) when compared to LPS-treated animals. However, a significant effect was recorded for kynurenine:tryptophan (p\u0026thinsp;=\u0026thinsp;0.0035). Moreover, HS68 was found to have no significant effect on the plasma levels of tryptophan (p\u0026thinsp;=\u0026thinsp;0.9991), kynurenine (p\u0026thinsp;=\u0026thinsp;0.7195), kynurenine:tryptophan (p\u0026thinsp;=\u0026thinsp;0.4029), kynurenic acid (p\u0026thinsp;=\u0026thinsp;0.9485), quinolinic acid (p\u0026thinsp;=\u0026thinsp;0.9752), quinolinic acid:kynurenic acid (p\u0026thinsp;=\u0026thinsp;0.8721) or serotonin (p\u0026thinsp;=\u0026thinsp;0.8125). These experimental read-outs support the possibility that peripherally infused HUCPVC have an influence on the KP metabolism in the brain.\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTRY, tryptophan; KYN, kynurenine; KYNA, kynurenic acid; QUIN, quinolinic acid; 5-HT, serotonin; Glu, glutamate; Gln, glutamine, LPS, lipopolysaccharide; HUCPVC, human umbilical cord perivascular cells; HS68, human foreskin fibroblast cells. Data represents mean (\u0026plusmn;\u0026thinsp;SEM). n\u0026thinsp;=\u0026thinsp;5\u0026ndash;8 mice per group, (except HS68, n\u0026thinsp;=\u0026thinsp;3). \u003csup\u003ea\u003c/sup\u003eSignificant effect of LPS (versus Control), \u003csup\u003eb\u003c/sup\u003esignificant effect of HUCPVC (versus LPS), \u003csup\u003ec\u003c/sup\u003esignificant effect of HS68 (versus LPS), Ordinary One-way ANOVA with Tukey\u0026rsquo;s multiple comparison test. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.005, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMetabolite\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLPS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLPS\u0026thinsp;+\u0026thinsp;HUCPVC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLPS\u0026thinsp;+\u0026thinsp;HS68\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eBrain\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTRY (nM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e172 (8.09)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e209 (7.28)\u003csup\u003ea*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e170 (7.16)\u003csup\u003eb*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e236 (17.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKYN (nM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.8 (0.96)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.1 (3.46)\u003csup\u003ea****\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43 (5.97)\u003csup\u003eb*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.9 (9.93)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKYN:TRY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.077 (0.009)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.338 (0.01)\u003csup\u003ea****\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.238 (0.025)\u003csup\u003eb*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.173 (0.079)\u003csup\u003ec**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKYNA (nM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.257 (0.155)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.388 (0.060)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.513 (0.237)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.39 (0.206)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQUIN (nM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3044 (604)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7892 (362)\u003csup\u003ea***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4086 (651)\u003csup\u003eb**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8090 (1311)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQUIN:KYNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3081 (511)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5717 (330)\u003csup\u003ea*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3080 (676)\u003csup\u003eb*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5790 (229)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5-HT (nM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e385 (40.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e520 (50.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e464 (42.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e507 (3.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlu (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e909 (66)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e893 (36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e893 (43)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1103 (281)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGln (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1853 (179.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2025 (162.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2012 (138.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2374 (583)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ePlasma\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTRY (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.758 (0.030)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.756 (0.033)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.741 (0.046)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.746 (0.103)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKYN (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.824 (0.061)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.155 (0.168)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.155 (0.133)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.915 (0.040)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKYN:TRY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.106 (0.103)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.71 (0.212)\u003csup\u003ea*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.54 (0.104)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.28 (0.208)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKYNA (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.159 (0.005)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.14 (0.014)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.141 (0.010)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.151 (0.003)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQUIN (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.389 (0.392)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.751 (0.507)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.808 (0.465)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.403 (0.336)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQUIN:KYNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.9 (3.16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.3 (17.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.1 (4.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.2 (2.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5-HT (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.19 (0.411)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.24 (0.525)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.12 (0.494)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.47 (0.070)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlu (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.1 (1.82)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12 (2.19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.31 (1.88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.1 (3.12)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGln (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e170.1 (26.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e169.5 (24.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e135.1 (18.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e176.5 (20.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\u003cstrong\u003eAssessment of Kynurenine pathway metabolites, serotonin, glutamate and glutamine levels by LC-MS after 24h LPS treatment along with either HUCPVCs or HS68.\u003c/strong\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003eImpact of peripherally administered HUCPVC on LPS-induced neuroinflammation and glutamatergic neurotransmission\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eMicroglial activation by LPS and modulation by HUCPVC\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eCorroborating our previous study where we reported significant mitigation of LPS-induced proinflammatory cytokines in the brain by peripherally administered HUCPVC [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e], in this study we demonstrate the potential of HUCPVC potential to modulate neuroinflammation by measuring LPS-induced microglial activation in the hippocampus (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA,B,C\u003cstrong\u003e)\u003c/strong\u003e and cortex (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD,E,F), LPS significantly increased activation of microglia as assessed by increased positive staining for ionized calcium-binding adapter molecule 1 (IBA1) in the dentate gyrus region of the hippocampus (p\u0026thinsp;=\u0026thinsp;0.0028) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG) as well as the cortex (p\u0026thinsp;=\u0026thinsp;0.0003) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH). Significant modulation of this induction by HUCPVC was seen both in the dentate gyrus (p\u0026thinsp;=\u0026thinsp;0.0018) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG) as well as the cortex (p\u0026thinsp;=\u0026thinsp;0.0003) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eThe regulation of glutamate transporters \u0026amp; receptors by HUCPVC infusion\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eGlutamate-mediated excitotoxicity is linked to various neurodegenerative diseases and psychiatric disorders [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Since the synaptic levels of glutamate are predominantly maintained by excitatory amino acid transporters (EAATs), we tested the expression of EAAT2 in the brain in response to peripheral immune activation and HUCPVC infusion. In this experiment, EAAT2 was co-stained with glial fibrillary acidic protein (GFAP), a marker for astrocytes, where it is primarily localized [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. Overall EAAT2 expression was assessed by normalizing its positive signal with that of GFAP in the cerebral cortex and dentate gyrus (DG) region of the hippocampus. The results revealed that LPS significantly diminished EAAT2 expression levels in the cerebral cortex (p\u0026thinsp;=\u0026thinsp;0.0319, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, G) and hippocampus (p\u0026thinsp;=\u0026thinsp;0.0182, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE, H). Conversely, LPS in combination with HUCPVC significantly reinstated the transporter\u0026rsquo;s expression level both in the cerebral cortex (p\u0026thinsp;=\u0026thinsp;0.0102, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, G) and hippocampus (p\u0026thinsp;=\u0026thinsp;0.0180, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF, H). This differential EAAT2 protein expression in control, LPS and LPS\u0026thinsp;+\u0026thinsp;HUCPVC-treated animals was also demonstrated by Western blot. We observed an obvious loss of EAAT2 immunoreactivity due to LPS, which was regained close to the control level in the HUCPVC-treated group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI). The densitometric data analysis (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eJ) confirmed significant downregulation of EAAT2 expression by LPS (p\u0026thinsp;=\u0026thinsp;0.0010) and significant rescue by HUCPVC treatment (p\u0026thinsp;=\u0026thinsp;0.0034). NMDAR is one of the prominent glutamate receptors that mediate the activity of glutamate and 2 main subunits, NR1 and NR2A/B, are obligatory for the receptor\u0026rsquo;s activity and signaling [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. We tested the transcript levels of these NMDAR subunits in the mRNA isolated from whole brains of the control, LPS, and LPS\u0026thinsp;+\u0026thinsp;HUCPVC groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). LPS was found to significantly upregulate the transcript expression of \u003cem\u003eNR2A\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and \u003cem\u003eNR2B\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Conversely, HUCPVC significantly downregulated \u003cem\u003eNR2A\u003c/em\u003e (P\u0026thinsp;=\u0026thinsp;0.0006) and \u003cem\u003eNR2B\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) transcripts. However, no significant change was observed in the \u003cem\u003eNR1\u003c/em\u003e gene expression in both LPS (p\u0026thinsp;=\u0026thinsp;0.4128) and LPS\u0026thinsp;+\u0026thinsp;HUCPVC (p\u0026thinsp;=\u0026thinsp;0.3258) groups, when compared to control.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eExtended role of HUCPVC in regulating synaptosomal proteins linked to glutamate trafficking \u0026amp; receptor activity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe synaptosomal fraction is enriched with several proteins that impact glutamate receptor (NMDAR) activity and signaling. By Western blot, we tested some of these proteins such as NMDAR subunit- NR2B, postsynaptic density protein (PSD95), synaptophysin, and drebrin which are associated with glutamate trafficking and NMDAR function [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. A significant increase in NR2B expression by LPS (p\u0026thinsp;=\u0026thinsp;0.0386) was observed which was equally counteracted by HUCPVC intervention (p\u0026thinsp;=\u0026thinsp;0.0299) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). The expression level of drebrin was found to be significantly downregulated by LPS (p\u0026thinsp;=\u0026thinsp;0.0013) and rescued by HUCPVC (p\u0026thinsp;=\u0026thinsp;0.0386) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). However, no change in the expressions of either PSD95 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, D) or synaptophysin \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE, F\u003cstrong\u003e)\u003c/strong\u003e was found to be induced by LPS alone or in combination with HUCPVC.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eExploiting the well-known immunomodulatory potential of MSC, this study is the first, to our knowledge, to examine the influence of intravenously infused MSC on the activated kynurenine pathway (KP) and glutamate neurotransmission. Here, we report that peripherally administered HUCPVC regulate KP enzymes and metabolites in the LPS-activated CNS. Furthermore, these MSC were also found to exert a modulatory effect on the expression profile of glutamate receptor subunits and glutamate transporters. Thus, this study lends a novel approach to target aberrant signaling of KP and the subsequent glutamate excitotoxicity that are known to have diverse neuropathological consequences.\u003c/p\u003e \u003cp\u003eInflammation-associated upregulation and activation of IDO (product of \u003cem\u003eido1\u003c/em\u003e gene) in the brain is a critical step in the break down of tryptophan to kynurenine and initiation of the KP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Moreover, IDO activation is shown to be crucial for depressive-like behavior in mice treated with LPS for 24h [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We have previously reported that HUCPVC modulate neuroinflammation and depressive behavior after 24h of LPS injection in mice [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this study, we report that HUCPVC regulate \u003cem\u003eIDO\u003c/em\u003e at the transcriptional level and the enzyme\u0026rsquo;s activation in the brain, as assessed by kynurenine:tryptophan ratio. This finding informs us of the possibility that the modulation of inflammation-associated depressive behavior by HUCPVC could be due to their ability to influence the catalytic function of IDO. Kynurenine synthesized by IDO can be a favorable substrate for either astrocytic enzyme KAT leading to the production of neuroprotective KYNA or microglial enzymes KMO and HAAO leading to the production of neurotoxic QUIN. Consistent with other studies [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], we recorded an LPS-induced aberration in the brain mRNA levels of \u003cem\u003eKmo, Haao\u003c/em\u003e and \u003cem\u003eKat2\u003c/em\u003e, which is a predominant isomer of the KAT enzyme responsible for KYNA production [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Since the inadequate metabolism of QUIN by the enzyme QPRT contributes to increased neurotoxicity [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], we also tested the brain mRNA level of the enzyme, which was sharply downregulated by LPS. Thus, the recovery of an LPS-activated imbalance of the KP enzymes by HUCPVC treatment indicates the capability of MSC to influence the KP enzymatic machinery of glia governing the central production of KP metabolites.\u003c/p\u003e \u003cp\u003eKP metabolite levels in the brain have been intensely interrogated owing to their strong affiliation with many CNS disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In the current study, we evaluate the downstream immunomodulatory effect of intravenously administered HUCPVC on the neurotoxicity index, as assessed by the brain QUIN/KYNA ratio. Since the majority of brain kynurenine is peripherally derived during inflammation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], plasma levels of the KP metabolites were also evaluated. Furthermore, to delineate the potential MSC-associated immunomodulatory effect, human foreskin-derived fibroblast cells (HS68), which have a relatively low level of immunomodulatory potential compared to MSC [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], were independently injected into a group of animals. Our results indicate that compared to the brain, IDO activation in the plasma was not sufficient to increase the flux of circulating kynurenine. We hypothesize that this mild increase in kynurenine:tryptophan ratio, as shown in this study and by others [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], could merely be the statistical outcome of non-significant changes in tryptophan or kynurenine levels, which likely holds little metabolic or clinical significance. This lack of increase in plasma kynurenine may be due to its rapid clearance by the kidney and excretion of its metabolites in urine [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Sufficient IDO-induced tryptophan oxidation is required to exceed the effect of renal processing and result in appreciable levels of plasma kynurenine. Moreover, kynurenine being a substrate to KMO, the inflammation-induced expression and/or activity of KMO can counter the effect of IDO. In addition, since circulating proinflammatory cytokines are shown to peak within 1\u0026ndash;6 hours of LPS treatment [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], arguably, these early timepoints could correspond to a relatively higher plasma IDO activity, as shown by Wirthgen et. al [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Thus, the low plasma levels of other KP metabolites in the current study could be the downstream effect of this lack of increase in kynurenine. Conversely, in the brain, we noted significant aberration of KP metabolite levels due to LPS. Significantly altered KP metabolites in the brain due to LPS were rescued back to basal levels by HUCPVC treatment; an effect not seen for the most part with fibroblast cells. These findings reveal significant and specific immunomodulatory effect of MSC on KP metabolism in response to LPS; thus maintaining homeostasis between two functionally contradictory branches of the pathway. Interestingly, and in accord with other studies [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], we found an increase in tryptophan by LPS. This increase, which could be due to LPS-induced lipolysis resulting in increased availability of albumin-free tryptophan to cross the blood-brain barrier (BBB) [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], was reversed by HUCPVC. Cytokine-stimulated IDO activation is also known to have a negative impact on serotonin (5HT) turnover [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Since the level of 5-hydroxy indole acetic acid (5HIAA), the metabolite of 5HT was not examined in this study, the unchanged 5HT level does not reflect its actual turnover and thus does not preclude the possibility of inflammation-afflicted modulation of serotonergic neurotransmission.\u003c/p\u003e \u003cp\u003eThe nexus between activated cerebral KP metabolism and NMDAR activation, leading to enhanced glutamate function, is associated with many neuropathological conditions [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The observed HUCPVC-induced modulation of KP metabolites in this study, notably that of QUIN, an endogenous NMDAR agonist, provoked further investigation into the expression profile of the obligatory subunits of NMDAR, that mediate the receptor\u0026rsquo;s activity, and whose upregulation is implicated in various brain pathologies, including inflammation-related depressive phenotype [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In this study, we report a significant increase in the transcript levels of the subunits, \u003cem\u003eNR2A\u003c/em\u003e and \u003cem\u003eNR2B\u003c/em\u003e, in response to LPS, which resonates with similar observations by others [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The demonstrated ability of HUCPVC to modulate these subunits may suggest a novel and relatively safer therapeutic alternative to the NMDAR subunit-targeting antidepressants that are shown to have psychoactive side effects and cardiovascular toxicity [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Glutamatergic circuitry is also negatively impacted by the perturbed transport mechanism responsible for the clearance of synaptic glutamate, leading to glutamate excitotoxicity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our study, for the first time, illustrates the potential of MSC, specifically HUCPVC, in regaining the LPS-induced decline in the astrocytic glutamate transporter EAAT2. Since the levels of glutamate (Glu) and glutamine (Gln) in the blood and brain also reflect glutamate excitotoxicity [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], we tested these metabolites in the plasma and whole-brain homogenate by LCMS. However, levels of Glu and Gln showed no significant changes between the control and LPS treatment groups. Since Glu and Gln cross BBB [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] and are expressed differentially in different brain regions [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], the lack of modulation in Glu and Gln levels reported in this study could be the consequence of the limitations of the methodology, which is unable to distinguish between the source of the metabolites in the plasma (central vs peripheral) or to delineate the region-specific expression of Glu and Gln in the brain. We further extended the scope of this study to investigate the immunomodulatory effect of systemically infused MSC on the neuroinflammation-associated synaptic imbalance implicated in neurodegenerative and psychiatric illnesses, including depression [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The effect of peripherally administered HUCPVC on LPS-induced dysregulation of synaptic markers such as drebrin, synaptophysin, PSD95, and NMDAR regulatory subunit NR2B, was tested in a synaptosomal isolate. Drebrin, an actin-binding protein in dendritic spines and one of the key players in the NMDAR-dependent synaptic neurotransmission, has been shown to be negatively regulated by the neuroinflammatory cascade associated with neurodegenerative diseases and psychiatric disorders [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The HUCPVC-mediated restoration of LPS-induced downregulation of drebrin expression suggests a novel potential application of MSC in restoration of synaptic loss. Furthermore, we report the upregulation of NR2B expression in response to LPS. This corroborates the previous findings that the proinflammatory cytokines in the brain facilitate the activation of the NMDAR subunit [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Pharmacological suppression of NR2B has been achieved using various selective antagonists of the NMDAR subunit in various brain pathologies [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. However, these pharmacological agents cause undesirable side effects including neurotoxicity and hypertension [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Thus, our findings support the possibility that MSC may be a potentially safer and more efficient therapeutic alternative to target NR2B.\u003c/p\u003e \u003cp\u003eThere has been a considerable lack of understanding of the basis of systemically administered MSC\u0026rsquo;s ability to provide neuroprotection in many diseases and injury models. In our previous study using an LPS-induced mouse model of neuroinflammation and depression, we have demonstrated phagocytosis-driven immunomodulation by peripherally infused HUCPVCs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The resulting systemic innate immune alteration from pro- to anti-inflammatory phenotype was plausibly correlated to the mitigation of LPS-induced neuroinflammatory response and depressive symptoms. In the current study, modulation of LPS-activated microglia via the kynurenine pathway in the CNS by HUCPVC may be another mechanism for peripheral immune modulation by the MSCs. Considering our previous findings that peripherally infused MSC fail to cross BBB [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], an alternate mechanism of MSC neuroprotective potential is its paracrine action, by which secreted factors are shown to mediate neuroprotection [\u003cspan additionalcitationids=\"CR74\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Contextually, studies showing the interplay between the kynurenine pathway and MSC support the involvement of IDO in the immunosuppressive effect of MSC [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Moreover, KYNA is also shown to regulate the expression of TNF-stimulated gene 6 (TSG-6), a paracrine factor, and promote TSG-6-mediated immunosuppressive and anti-inflammatory effects of MSC [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. However, the modulation of a broad spectrum of activated kynurenine metabolites and the downstream signaling consequences vis-\u0026agrave;-vis the glutamatergic system by MSC, as shown in this study, represents a novel finding.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis is the first study to examine and demonstrate the capability of intravenously injected MSC to regulate KP metabolites in a neuroinflammatory context, thus maintaining an optimal balance between \u0026lsquo;neuroprotective\u0026rsquo; and \u0026lsquo;neurotoxic\u0026rsquo; branches of the pathway. The novelty of this study is also marked by the demonstrated potential role of MSC in curbing glutamate excitotoxicity. In conclusion, our research findings further our knowledge to understand the mechanistic aspect of MSC neuroprotection, specifically through the KP. In addition, the specific anti-inflammatory properties we observed support the potential for use of MSC, particularly HUCPVC, as a therapeutic option for targeting inflammation-driven activated KP metabolites and glutamatergic systems linked to neurological diseases and affective disorders.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003cp\u003eACN: Acetonitrile\u003c/p\u003e\n \u003cp\u003eBBB: Blood-brain barrier\u003c/p\u003e\n \u003cp\u003eCNS: Central nervous system\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eDG: Dentate gyrus\u003c/p\u003e\n \u003cp\u003eEAAT: Excitatory amino acid transporter\u003c/p\u003e\n \u003cp\u003eGFAP: Glial fibrillary acidic protein\u003c/p\u003e\n \u003cp\u003eGln: Glutamine\u003c/p\u003e\n \u003cp\u003eGlu: Glutamate\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3HAA: 3-Hydroxy anthranilic acid\u003c/p\u003e\n \u003cp\u003e3HAAO: 3-Hydroxy anthranilic acid dioxygenase\u003c/p\u003e\n \u003cp\u003e5HIAA: 5-Hydroxy \u0026iacute;ndole acetic acid\u003c/p\u003e\n \u003cp\u003e3HK: 3-Hydroxykynurenine\u003c/p\u003e\n \u003cp\u003eHUCPVC: Human Umbilical Cord Perivascular cells\u003c/p\u003e\n \u003cp\u003eIDO: Indoleamine 2,3- dioxygenase\u003c/p\u003e\n \u003cp\u003eIP: Intraperitoneal\u003c/p\u003e\n \u003cp\u003eIV: Intravenous\u003c/p\u003e\n \u003cp\u003eKAT: Kynurenine aminotransferase\u003c/p\u003e\n \u003cp\u003eKMO: Kynurenine monooxygenase\u003c/p\u003e\n \u003cp\u003eKP: Kynurenine Pathway\u003c/p\u003e\n \u003cp\u003eKYN: Kynurenine\u003c/p\u003e\n \u003cp\u003eKYNA: Kynurenic acid\u003c/p\u003e\n \u003cp\u003eLC-MS: Liquid Chromatography with tandem mass spectrometry\u003c/p\u003e\n \u003cp\u003eLPS: Lipopolysaccharide\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMeOH: Methanol\u003c/p\u003e\n \u003cp\u003eMSC: Mesenchymal stromal cell\u003c/p\u003e\n \u003cp\u003eNAD: Nicotinamide adenine dinucleotide\u003c/p\u003e\n \u003cp\u003eNMDAR: N-methyl-D-aspartate receptor\u003c/p\u003e\n \u003cp\u003eOCT: Optimum cutting temperature\u003c/p\u003e\n \u003cp\u003ePFA: Paraformaldehyde\u003c/p\u003e\n \u003cp\u003eQPRT: Quinolinate phosphoribosyl transferase\u003c/p\u003e\n \u003cp\u003eQUIN: Quinolinic acid\u003c/p\u003e\n \u003cp\u003e5HT: Serotonin\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTRY: Tryptophan\u003c/p\u003e\n \u003cp\u003eTSG6: TNF-stimulated gene 6\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\u003cbr\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll use and animal care were conducted and reported in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and approved by the Animal Care Committee of the University Health Network (AUP 5232.4, University of Toronto, Canada).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the CReATe Fertility Centre\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFyyaz Siddiqui:\u003c/strong\u003e participated in study conception, animal handling, experimental design \u0026amp; execution, cell culture, imaging and data collection, data analysis, and manuscript preparation; \u003cstrong\u003eDenis Gallagher:\u003c/strong\u003e participated in study conception, supervision and manuscript preparation; \u003cstrong\u003eHannah Shuster-Hyman:\u003c/strong\u003e participated in image analysis and reviewed the manuscript; \u003cstrong\u003eLianet Lopez:\u003c/strong\u003e participated in cell culture and reviewed the manuscript; \u003cstrong\u003eAndr\u0026eacute;e Gauthier-Fisher:\u003c/strong\u003e participated in study design, data interpretation and preparation of the manuscript; \u003cstrong\u003eClifford Librach:\u003c/strong\u003e participated in study design, data interpretation and preparation of the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Fatima Sultani, Ashley St. Pierre, and the directors of the Analytical Facility for Bioactive Molecules, The Hospital for Sick Children, \u0026nbsp;Toronto, Canada for assistance with the samples\u0026rsquo; extraction/ preparation and LC-MS/MS analysis. The authors also acknowledge Andrea Archila, William Xiao and Roberto Lopez, Animal Resource Centre, University Health Network, Toronto, for their excellent technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHaroon E, Raison CL, Miller AH. Psychoneuroimmunology meets neuropsychopharmacology: translational implications of the impact of inflammation on behavior. \u003cstrong\u003eNeuropsychopharmacol.\u003c/strong\u003e 2012; 37:137\u0026ndash;162\u003c/li\u003e\n\u003cli\u003eRaison CL, Miller AH. Malaise, melancholia, and madness: the evolutionary legacy of an inflammatory bias. \u003cstrong\u003eBrain Behav Immunol.\u003c/strong\u003e 2013; 31:1\u0026ndash;8\u003c/li\u003e\n\u003cli\u003eRaison CL, Capuron L, Miller AH. Cytokines sing the blues: inflammation and the pathogenesis of depression. \u003cstrong\u003eTrends Immunol\u003c/strong\u003e. 2006; 27:24\u0026ndash;31\u003c/li\u003e\n\u003cli\u003eMiller AH, Maletic V, Raison CL. 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Cell Death Differ. 2018; 25 (7):1209-1223.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Kynurenine pathway, neuroinflammation, mesenchymal stromal cells, HUCPVC","lastPublishedDoi":"10.21203/rs.3.rs-2238679/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2238679/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe Kynurenine Pathway (KP) of tryptophan degradation and glutamate toxicity is implicated in several neurological disorders, including depression. Although mesenchymal stromal cells (MSC)-mediated immunomodulation and neuroprotection have been studied in many of these disorders, their potential to influence KP and the glutamatergic system has not yet been investigated. Hence, this study sought to investigate the effect of HUCPVC, a rich and potent source of MSC, on Lipopolysaccharide (LPS)-activated KP metabolites, KP enzymes, and key components of glutamate neurotransmission.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe immunomodulatory effect of peripherally administered HUCPVC on the expression profile of kynurenine pathway enzymes and metabolites was assessed in the plasma and brain of mice treated with LPS. An assessment of the glutamatergic system, including selected receptors, transporters and proteins was also conducted.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHUCPVC were found to modulate LPS-induced activation of KP enzymes and metabolites in the brain associated with neurotoxicity. Moreover, the reduced expression of the glutamatergic components due to LPS was also found to be significantly improved by HUCPVC.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe immunomodulatory properties of HUCPVC appear to confer neuroprotection, at least in part, through their ability to modulate the KP in the brain. This KP modulation enhances neuroprotective regulators and downregulates neurotoxic consequences, including glutamate neurotoxicity, which is associated with neuroinflammation and depressive behavior.\u003c/p\u003e","manuscriptTitle":"First Trimester Human Umbilical Cord Perivascular cells (HUCPVC) Modulate the Kynurenine Pathway and Glutamate Neurotransmission in an LPS-induced Mouse Model of Neuroinflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-18 20:22:11","doi":"10.21203/rs.3.rs-2238679/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6b1781e-4142-473c-be86-11eba79af209","owner":[],"postedDate":"November 18th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-02-08T01:29:25+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-18 20:22:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2238679","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2238679","identity":"rs-2238679","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}