Fluvoxamine attenuates inflammation in experimental sepsis via novel non-canonical pathways

Fluvoxamine attenuates inflammation in experimental sepsis via novel non-canonical pathways | 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 Fluvoxamine attenuates inflammation in experimental sepsis via novel non-canonical pathways Luis HA Costa, Isis P Trajano, Wanderson S Santos, Katiuscia M Araujo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7142228/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Dec, 2025 Read the published version in Inflammopharmacology → Version 1 posted 5 You are reading this latest preprint version Abstract Sepsis is characterized by a dysregulated systemic inflammatory response to infection and remains a major global health challenge, underscoring the need for novel therapeutic strategies. Drug repurposing offers a promising strategy, and fluvoxamine (FLV), a selective serotonin reuptake inhibitor (SSRI) widely used in psychiatric treatment, has been reported to exhibit anti-inflammatory properties. Here, we investigated the effects of FLV in a murine model of sepsis induced by cecal ligation and puncture (CLP). Oral pretreatment with FLV for seven days significantly increased the anti-inflammatory cytokine IL-10 in both plasma and peritoneal fluid. To assess central nervous system involvement, FLV was administered intracerebroventricularly, resulting in a broad reduction in circulating cytokines, including both pro- and anti-inflammatory mediators. In vitro , FLV suppressed inflammatory cytokine production in LPS-stimulated macrophages, indicating a direct effect on immune cells. Notably, these immunomodulatory effects were independent of serotonin signaling and sigma-1 receptor activation—pathways traditionally associated with SSRI mechanisms. These findings provide new insights into the immunomodulatory actions of FLV and support its potential repurposing as an adjunctive therapy for inflammatory diseases such as sepsis. selective serotonin reuptake inhibitor sigma-1 receptor serotonin inflammation macrophages cytokines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Despite significant recent advances in patient care and treatment, sepsis remains a leading cause of morbidity and mortality worldwide, including in developed countries (Fleischmann-Struzek et al. 2018 ; Rhee et al. 2019 ). The most recent consensus defines sepsis as a life- threatening dysregulated host response to infection (Singer et al. 2016 ), with inflammation playing a central role in its pathophysiology. Following infection, immune cells initiate the synthesis of numerous inflammatory mediators, a phenomenon commonly referred to as a “cytokine storm.” This excessive production of pro- and anti-inflammatory cytokines can impair organ function by disrupting microcirculation, reducing tissue oxygenation, and ultimately leading to cell death (Stearns-Kurosawa et al. 2011 ). The clinical management of septic patients focuses on mitigating this pathological response and typically involves three main components: hemodynamic management, infection control and modulation of the host response (Vincent 2022 ). While the first two are addressed through relatively well-established interventions such as fluid resuscitation, vasoactive agents, and antibiotic therapy, regulating the immune response of the host presents a more complex challenge. Given the heterogeneity of sepsis—varying in etiology, patient age, sex, and comorbidities—therapeutic approaches focusing on single molecules or pathways have often proven ineffective. Indeed, several clinical trials targeting specific cytokines such as TNF-α (Abraham et al. 1995 ; Gallagher et al. 2001 ), IL-1(Fisher 1994 ; Opal et al. 1997 ) or nitric oxide(López et al. 2004 ) have failed to demonstrate a benefit in septic patients. In contrast, the use of glucocorticoids—which exert broad immunosuppressive effects by modulating multiple inflammatory pathways—continues to be among the most recommended pharmacological interventions for sepsis (Annane 2011 ). The complexity of sepsis pathophysiology presents ongoing challenges for both clinical and translational research aimed at identifying effective new therapies. In this context, drug repurposing—the strategy of applying existing medications to new therapeutic indications—has emerged as an attractive alternative, offering advantages in terms of time, cost, and safety. One promising class of drugs in this regard is the selective serotonin reuptake inhibitors (SSRIs), commonly prescribed for psychiatric disorders but increasingly recognized for their anti-inflammatory properties (Patel et al., 2023 ). These immunomodulatory effects raise the possibility that SSRIs could serve as adjuvant therapies in the treatment of inflammatory conditions such as sepsis. Among SSRIs, fluvoxamine (FLV), a drug approved for the treatment of major depressive and obsessive-compulsive disorders, has shown particular promise. Clinical and preclinical studies have reported beneficial effects of FLV in systemic inflammatory conditions, including COVID-19 (Lenze et al. 2020 ; Reis et al. 2022 ; Kirenga et al. 2023 ) and in murine models of lethal septic shock (Rosen et al. 2019 ). In this work we aimed to further investigate the immunomodulatory mechanisms of FLV in sepsis, with a focus on how it regulates the host inflammatory response. Materials and methods Animals Male Wistar rats (280 ± 20 g) from the Animal Care Facility of the University of São Paulo, Campus of Ribeirão Preto, Brazil, were used in the experiments. The animals were maintained at a controlled ambient temperature of 23 ± 1°C with a 12 hours light/dark cycle with food and water ad libitum. All experiments were approved by the Animal Ethical Committee of the Dental School of Ribeirão Preto (2020.1.391.58.3). Cecal ligation and puncture (CLP) Following anesthesia using isoflurane (4% for induction and maintained at 1.5% during the surgery), the surgical site was shaved and disinfected with a povidone-iodine solution. Surgical procedure was performed as described elsewhere (Costa et al., 2024 ). Briefly, after a midline abdominal incision, cecum was exposed and loosely ligated just above the ileocecal valve and then perforated four times using a 16G needle. After gentle squeeze to allow feces extravasation, cecum was returned to the peritoneal cavity and the incision was sutured. Saline (10ml/kg) was injected subcutaneously immediately after surgery. All animals exhibited typical signs of infection (reduced locomotor activity, piloerection, tachypnea). Sham animals underwent the same procedure but did not have their cecum ligated or perforated. Lateral ventricle cannulation Animals were anesthetized with a mixture of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively) and fixed in a stereotaxic frame. A guide cannula (0.7 OD) was introduced in the left lateral ventricle using the following coordinates: AP -0.8mm / L + 1.4mm/ DV -3.2 to -3.5 mm from bregma. Two screws were attached to the skull and dental cement were used to keep the cannula in position. Animals were allowed to recover from the surgery during 7 days before the intracerebroventricular injection. Cytokines measurement Blood was collected from trunk after decapitation, centrifuged (3500 rpm, 20 min, 4°C) and plasma was separated for cytokines analysis. For peritoneal lavage, 5ml of cold PBS was injected in the peritoneal cavity and, after gentle local massage, an abdominal incision was made and 2ml of the lavage was collected using a plastic Pasteur pipette. It was then centrifugated (3500 rpm, 20 min, 4°C) and the supernatant was collected. Cytokines measurements were carried out using commercial ELISA kits and following the manufacturers’ instructions (IL-1β [DY510, R&D Systems], IL-6 [DY501, R&D Systems] and IL-10 [DY522, R&D Systems]). Serotonin (5-HT) quantification Plasma and hypothalamic 5-HT were quantified by ELISA (ab133053, Abcam), following the manufactures’ protocol. Plasma samples were prepared as described for the cytokine measurements. The hypothalamus was dissected and homogenized in RIPA buffer containing 10% protease cocktail inhibitor and kept under agitation in ice during 2 hours. Samples were centrifuged (4300 rpm, 20min, 4℃) and the supernatant was collected. Total protein quantification was accessed using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, 10741395). The results were expressed as ng/mL and ng/mg of protein. Peritoneal macrophages cell culture For the in vitro experiments using peritoneal macrophages, rats were injected intraperitoneally with 3% (w/v) Brewer thioglycollate medium. After three days, under deep anesthesia, cold RPMI medium was injected in the peritoneal cavity, the abdomen was gently massaged and an incision was made to carefully collect the fluid using a sterile Pasteur pipette. The peritoneal medium with cells was centrifuged (1500 rpm, 8 min, 4℃) and resuspended/ washed two times with cold medium, centrifuged again and the supernatant was discarded. Cells were resuspended in complete RPMI medium containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin. 1x10 6 cells were seeded per well in a 24-wells plate and kept in an incubator (37 ◦ C, 5% CO 2 ) for 2 h. After this period, wells were washed two times with RPMI to removed non-adherent macrophages and cells were incubated overnight in supplemented medium. Cells were stimulated with LPS only (1µg/ml, 0111: B4, Sigma Aldrich) or FLV (20µM, 15617, Cayman), sigma-1 receptor antagonist (30µM, E-52862, Cayman) or serotonin (10µM, H9523, Sigma-Aldrich) 30 minutes before LPS. Twenty-four hours after LPS the supernatant was collected and frozen until cytokine quantification by ELISA. Experiments were performed at least three times. Statistical analysis The data are presented as mean ± SD and individual values are presented in the bar graphs. The normality of data was tested using the Shapiro–Wilk normality test. Variables with normal distribution were analyzed by one-way analysis of variance (ANOVA), followed by post hoc Tukey tests. For those with nonparametric distribution, a Kruskal–Wallis test was performed, followed by Dunn’s post hoc tests. At-test as used to compare two groups, when required. The statistical test used and the number of mouse subjects per group are reported in the figure legends. Values of p ≤ 0.05 were considered statistically significant. Results Systemic and central fluvoxamine administration modulates cytokines production during sepsis To investigate if fluvoxamine pretreatment modulates inflammation during sepsis, rats received FLV (30mg/kg; Luvox , Abott) or vehicle (saline) by gavage during seven days before being subjected to sepsis by CLP. This dose is equivalent to the recommendation for humans (100-300mg/ day) and it was converted to rats based on the body surface area (Center for Drug Evaluation and Research 2005 ). As expected, sepsis promoted a sustained increase in the levels of cytokines in the circulation (Fig. 1 a-c) and in the infection site, indicated by measurements in the peritoneal fluid (Fig. 1 d-f), 6 and 12 hours after CLP. These timepoints represent the acute phase of the disease, when the highest levels of inflammatory mediators are observed and when the immunomodulatory actions of FLV are likely to be observed. Interestingly, seven days treatment with fluvoxamine did not alter the levels of the pro-inflammatory cytokines IL-6 and IL-1β in septic animals, but it further increased both plasma and peritoneal levels of the anti-inflammatory cytokine IL-10. FLV pretreatment did not change cytokine levels in sham animals, which remained below detection levels using our ELISA kits. Given that FLV is able to cross the blood-brain barrier (BBB) and knowing that the brain orchestrates mechanisms that modulate systemic inflammatory response, we administered FLV intracerebroventricularly (i.c.v) to investigate the potential central action of this drug during sepsis. 22.8µg of fluvoxamine (Cayman) or its vehicle (20% DMSO) were infused in a volume of 5µl in each animal, 60 minutes before CLP surgery. This dose allows us to mimic the brain steady-steady state concentration of FLV (12µM) observed in patients with long-term use of this drug (Bolo et al. 2000 ). Central FLV significantly reduced plasma levels of all the cytokines analyzed at 6 hours after CLP also decreased IL-1β at 12 hours (Fig. 2 a-c). Differently from the systemic pretreatment, it did not have any effect on the production of cytokines in the site of infection (Fig. 2 d-f). Fluvoxamine attenuates cytokine production by peritoneal macrophages in vitro We next investigated whether fluvoxamine modulates the inflammatory response through a direct action on immune cells. To this end, peritoneal macrophages were pretreated in vitro with FLV or vehicle (XXX) diluted in culture medium 30 minutes before the inflammatory stimulus with LPS. After 24 hours, cytokine production was quantified by ELISA. FLV treatment significantly reduced the levels of IL-1β, IL-6, and IL-10 (Fig. 3 a). Given that FLV may exert anti-inflammatory effects via sigma-1 receptor (S1R) agonism, we evaluated whether a selective S1R antagonist (S1RA) could alter FLV's immunomodulatory activity. Interestingly, S1RA alone reduced IL-6 and IL-10 levels in LPS-stimulated cells and did not block FLV’s effects. Instead, co-treatment with S1RA and FLV further enhanced the anti-inflammatory response (Fig. 3 b). Immunomodulatory actions of systemic fluvoxamine are serotonin- independent To further elucidate the immunomodulatory mechanisms of fluvoxamine, we investigated the involvement of serotonin (5-HT), given that FLV is a selective serotonin reuptake inhibitor (SSRI). To assess the role of 5-HT in vivo , serotonin was depleted using para-chlorophenylalanine (PCPA, 100 mg/kg; Sigma-Aldrich), administered intraperitoneally once daily in parallel with FLV pretreatment. PCPA was diluted in a 2% Tween 80 saline solution. Serotonin depletion alone significantly reduced circulating levels of all cytokines analyzed in septic animals and did not block the anti-inflammatory effects of FLV pretreatment (Fig. 4 a). Measurement of plasma 5-HT levels revealed that FLV treatment did not elevate serotonin concentrations in either sham or septic animals (Fig. 4 b), and sepsis itself did not alter plasma 5-HT levels. To investigate the direct effects of serotonin on immune cells, we treated peritoneal macrophages in vitro with 5-HT prior to LPS stimulation. Serotonin treatment alone did not significantly alter cytokine production. However, it partially reversed the inhibitory effects of FLV on IL-1β and IL-6 levels, while having no impact on FLV-mediated IL-10 modulation (Fig. 4 c) Mechanisms of immune modulation induced by central fluvoxamine administration To investigate the brain mechanisms underlying the systemic anti-inflammatory effects of centrally administered fluvoxamine, we examined the involvement of previously studied pathways. First, we assessed the role of the sigma-1 receptor by administering S1RA (160µg/ animals) intracerebroventricularly 30 minutes prior to FLV administration. Blocking this pathway did not reverse the anti-inflammatory effects of FLV, as IL-6 and IL-10 levels remained significantly lower than in animals treated with FLV (Fig. 5 a). We then evaluated the potential contribution of serotonin signaling using WAY-100635 (3µg/ animal), a selective 5-HT₁A receptor antagonist. This receptor subtype was chosen based on its involvement in central circuits responding to peripheral inflammation (Kim et al. 2015 ). Pretreatment with WAY-100635, administered i.c.v. 30 minutes before FLV, did not affect the cytokine modulation induced by central FLV administration (Fig. 5 a). To further explore serotonin involvement, we measured 5-HT levels in the hypothalamus, a key brain region involved in the regulation of peripheral physiological responses, following seven days of oral FLV pretreatment. Septic animals exhibited a reduction in hypothalamic 5-HT compared to sham controls; however, FLV had no effect on 5-HT levels in either condition (Fig. 5 b). Discussion Previous investigations have suggested that drugs commonly used in the treatment of psychiatric disorders, like fluvoxamine, have anti-inflammatory properties (Nykamp et al. 2022 ; Patel et al. 2023 ). In the current study, the analysis of cytokine production demonstrates that central and peripheral actions of FLV have distinct immunomodulatory effects. Oral pretreatment with FLV significantly upregulated IL-10 levels during sepsis, both in plasma and peritoneal lavage fluid. This finding is consistent with previous work showing that fluoxetine, another SSRI, also enhances IL-10 production during polymicrobial infection, and that this anti-inflammatory cytokine mediates fluoxetine-induced protection in sepsis (Gallant et al., 2025 ). In contrast, intracerebroventricular administration of FLV had a broader effect, reducing all cytokines analyzed, including IL-10, in the plasma but not in the peritoneal lavage. These divergent effects suggest that systemic FLV may act directly at the site of infection, as indicated by the modulation of peritoneal exudate cytokines, while central FLV likely engages pathways that primarily act on circulating components. In addition to differences in the site of action, distinct cytokine regulation patterns were observed, particularly in the differential modulation of IL-10 compared to other pro-inflammatory mediators, indicating distinct pathways depending on the administration route and evidencing the complexity of the mechanisms of action of FLV in immune modulation. To investigate the potential mechanisms underlying fluvoxamine’s immunomodulatory effects during inflammation, we examined its direct action on immune cells. FLV significantly reduced cytokine production in LPS-stimulated macrophages in vitro , indicating a direct anti-inflammatory effect. However, in contrast to the in vivo findings, FLV did not increase IL-10 levels. One possible explanation is that the immunomodulatory effects of FLV depend on long-term exposure, which cannot be temporally replicated in cell culture conditions. Supporting this hypothesis, a previous study has shown that repeated administration of fluoxetine increases IL-10 production following LPS challenge in vivo , whereas a single dose has no effect on plasma IL-10 levels (Kostadinov et al. 2015 ). Although macrophages and circulating monocytes are key contributors to the acute immune response in sepsis, other immune cell populations may also be targets of FLV. Neutrophils, for example, are major producers of IL-10 during CLP-induced sepsis, particularly at the site of infection (Kasten et al. 2010 ). Further studies are needed to determine whether FLV acts directly on these cells or modulates intercellular communication among different immune cell types during sepsis. Previous studies have demonstrated that FLV attenuates inflammation during sepsis via activation of the sigma-1 receptor (S1R), a chaperone protein that interacts with IRE1 (inositol-requiring enzyme 1α), an endoplasmic reticulum stress sensor (Rosen et al. 2019 ). While all SSRIs function as S1R agonists, fluvoxamine has the highest binding affinity for this receptor (Narita et al. 1996 ), making it a potential therapeutic target. However, in our in vitro experiments, the use of a selective S1R antagonist (S1RA) did not abolish the anti-inflammatory effects of FLV in LPS-stimulated macrophages, suggesting that FLV modulates cytokine production through an S1R-independent mechanism. Interestingly, S1RA alone also reduced cytokine expression and exhibited an additive anti-inflammatory effect when co-administered with FLV. The literature has conflicting data regarding the role of S1R activation in inflammation, with both pro- (Bravo-Caparrós et al. 2020 ; Denaro et al. 2024 ) and anti-inflammatory (Szabo et al. 2014 ; Shanmugam et al. 2015 ) effects being reported. Such discrepancies may be attributed to differences in experimental approaches—including pharmacological versus genetic manipulation—and to the diversity of cell types studied, such as macrophages, neutrophils, neurons, and astrocytes. These findings indicate that FLV modulates inflammation in macrophages independently of sigma-1 receptor activation. Another possible mechanism of action of FLV may involve serotonin, given that this drug is a 5-HT reuptake inhibitor, and numerous studies have suggested that the immunomodulatory effects of SSRIs are mediated by neurotransmitter. Immune cells express various subtypes of serotonin receptors, the serotonin transporter (SERT)- the primary target for SSRIs- and tryptophan hydroxylase (TPH), he rate-limiting enzyme in serotonin (5-HT) synthesis (Herr et al. 2017 ). Administration of a TPH inhibitor led to reduced cytokine production in septic rats and the addition of 5-HT to peritoneal macrophage cultures abolished anti-inflammatory effect of FLV, indicating a serotonin-independent mechanism. Furthermore, TPH1 knockout mice—which have significantly reduced circulating 5-HT—showed lower mortality and decreased organ damage following sepsis (Zhang et al. 2017 ). Another study using the same TPH1-deficient model confirmed that the anti-inflammatory effects of fluoxetine are also independent of serotonin (Gallant et al, 2025 ). Together with the observation that FLV pre-treatment does not alter plasma serotonin levels, these findings reinforce the hypothesis that the immunomodulatory actions of circulating FLV and other SSRIs are not mediated by serotonin. To the best of our knowledge, this is the first study to demonstrate that fluvoxamine modulates systemic inflammation via central mechanisms. Previous work has shown that i.c.v. administration of amitriptyline, a serotonin and norepinephrine reuptake inhibitor, reduces paw edema in rats in a dose-dependent manner (Hajhashemi et al. 2010 ), but no studies to date have investigated the central anti-inflammatory effects of SSRIs during sepsis. Despite being expressed in both neurons (Kourrich et al. 2012 ) and glial cells (Wang et al., 2024 ), sigma-1 receptor does not mediate the anti-inflammatory effects of FLV. Our group previously reported that central serotonin administration attenuates systemic inflammation (Mota et al. 2019 ) but this mechanism is unlikely to explain FLV's central effects. First, the anti-inflammatory actions of FLV were not abolished by pretreatment with the 5-HT1A receptor antagonist WAY-100635. Second, prolonged FLV administration did not elevate brain serotonin levels, regardless of sepsis. While the precise molecular target of central FLV remains to be identified, we can speculate the pathway through which it may exert its immunomodulatory actions during sepsis. One plausible mechanism involves modulation of the vagal anti-inflammatory reflex—a key component of brain-to-immune system communication. In this pathway, afferent vagal signals convey peripheral inflammation to the brain, which in turn activates descending efferent vagal fibers that signal to the spleen and ultimately suppresses cytokine production (Tracey 2002 ). It is possible that central FLV enhances the activity of these neural circuits, thereby amplifying the anti-inflammatory vagal reflex. Additionally, central FLV may act via humoral pathways such as activation of the hypothalamic–pituitary–adrenal (HPA) axis, leading to increased corticosteroid release—potent endogenous regulators of inflammation. Further investigation is needed to delineate the specific neural substrates and signaling pathways involved in the central anti-inflammatory action of FLV during sepsis. In summary, we demonstrate that fluvoxamine exerts anti-inflammatory effects during sepsis through both peripheral and central mechanisms that are independent of commonly pathways attributed to SSRIs, like serotonin and sigma-1 receptor activation. Our results expand the understanding of the immunomodulatory action of this drug beyond its conventional use as an antidepressant and support its potential repurposing for the treatment of inflammatory conditions. Declarations Ethics approval All experiments were approved by the Animal Ethical Committee of the Dental School of Ribeirão Preto (2020.1.391.58.3). 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. Conflict of interest The authors declare that they have no competing interests Funding This work was supported by Grants 2016/17681–9 (LGSB) and 2019/27231–9 (LHAC) from São Paulo Research Foundation (FAPESP). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. Financial support from the National Council for Scientific and Technological Development (CNPq) is acknowledged. Authors' contributions LHAC and LGSB designed the study. LHAC, IPT, WSS and KMA conducted the experiments. LHAC drafted the manuscript. All authors read and approved the final manuscript. 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JAMA - Journal of the American Medical Association 315 Stearns-Kurosawa DJ, Osuchowski MF, Valentine C, et al (2011) The pathogenesis of sepsis. Annual Review of Pathology: Mechanisms of Disease 6:. https://doi.org/10.1146/annurev-pathol-011110-130327 Szabo A, Kovacs A, Frecska E, Rajnavolgyi E (2014) Psychedelic N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine modulate innate and adaptive inflammatory responses through the sigma-1 receptor of human monocyte-derived dendritic cells. PLoS One 9:. https://doi.org/10.1371/journal.pone.0106533 Tracey KJ (2002) The inflammatory reflex. Nature 420 Vincent JL (2022) Current sepsis therapeutics. EBioMedicine 86 Wang JY, Ren P, Cui LY,et al(2024) Astrocyte-specific activation of sigma-1 receptors in mPFC mediates the faster onset antidepressant effect by inhibiting NF-κB-induced neuroinflammation. Brain Behav Immun. Aug;120:256-274. doi: 10.1016/j.bbi.2024.06.008. Zhang J, Bi J, Liu S, et al (2017) 5-HT Drives Mortality in Sepsis Induced by Cecal Ligation and Puncture in Mice. Mediators Inflamm 2017:. https://doi.org/10.1155/2017/6374283 Cite Share Download PDF Status: Published Journal Publication published 15 Dec, 2025 Read the published version in Inflammopharmacology → Version 1 posted Reviewers agreed at journal 24 Jul, 2025 Reviewers invited by journal 24 Jul, 2025 Editor invited by journal 23 Jul, 2025 Editor assigned by journal 17 Jul, 2025 First submitted to journal 16 Jul, 2025 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. 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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-7142228","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":490428343,"identity":"3d90354a-0c8b-43a5-85a0-4cd61a061e42","order_by":0,"name":"Luis HA Costa","email":"","orcid":"","institution":"USP: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"HA","lastName":"Costa","suffix":""},{"id":490428344,"identity":"0d4d2d61-6588-4596-b9ac-fc6df287e663","order_by":1,"name":"Isis P Trajano","email":"","orcid":"","institution":"USP: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Isis","middleName":"P","lastName":"Trajano","suffix":""},{"id":490428345,"identity":"4046276a-447d-471b-8416-11b11e7f8499","order_by":2,"name":"Wanderson S Santos","email":"","orcid":"","institution":"USP: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Wanderson","middleName":"S","lastName":"Santos","suffix":""},{"id":490428346,"identity":"d273ce52-3740-4079-bea2-07e0a064519b","order_by":3,"name":"Katiuscia M Araujo","email":"","orcid":"","institution":"USP: Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Katiuscia","middleName":"M","lastName":"Araujo","suffix":""},{"id":490428347,"identity":"30e8f7df-9a0b-4b83-9e53-f5294ce61a29","order_by":4,"name":"Luiz Guilherme de Siqueira Branco","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBACAwhlw48uQlBLmmQDqVoOk6DFXPrwswc/Ks5L8EufPcDM8+cOUOQAfi2WfWnmhj1nbktI9uUlMPO2PQOKJBBw2BkGM2nGttt1Bmd4DJh5Gw6DRAhpYf8G1HJOAqyF5w9RWnhAthyAamEjQotlD0+ZZM+ZZAnJHr6Eg3PbnvFY9hDQYs7Dvk3iR4WdBD8P78EHb/7ckTPnIaAFCfAwHGBgOECCBpAWIDhAio5RMApGwSgYIQAAz447rUsIVSkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-0292-4947","institution":"University of Sao Paulo: Universidade de Sao Paulo","correspondingAuthor":true,"prefix":"","firstName":"Luiz","middleName":"Guilherme de Siqueira","lastName":"Branco","suffix":""}],"badges":[],"createdAt":"2025-07-16 16:54:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7142228/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7142228/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10787-025-02064-7","type":"published","date":"2025-12-15T15:58:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87724365,"identity":"dd436a19-da68-41f4-b91d-c164af75c90b","added_by":"auto","created_at":"2025-07-28 10:24:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33697,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine levels in plasma (a–c) and peritoneal fluid (d–f) of control (sham) and septic (CLP) animals pretreated with fluvoxamine (FLV) or vehicle (VEH; saline) by oral gavage for seven consecutive days. Concentrations of IL-1β (a, d), IL-6 (b, e), and IL-10 (c, f) were measured at 6 and 12 hours post-CLP surgery. nd = not detectable. Data are expressed as mean ± SD. *p \u0026lt; 0.05 by one-way ANOVA; n = 4–7 per group\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7142228/v1/915da0cf743d52d8b3081100.png"},{"id":87724445,"identity":"9e614183-b890-4e47-9578-0495a45e8243","added_by":"auto","created_at":"2025-07-28 10:24:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35641,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine levels in plasma (a–c) and peritoneal fluid (d–f) of control (sham) and septic (CLP) animals following intracerebroventricular administration of fluvoxamine (FLV) or vehicle (VEH; 20% DMSO). Concentrations of IL-1β (a, d), IL-6 (b, e), and IL-10 (c, f) were measured at 6 and 12 hours after CLP surgery. nd = not detectable. Data are expressed as mean ± SD.*p \u0026lt; 0.05 using one-way ANOVA. n =4-7\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7142228/v1/0514b6dff2a4e0ae5baee664.png"},{"id":87724441,"identity":"171af090-7223-4776-be2e-bb5bbdff335c","added_by":"auto","created_at":"2025-07-28 10:24:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27841,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine production by peritoneal macrophages in vitro. (a) Quantification of IL-1β, IL-6, and IL-10 levels in the culture supernatant of peritoneal macrophages stimulated with LPS for 24 hours and pretreated with fluvoxamine (FLV) or vehicle (Veh). p \u0026lt; 0.05 by paired t-test; n = 5–6. (b) IL-6 and IL-10 levels in the culture supernatant of peritoneal macrophages treated with a sigma-1 receptor antagonist (S1RA) or vehicle and stimulated with LPS for 24 hours. Data are expressed as variation relative to the control group (Veh). *p \u0026lt; 0.05 by one-way ANOVA; n = 4–6\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7142228/v1/53adbeaeffe0ce4311d07e5d.png"},{"id":87724372,"identity":"44d2514d-7a02-4bd0-affd-ea66093c9f66","added_by":"auto","created_at":"2025-07-28 10:24:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33210,"visible":true,"origin":"","legend":"\u003cp\u003eRole of serotonin in fluvoxamine-mediated immunomodulation. (a) Quantification of IL-1β, IL-6, and IL-10 levels in the supernatant of peritoneal macrophages stimulated with LPS for 24 hours and pretreated with serotonin (5-HT), fluvoxamine (FLV), or vehicle (Veh); n = 4–5. (b) Plasma serotonin levels in control (sham) and septic (CLP) animals pretreated with FLV or vehicle (VEH; saline) by oral gavage for seven days; n = 4–7. (c) Plasma cytokine levels in septic animals pretreated with FLV or vehicle (gavage) in combination with para-chlorophenylalanine (PCPA; intraperitoneal), a serotonin synthesis inhibitor. nd = not detectable. *p \u0026lt; 0.05 by one-way ANOVA; n = 4–8\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7142228/v1/6fae2c28d02274f6b6586c5b.png"},{"id":87724356,"identity":"63dc1410-b8ec-4f3f-acb1-65024cec4a0e","added_by":"auto","created_at":"2025-07-28 10:24:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30896,"visible":true,"origin":"","legend":"\u003cp\u003eCentral modulation of immune response by fluvoxamine. (a) Plasma levels of IL-6 and IL-10 in septic animals that received intracerebroventricular injections of fluvoxamine (FLV), WAY-100635 (WAY; a 5-HT1A receptor antagonist), sigma-1 receptor antagonist (S1RA), or vehicle (Veh) prior to sepsis induction. Blood samples were collected 12 hours after CLP surgery. *p \u0026lt; 0.05 by one-way ANOVA; n = 4–7. (b) Hypothalamic serotonin levels in control (sham) and septic (CLP) animals pretreated with FLV or vehicle (VEH; saline) by oral gavage for seven days. *p \u0026lt; 0.05 by one-way ANOVA; n = 4–6\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7142228/v1/607ec50aac284ead03790420.png"},{"id":98813980,"identity":"a229bb7b-3006-40fd-bd86-37e3ff05148d","added_by":"auto","created_at":"2025-12-22 16:09:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":548732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7142228/v1/4d8169b7-d9cb-407f-b6a5-c56feb7a1792.pdf"}],"financialInterests":"","formattedTitle":"Fluvoxamine attenuates inflammation in experimental sepsis via novel non-canonical pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDespite significant recent advances in patient care and treatment, sepsis remains a leading cause of morbidity and mortality worldwide, including in developed countries (Fleischmann-Struzek et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rhee et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The most recent consensus defines sepsis as a life- threatening dysregulated host response to infection (Singer et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), with inflammation playing a central role in its pathophysiology. Following infection, immune cells initiate the synthesis of numerous inflammatory mediators, a phenomenon commonly referred to as a \u0026ldquo;cytokine storm.\u0026rdquo; This excessive production of pro- and anti-inflammatory cytokines can impair organ function by disrupting microcirculation, reducing tissue oxygenation, and ultimately leading to cell death (Stearns-Kurosawa et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe clinical management of septic patients focuses on mitigating this pathological response and typically involves three main components: hemodynamic management, infection control and modulation of the host response (Vincent \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While the first two are addressed through relatively well-established interventions such as fluid resuscitation, vasoactive agents, and antibiotic therapy, regulating the immune response of the host presents a more complex challenge. Given the heterogeneity of sepsis\u0026mdash;varying in etiology, patient age, sex, and comorbidities\u0026mdash;therapeutic approaches focusing on single molecules or pathways have often proven ineffective. Indeed, several clinical trials targeting specific cytokines such as TNF-α (Abraham et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Gallagher et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), IL-1(Fisher \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Opal et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) or nitric oxide(L\u0026oacute;pez et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) have failed to demonstrate a benefit in septic patients. In contrast, the use of glucocorticoids\u0026mdash;which exert broad immunosuppressive effects by modulating multiple inflammatory pathways\u0026mdash;continues to be among the most recommended pharmacological interventions for sepsis (Annane \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe complexity of sepsis pathophysiology presents ongoing challenges for both clinical and translational research aimed at identifying effective new therapies. In this context, drug repurposing\u0026mdash;the strategy of applying existing medications to new therapeutic indications\u0026mdash;has emerged as an attractive alternative, offering advantages in terms of time, cost, and safety. One promising class of drugs in this regard is the selective serotonin reuptake inhibitors (SSRIs), commonly prescribed for psychiatric disorders but increasingly recognized for their anti-inflammatory properties (Patel et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These immunomodulatory effects raise the possibility that SSRIs could serve as adjuvant therapies in the treatment of inflammatory conditions such as sepsis.\u003c/p\u003e\u003cp\u003eAmong SSRIs, fluvoxamine (FLV), a drug approved for the treatment of major depressive and obsessive-compulsive disorders, has shown particular promise. Clinical and preclinical studies have reported beneficial effects of FLV in systemic inflammatory conditions, including COVID-19 (Lenze et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Reis et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kirenga et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and in murine models of lethal septic shock (Rosen et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this work we aimed to further investigate the immunomodulatory mechanisms of FLV in sepsis, with a focus on how it regulates the host inflammatory response.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/p\u003e\u003cp\u003e Male Wistar rats (280\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) from the Animal Care Facility of the University of S\u0026atilde;o Paulo, Campus of Ribeir\u0026atilde;o Preto, Brazil, were used in the experiments. The animals were maintained at a controlled ambient temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a 12 hours light/dark cycle with food and water ad libitum. All experiments were approved by the Animal Ethical Committee of the Dental School of Ribeir\u0026atilde;o Preto (2020.1.391.58.3).\u003c/p\u003e\u003cp\u003e\u003cem\u003eCecal ligation and puncture (CLP)\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFollowing anesthesia using isoflurane (4% for induction and maintained at 1.5% during the surgery), the surgical site was shaved and disinfected with a povidone-iodine solution. Surgical procedure was performed as described elsewhere (Costa et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Briefly, after a midline abdominal incision, cecum was exposed and loosely ligated just above the ileocecal valve and then perforated four times using a 16G needle. After gentle squeeze to allow feces extravasation, cecum was returned to the peritoneal cavity and the incision was sutured. Saline (10ml/kg) was injected subcutaneously immediately after surgery. All animals exhibited typical signs of infection (reduced locomotor activity, piloerection, tachypnea). Sham animals underwent the same procedure but did not have their cecum ligated or perforated.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLateral ventricle cannulation\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAnimals were anesthetized with a mixture of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively) and fixed in a stereotaxic frame. A guide cannula (0.7 OD) was introduced in the left lateral ventricle using the following coordinates: AP -0.8mm / L\u0026thinsp;+\u0026thinsp;1.4mm/ DV -3.2 to -3.5 mm from bregma. Two screws were attached to the skull and dental cement were used to keep the cannula in position. Animals were allowed to recover from the surgery during 7 days before the intracerebroventricular injection.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCytokines measurement\u003c/em\u003e\u003c/p\u003e\u003cp\u003eBlood was collected from trunk after decapitation, centrifuged (3500 rpm, 20 min, 4\u0026deg;C) and plasma was separated for cytokines analysis. For peritoneal lavage, 5ml of cold PBS was injected in the peritoneal cavity and, after gentle local massage, an abdominal incision was made and 2ml of the lavage was collected using a plastic Pasteur pipette. It was then centrifugated (3500 rpm, 20 min, 4\u0026deg;C) and the supernatant was collected. Cytokines measurements were carried out using commercial ELISA kits and following the manufacturers\u0026rsquo; instructions (IL-1β [DY510, R\u0026amp;D Systems], IL-6 [DY501, R\u0026amp;D Systems] and IL-10 [DY522, R\u0026amp;D Systems]).\u003c/p\u003e\u003cp\u003e\u003cem\u003eSerotonin (5-HT) quantification\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePlasma and hypothalamic 5-HT were quantified by ELISA (ab133053, Abcam), following the manufactures\u0026rsquo; protocol. Plasma samples were prepared as described for the cytokine measurements. The hypothalamus was dissected and homogenized in RIPA buffer containing 10% protease cocktail inhibitor and kept under agitation in ice during 2 hours. Samples were centrifuged (4300 rpm, 20min, 4℃) and the supernatant was collected. Total protein quantification was accessed using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, 10741395). The results were expressed as ng/mL and ng/mg of protein.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePeritoneal macrophages cell culture\u003c/em\u003e\u003c/p\u003e\u003cp\u003eFor the \u003cem\u003ein vitro\u003c/em\u003e experiments using peritoneal macrophages, rats were injected intraperitoneally with 3% (w/v) Brewer thioglycollate medium. After three days, under deep anesthesia, cold RPMI medium was injected in the peritoneal cavity, the abdomen was gently massaged and an incision was made to carefully collect the fluid using a sterile Pasteur pipette. The peritoneal medium with cells was centrifuged (1500 rpm, 8 min, 4℃) and resuspended/ washed two times with cold medium, centrifuged again and the supernatant was discarded. Cells were resuspended in complete RPMI medium containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin. 1x10\u003csup\u003e6\u003c/sup\u003e cells were seeded per well in a 24-wells plate and kept in an incubator (37 ◦ C, 5% CO\u003csub\u003e2\u003c/sub\u003e) for 2 h. After this period, wells were washed two times with RPMI to removed non-adherent macrophages and cells were incubated overnight in supplemented medium. Cells were stimulated with LPS only (1\u0026micro;g/ml, 0111: B4, Sigma Aldrich) or FLV (20\u0026micro;M, 15617, Cayman), sigma-1 receptor antagonist (30\u0026micro;M, E-52862, Cayman) or serotonin (10\u0026micro;M, H9523, Sigma-Aldrich) 30 minutes before LPS. Twenty-four hours after LPS the supernatant was collected and frozen until cytokine quantification by ELISA. Experiments were performed at least three times.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and individual values are presented in the bar graphs. The normality of data was tested using the Shapiro\u0026ndash;Wilk normality test. Variables with normal distribution were analyzed by one-way analysis of variance (ANOVA), followed by post hoc Tukey tests. For those with nonparametric distribution, a Kruskal\u0026ndash;Wallis test was performed, followed by Dunn\u0026rsquo;s post hoc tests. At-test as used to compare two groups, when required. The statistical test used and the number of mouse subjects per group are reported in the figure legends. Values of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eSystemic and central fluvoxamine administration modulates cytokines production during sepsis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo investigate if fluvoxamine pretreatment modulates inflammation during sepsis, rats received FLV (30mg/kg; \u003cem\u003eLuvox\u003c/em\u003e, Abott) or vehicle (saline) by gavage during seven days before being subjected to sepsis by CLP. This dose is equivalent to the recommendation for humans (100-300mg/ day) and it was converted to rats based on the body surface area (Center for Drug Evaluation and Research \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). As expected, sepsis promoted a sustained increase in the levels of cytokines in the circulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c) and in the infection site, indicated by measurements in the peritoneal fluid (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f), 6 and 12 hours after CLP. These timepoints represent the acute phase of the disease, when the highest levels of inflammatory mediators are observed and when the immunomodulatory actions of FLV are likely to be observed. Interestingly, seven days treatment with fluvoxamine did not alter the levels of the pro-inflammatory cytokines IL-6 and IL-1β in septic animals, but it further increased both plasma and peritoneal levels of the anti-inflammatory cytokine IL-10. FLV pretreatment did not change cytokine levels in sham animals, which remained below detection levels using our ELISA kits.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven that FLV is able to cross the blood-brain barrier (BBB) and knowing that the brain orchestrates mechanisms that modulate systemic inflammatory response, we administered FLV intracerebroventricularly (i.c.v) to investigate the potential central action of this drug during sepsis. 22.8\u0026micro;g of fluvoxamine (Cayman) or its vehicle (20% DMSO) were infused in a volume of 5\u0026micro;l in each animal, 60 minutes before CLP surgery. This dose allows us to mimic the brain steady-steady state concentration of FLV (12\u0026micro;M) observed in patients with long-term use of this drug (Bolo et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Central FLV significantly reduced plasma levels of all the cytokines analyzed at 6 hours after CLP also decreased IL-1β at 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). Differently from the systemic pretreatment, it did not have any effect on the production of cytokines in the site of infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eFluvoxamine attenuates cytokine production by peritoneal macrophages in vitro\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe next investigated whether fluvoxamine modulates the inflammatory response through a direct action on immune cells. To this end, peritoneal macrophages were pretreated \u003cem\u003ein vitro\u003c/em\u003e with FLV or vehicle (XXX) diluted in culture medium 30 minutes before the inflammatory stimulus with LPS. After 24 hours, cytokine production was quantified by ELISA. FLV treatment significantly reduced the levels of IL-1β, IL-6, and IL-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Given that FLV may exert anti-inflammatory effects via sigma-1 receptor (S1R) agonism, we evaluated whether a selective S1R antagonist (S1RA) could alter FLV's immunomodulatory activity. Interestingly, S1RA alone reduced IL-6 and IL-10 levels in LPS-stimulated cells and did not block FLV\u0026rsquo;s effects. Instead, co-treatment with S1RA and FLV further enhanced the anti-inflammatory response (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eImmunomodulatory actions of systemic fluvoxamine are serotonin- independent\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo further elucidate the immunomodulatory mechanisms of fluvoxamine, we investigated the involvement of serotonin (5-HT), given that FLV is a selective serotonin reuptake inhibitor (SSRI). To assess the role of 5-HT \u003cem\u003ein vivo\u003c/em\u003e, serotonin was depleted using para-chlorophenylalanine (PCPA, 100 mg/kg; Sigma-Aldrich), administered intraperitoneally once daily in parallel with FLV pretreatment. PCPA was diluted in a 2% Tween 80 saline solution. Serotonin depletion alone significantly reduced circulating levels of all cytokines analyzed in septic animals and did not block the anti-inflammatory effects of FLV pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Measurement of plasma 5-HT levels revealed that FLV treatment did not elevate serotonin concentrations in either sham or septic animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), and sepsis itself did not alter plasma 5-HT levels. To investigate the direct effects of serotonin on immune cells, we treated peritoneal macrophages in vitro with 5-HT prior to LPS stimulation. Serotonin treatment alone did not significantly alter cytokine production. However, it partially reversed the inhibitory effects of FLV on IL-1β and IL-6 levels, while having no impact on FLV-mediated IL-10 modulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eMechanisms of immune modulation induced by central fluvoxamine administration\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo investigate the brain mechanisms underlying the systemic anti-inflammatory effects of centrally administered fluvoxamine, we examined the involvement of previously studied pathways. First, we assessed the role of the sigma-1 receptor by administering S1RA (160\u0026micro;g/ animals) intracerebroventricularly 30 minutes prior to FLV administration. Blocking this pathway did not reverse the anti-inflammatory effects of FLV, as IL-6 and IL-10 levels remained significantly lower than in animals treated with FLV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). We then evaluated the potential contribution of serotonin signaling using WAY-100635 (3\u0026micro;g/ animal), a selective 5-HT₁A receptor antagonist. This receptor subtype was chosen based on its involvement in central circuits responding to peripheral inflammation (Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Pretreatment with WAY-100635, administered i.c.v. 30 minutes before FLV, did not affect the cytokine modulation induced by central FLV administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). To further explore serotonin involvement, we measured 5-HT levels in the hypothalamus, a key brain region involved in the regulation of peripheral physiological responses, following seven days of oral FLV pretreatment. Septic animals exhibited a reduction in hypothalamic 5-HT compared to sham controls; however, FLV had no effect on 5-HT levels in either condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious investigations have suggested that drugs commonly used in the treatment of psychiatric disorders, like fluvoxamine, have anti-inflammatory properties (Nykamp et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Patel et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the current study, the analysis of cytokine production demonstrates that central and peripheral actions of FLV have distinct immunomodulatory effects. Oral pretreatment with FLV significantly upregulated IL-10 levels during sepsis, both in plasma and peritoneal lavage fluid. This finding is consistent with previous work showing that fluoxetine, another SSRI, also enhances IL-10 production during polymicrobial infection, and that this anti-inflammatory cytokine mediates fluoxetine-induced protection in sepsis (Gallant et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In contrast, intracerebroventricular administration of FLV had a broader effect, reducing all cytokines analyzed, including IL-10, in the plasma but not in the peritoneal lavage. These divergent effects suggest that systemic FLV may act directly at the site of infection, as indicated by the modulation of peritoneal exudate cytokines, while central FLV likely engages pathways that primarily act on circulating components. In addition to differences in the site of action, distinct cytokine regulation patterns were observed, particularly in the differential modulation of IL-10 compared to other pro-inflammatory mediators, indicating distinct pathways depending on the administration route and evidencing the complexity of the mechanisms of action of FLV in immune modulation.\u003c/p\u003e\u003cp\u003eTo investigate the potential mechanisms underlying fluvoxamine\u0026rsquo;s immunomodulatory effects during inflammation, we examined its direct action on immune cells. FLV significantly reduced cytokine production in LPS-stimulated macrophages \u003cem\u003ein vitro\u003c/em\u003e, indicating a direct anti-inflammatory effect. However, in contrast to the \u003cem\u003ein vivo\u003c/em\u003e findings, FLV did not increase IL-10 levels. One possible explanation is that the immunomodulatory effects of FLV depend on long-term exposure, which cannot be temporally replicated in cell culture conditions. Supporting this hypothesis, a previous study has shown that repeated administration of fluoxetine increases IL-10 production following LPS challenge \u003cem\u003ein vivo\u003c/em\u003e, whereas a single dose has no effect on plasma IL-10 levels (Kostadinov et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Although macrophages and circulating monocytes are key contributors to the acute immune response in sepsis, other immune cell populations may also be targets of FLV. Neutrophils, for example, are major producers of IL-10 during CLP-induced sepsis, particularly at the site of infection (Kasten et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Further studies are needed to determine whether FLV acts directly on these cells or modulates intercellular communication among different immune cell types during sepsis.\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that FLV attenuates inflammation during sepsis via activation of the sigma-1 receptor (S1R), a chaperone protein that interacts with IRE1 (inositol-requiring enzyme 1α), an endoplasmic reticulum stress sensor (Rosen et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). While all SSRIs function as S1R agonists, fluvoxamine has the highest binding affinity for this receptor (Narita et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), making it a potential therapeutic target. However, in our \u003cem\u003ein vitro\u003c/em\u003e experiments, the use of a selective S1R antagonist (S1RA) did not abolish the anti-inflammatory effects of FLV in LPS-stimulated macrophages, suggesting that FLV modulates cytokine production through an S1R-independent mechanism. Interestingly, S1RA alone also reduced cytokine expression and exhibited an additive anti-inflammatory effect when co-administered with FLV. The literature has conflicting data regarding the role of S1R activation in inflammation, with both pro- (Bravo-Caparr\u0026oacute;s et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Denaro et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and anti-inflammatory (Szabo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Shanmugam et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) effects being reported. Such discrepancies may be attributed to differences in experimental approaches\u0026mdash;including pharmacological versus genetic manipulation\u0026mdash;and to the diversity of cell types studied, such as macrophages, neutrophils, neurons, and astrocytes. These findings indicate that FLV modulates inflammation in macrophages independently of sigma-1 receptor activation.\u003c/p\u003e\u003cp\u003eAnother possible mechanism of action of FLV may involve serotonin, given that this drug is a 5-HT reuptake inhibitor, and numerous studies have suggested that the immunomodulatory effects of SSRIs are mediated by neurotransmitter. Immune cells express various subtypes of serotonin receptors, the serotonin transporter (SERT)- the primary target for SSRIs- and tryptophan hydroxylase (TPH), he rate-limiting enzyme in serotonin (5-HT) synthesis (Herr et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Administration of a TPH inhibitor led to reduced cytokine production in septic rats and the addition of 5-HT to peritoneal macrophage cultures abolished anti-inflammatory effect of FLV, indicating a serotonin-independent mechanism. Furthermore, TPH1 knockout mice\u0026mdash;which have significantly reduced circulating 5-HT\u0026mdash;showed lower mortality and decreased organ damage following sepsis (Zhang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Another study using the same TPH1-deficient model confirmed that the anti-inflammatory effects of fluoxetine are also independent of serotonin (Gallant et al, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Together with the observation that FLV pre-treatment does not alter plasma serotonin levels, these findings reinforce the hypothesis that the immunomodulatory actions of circulating FLV and other SSRIs are not mediated by serotonin.\u003c/p\u003e\u003cp\u003eTo the best of our knowledge, this is the first study to demonstrate that fluvoxamine modulates systemic inflammation via central mechanisms. Previous work has shown that i.c.v. administration of amitriptyline, a serotonin and norepinephrine reuptake inhibitor, reduces paw edema in rats in a dose-dependent manner (Hajhashemi et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), but no studies to date have investigated the central anti-inflammatory effects of SSRIs during sepsis. Despite being expressed in both neurons (Kourrich et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and glial cells (Wang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), sigma-1 receptor does not mediate the anti-inflammatory effects of FLV. Our group previously reported that central serotonin administration attenuates systemic inflammation (Mota et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) but this mechanism is unlikely to explain FLV's central effects. First, the anti-inflammatory actions of FLV were not abolished by pretreatment with the 5-HT1A receptor antagonist WAY-100635. Second, prolonged FLV administration did not elevate brain serotonin levels, regardless of sepsis. While the precise molecular target of central FLV remains to be identified, we can speculate the pathway through which it may exert its immunomodulatory actions during sepsis.\u003c/p\u003e\u003cp\u003eOne plausible mechanism involves modulation of the vagal anti-inflammatory reflex\u0026mdash;a key component of brain-to-immune system communication. In this pathway, afferent vagal signals convey peripheral inflammation to the brain, which in turn activates descending efferent vagal fibers that signal to the spleen and ultimately suppresses cytokine production (Tracey \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). It is possible that central FLV enhances the activity of these neural circuits, thereby amplifying the anti-inflammatory vagal reflex. Additionally, central FLV may act via humoral pathways such as activation of the hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal (HPA) axis, leading to increased corticosteroid release\u0026mdash;potent endogenous regulators of inflammation. Further investigation is needed to delineate the specific neural substrates and signaling pathways involved in the central anti-inflammatory action of FLV during sepsis.\u003c/p\u003e\u003cp\u003eIn summary, we demonstrate that fluvoxamine exerts anti-inflammatory effects during sepsis through both peripheral and central mechanisms that are independent of commonly pathways attributed to SSRIs, like serotonin and sigma-1 receptor activation. Our results expand the understanding of the immunomodulatory action of this drug beyond its conventional use as an antidepressant and support its potential repurposing for the treatment of inflammatory conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003eAll experiments were approved by the Animal Ethical Committee of the Dental School of Ribeir\u0026atilde;o Preto (2020.1.391.58.3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by Grants 2016/17681\u0026ndash;9 (LGSB) and 2019/27231\u0026ndash;9 (LHAC) from S\u0026atilde;o Paulo Research Foundation (FAPESP). This study was financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brasil (CAPES) - Finance Code 001. Financial support from the National Council for Scientific and Technological Development (CNPq) is acknowledged.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003eLHAC and LGSB designed the study. LHAC, IPT, WSS and KMA conducted the experiments. LHAC drafted the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eThe authors thank Nadir Fernandes and Mauro Silva for excellent technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbraham E, Wunderink R, Silverman H, et al (1995) Efficacy and Safety of Monoclonal Antibody to Human Tumor Necrosis Factor \u0026alpha; in Patients With Sepsis Syndrome: A Randomized, Controlled, Double-blind, Multicenter Clinical Trial. JAMA: The Journal of the American Medical Association 273:. https://doi.org/10.1001/jama.1995.03520360048038\u003c/li\u003e\n\u003cli\u003eAnnane D (2011) Corticosteroids for severe sepsis: An evidence-based guide for physicians. 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Aug;120:256-274. doi: 10.1016/j.bbi.2024.06.008.\u003c/li\u003e\n\u003cli\u003eZhang J, Bi J, Liu S, et al (2017) 5-HT Drives Mortality in Sepsis Induced by Cecal Ligation and Puncture in Mice. Mediators Inflamm 2017:. https://doi.org/10.1155/2017/6374283\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"selective serotonin reuptake inhibitor, sigma-1 receptor, serotonin, inflammation, macrophages, cytokines","lastPublishedDoi":"10.21203/rs.3.rs-7142228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7142228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSepsis is characterized by a dysregulated systemic inflammatory response to infection and remains a major global health challenge, underscoring the need for novel therapeutic strategies. Drug repurposing offers a promising strategy, and fluvoxamine (FLV), a selective serotonin reuptake inhibitor (SSRI) widely used in psychiatric treatment, has been reported to exhibit anti-inflammatory properties. Here, we investigated the effects of FLV in a murine model of sepsis induced by cecal ligation and puncture (CLP). Oral pretreatment with FLV for seven days significantly increased the anti-inflammatory cytokine IL-10 in both plasma and peritoneal fluid. To assess central nervous system involvement, FLV was administered intracerebroventricularly, resulting in a broad reduction in circulating cytokines, including both pro- and anti-inflammatory mediators. \u003cem\u003eIn vitro\u003c/em\u003e, FLV suppressed inflammatory cytokine production in LPS-stimulated macrophages, indicating a direct effect on immune cells. Notably, these immunomodulatory effects were independent of serotonin signaling and sigma-1 receptor activation\u0026mdash;pathways traditionally associated with SSRI mechanisms. These findings provide new insights into the immunomodulatory actions of FLV and support its potential repurposing as an adjunctive therapy for inflammatory diseases such as sepsis.\u003c/p\u003e","manuscriptTitle":"Fluvoxamine attenuates inflammation in experimental sepsis via novel non-canonical pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 10:23:22","doi":"10.21203/rs.3.rs-7142228/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-07-24T15:52:49+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-24T15:46:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Inflammopharmacology","date":"2025-07-23T11:19:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-17T11:45:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammopharmacology","date":"2025-07-16T12:53:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1537a842-facc-4af7-b354-13bab9494c61","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:02:57+00:00","versionOfRecord":{"articleIdentity":"rs-7142228","link":"https://doi.org/10.1007/s10787-025-02064-7","journal":{"identity":"inflammopharmacology","isVorOnly":false,"title":"Inflammopharmacology"},"publishedOn":"2025-12-15 15:58:16","publishedOnDateReadable":"December 15th, 2025"},"versionCreatedAt":"2025-07-28 10:23:22","video":"","vorDoi":"10.1007/s10787-025-02064-7","vorDoiUrl":"https://doi.org/10.1007/s10787-025-02064-7","workflowStages":[]},"version":"v1","identity":"rs-7142228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7142228","identity":"rs-7142228","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}