Toll-like receptor 4 deficiency mitigates acute DNBS-induced enteric neurogliopathy via modulation of excitatory and 5-HTergic pathways

Toll-like receptor 4 deficiency mitigates acute DNBS-induced enteric neurogliopathy via modulation of excitatory and 5-HTergic pathways | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 21 June 2025 V1 Latest version Share on Toll-like receptor 4 deficiency mitigates acute DNBS-induced enteric neurogliopathy via modulation of excitatory and 5-HTergic pathways Authors : Sofia Faggin 0000-0003-4813-2259 , Silvia Cerantola , Gloria Carrossa , Annalisa Bosi , Alessandra Ponti , Eleonora Napoli , Elena Stocco , Andrea Porzionato , Edoardo Savarino 0000-0002-3187-2894 , Valentina Caputi , Cristina Giaroni , and Maria Cecilia Giron 0000-0002-0825-7965 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175050561.11108548/v1 268 views 186 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Toll-like receptor 4 (TLR4) polymorphisms, dysregulated enteric neuro-glia network, and dysmotility have been observed in patients with Crohn’s Disease (CD) and related experimental animal models. However, the poorly understood pathological role of TLR4, combined with the absence of effective therapies for CD-associated gastrointestinal motor disorders, highlights the need for a deeper morpho-functional characterization of the underlying neuropathy. Experimental Approach: Male C57BL/6J (WT) and TLR4 -/- mice (10±2 weeks old) received intrarectally a single dose of 2.5% DNBS. After eight days, pro-inflammatory cytokines’ mRNA levels and histological anomalies were measured in ileal samples. Gut motility and ileal contractility were evaluated by changes in fluorescent-labelled dextran transit and by neuromuscular responses of ileal full-thickness to receptor/non-receptor-mediated stimuli, respectively. Enteric nervous system (ENS) neuroglial network was determined by confocal immunofluorescence microscopy in longitudinal muscle-myenteric plexus whole-mount preparations. Key Results: DNBS-induced ileitis led to prolonged gastrointestinal transit, reduced excitatory neuromuscular responses, accompanied by reactive gliosis, neurodegeneration and increased infiltration of IBA1 + macrophages in the ENS of WT mice. TLR4 deficiency elicits a protective effect towards DNBS-mediated damage on the morpho-functional integrity of ENS by modulating excitatory cholinergic and tachykinergic neurotransmissions as well as counteracting neuroglial alterations. An altered 5-HT-mediated neuromuscular response and 5-HT 4 R transcripts were identified during ileitis which was reversed by TLR4 deficiency. Conclusion and Implications: These outcomes reveal TLR4 signaling as a potential therapeutic target for addressing ENS-immunity axis anomalies, implicated in CD onset/progression. Original Article Toll-like receptor 4 deficiency mitigates acute DNBS-induced enteric neurogliopathy via modulation of excitatory and 5-HTergic pathways Sofia Faggin 1 , Silvia Cerantola 1 , Gloria Carrossa 1 , Annalisa Bosi 2 , Alessandra Ponti 2 , Eleonora Napoli 3 , Elena Stocco 4 , Andrea Porzionato 4 , Edoardo V. Savarino 5 , Valentina Caputi 6* , Cristina Giaroni 2# , Maria Cecilia Giron 1*# 1 Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy 2 Department of Medicine and Surgery, University of Insubria, Varese, Italy 3 Department of Neurology, School of Medicine, University of California Davis, Sacramento, CA, USA 4 Department of Neuroscience, University of Padova, Padova, Italy 5 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy 6 Department of Poultry Science, University of Arkansas, Fayetteville, AR, USA # Maria Cecilia Giron and # Cristina Giaroni share the last authorship *Maria Cecilia Giron and *Valentina Caputi are both corresponding authors: Maria Cecilia Giron, PharmD, PhD Pharmacology Building, Department of Pharmaceutical and Pharmacological Sciences University of Padova, Largo Meneghetti, 2 – 35131 Padova, Italy email: [email protected] Valentina Caputi, PhD Poultry Production and Product Safety Research Unit, Agricultural Research Service, USDA Department of Poultry Science, University of Arkansas, Fayetteville Fayetteville, AR, United States. email: [email protected] Word count: Abstract: 234 words Introduction: 526 words Results: 1982 words Discussion: 1483 words Total: 3991 words Background and Purpose: Toll-like receptor 4 (TLR4) polymorphisms, dysregulated enteric neuro-glia network, and dysmotility have been observed in patients with Crohn’s Disease (CD) and related experimental animal models. However, the poorly understood pathological role of TLR4, combined with the absence of effective therapies for CD-associated gastrointestinal motor disorders, highlights the need for a deeper morpho-functional characterization of the underlying neuropathy. Experimental Approach: Male C57BL/6J (WT) and TLR4 -/- mice (10±2 weeks old) received intrarectally a single dose of 2.5% DNBS. After eight days, pro-inflammatory cytokines’ mRNA levels and histological anomalies were measured in ileal samples. Gut motility and ileal contractility were evaluated by changes in fluorescent-labelled dextran transit and by neuromuscular responses of ileal full-thickness to receptor/non-receptor-mediated stimuli, respectively. Enteric nervous system (ENS) neuroglial network was determined by confocal immunofluorescence microscopy in longitudinal muscle-myenteric plexus whole-mount preparations. Key Results: DNBS-induced ileitis led to prolonged gastrointestinal transit, reduced excitatory neuromuscular responses, accompanied by reactive gliosis, neurodegeneration and increased infiltration of IBA1 + macrophages in the ENS of WT mice. TLR4 deficiency elicits a protective effect towards DNBS-mediated damage on the morpho-functional integrity of ENS by modulating excitatory cholinergic and tachykinergic neurotransmissions as well as counteracting neuroglial alterations. An altered 5-HT-mediated neuromuscular response and 5-HT 4 R transcripts were identified during ileitis which was reversed by TLR4 deficiency. Conclusion and Implications: These outcomes reveal TLR4 signaling as a potential therapeutic target for addressing ENS-immunity axis anomalies, implicated in CD onset/progression. Keywords: enteric nervous system, Toll-like receptor 4, Ileitis, α-synuclein, serotonin, intestinal motility, cholinergic neurotransmission Abbreviations α-syn, α-synuclein 5-HT, serotonin 5-HTRs, serotonergic receptors CD, Crohn’s disease ChAT, choline acetyltransferase DNBS, dinitrobenzene sulfonic acid EFS, electric field stimulation EGCs, enteric glial cells ENS, enteric nervous system GFAP, glial fibrillary acidic protein H&E, hematoxylin/eosin IBA1 ionized calcium-binding adapter molecule 1 IBD, inflammatory bowel disease iNOS, inducible NOS LMMP, longitudinal-muscle myenteric plexus nNOS, neuronal NOS SIDS, small intestine damage score SP, substance P TLRs, toll-like receptors UC, ulcerative colitis What is already known TLR4 signaling in the intestinal tract is a critical regulator of neuroimmunity. TLR4 polymorphisms are associated with the development of Crohn’s disease. What does this study add TLR4 drives DNBS-induced imbalance of excitatory/inhibitory neurotransmissions and shapes enteric neuroplasticity and neuro-immune crosstalk. TLR4 modulates 5-HTergic signaling and 5-HT 4 receptor expression during DNBS-induced ileitis. What is the clinical significance Our findings uncover TLR4 impact on excitatory and 5-HTergic pathways on DNBS-mediated small intestine dysmotility. By targeting/inhibiting TLR4, the outcome of ileitis and its consequence may be alleviated or even prevented. 1. INTRODUCTION The innate immune response relies on Toll-like receptors (TLRs), the most important class of pattern recognition receptors (PRRs), critically involved in initiating inflammatory responses and promoting adaptive immune reactions due to their ability to recognize a wide range of conserved molecular motifs, found in microbes (microbial-associated molecular patterns, MAMPs) or damaged cells (damage-associated molecular patterns, DAMPs) (Caputi, Marsilio, Cerantola, et al., 2017). Among all TLRs, TLR4 specifically recognizes MAMPs (e.g., cell wall-derived lipopolysaccharides of Gram-negative bacteria) and/or DAMPs (e.g., extracellular matrix components and intracellular factors, including DNA-binding proteins)(Kim, Kim, Lee, & Hwangbo, 2023). The activation of TLR4 triggers the inflammatory cascade through both MyD88-dependent and independent signaling pathways, underscoring its protective role against harmful insults; however, its excessive activation might lead to tissue damage. Recently, several potential MAMPs- and/or DAMPs-derived signals have been identified as critical players in the modulation of the enteric nervous system (ENS)(Rakoff-Nahoum, Paglino, Eslami-Varzaneh, Edberg, & Medzhitov, 2004). By expressing TLR4, neurons possess the ability to directly respond to MAMPs/DAMPs, with consequent release of diverse neurotransmitters in response to potentially harmful triggers (Rakoff-Nahoum et al., 2004). Conversely, various single nucleotide polymorphisms (SNPs) in the human TLR4 gene have been identified and associated with TLR4 hyporesponsiveness or increased susceptibility to several diseases, including inflammatory bowel disease (IBD) (Vlk et al., 2023). IBD, which includes chronic relapsing immune-mediated disorders, such as Crohn’s disease (CD) and ulcerative colitis (UC), affects more than 2 million people in Europe with a marked increase in incidence in the last decades (Zhao, Gonczi, Lakatos, & Burisch, 2021). Current clinical management of IBD generally includes a combination of therapies aimed at achieving and maintaining remission, alleviating symptoms, and preventing complications. However, while significant research has focused on identifying therapeutic strategies aimed at controlling mucosal inflammation, mediated by various genetic, environmental, immunological and microbial triggers (Zhao et al., 2021), little attention has been given to the assessment of the efficacy of these treatments in addressing IBD-associated ENS dysfunction. Over time, IBD can degenerate to enteric neuro-gliopathy, further disrupting GI functions and leading to several digestive issues (Suman, 2024). Alterations in enteric neurons are evident in both CD and UC, but they are more pronounced in patients with CD, who often exhibit myenteric and submucosal plexitis in the ileum and colon, highlighting the impact of GI immune responses on structure and activity of the enteric neuronal network (Le Berre, Naveilhan, & Rolli-Derkinderen, 2023; Suman, 2024). Given the central role played by the ENS in neuroimmune communication, a detailed understanding of ENS alterations during CD could pave the way for the development of targeted therapeutic approaches to address these chronic disorders. Recently, our group has shown that TLR4 deficiency affects ENS homeostasis determining an altered susceptibility to gut inflammatory damage (Cerantola et al., 2020; Faggin et al., 2025), supporting the key contribution of TLR4-ENS axis in ensuring gut function. Therefore, the present investigation aimed at identifying the neuroimmune pathways affected by DNBS-induced ileitis in absence of TLR4 signaling. Our findings reveal for the first time that during small intestine inflammation TLR4 plays a major role in modulating the main excitatory responses (i.e., cholinergic and tachykinergic contraction) and 5-HTergic neurotransmission, being therefore highly involved in the course and severity of enteric neuropathy. 2. METHODS 2.1 Animals and housing Male TLR4 -/- (B6.B10ScN-Tlr4 lps-del /JthJ; 10 ± 2 weeks old; Charles River Laboratories, Italy) and age-matched wild-type (WT) C57BL/6J mice (Charles River Laboratories, Italy) were housed in groups of five animals in individually ventilated cages at the Animal Facility of the Department of Pharmaceutical and Pharmacological Sciences, University of Padova. All animals were maintained under controlled environmental conditions (temperature 21 ± 1°C; relative humidity 60–70%) with a regular 12/12 h light/dark cycle, free access to standard laboratory chow and tap water ad libitum . All experimental protocols and animal care were performed in accordance with national and EU guidelines for the handling and use of experimental animals, EU Directive 2010/63/EU for animal experiments, and approved by the University of Padova Animal Care and Use Ethics Committee and by the Italian Ministry of Health (authorization number: 1142/2015-PR and 624/2021-PR). The experimental design ( Figure S1 ), statistical analyses, and successive data reporting in animal studies were executed in compliance with the ARRIVE 2.0 guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020) to improve research transparency and reproducibility. 2.2 In vivo treatment To induce experimental ileitis, animals were pre-sensitized applying 1% dinitrobenzene sulfonic acid (DNBS) in acetone/olive oil mixture (4:1 v/v ratio) onto mouse shaved dorsal skin, as previously described (Brun et al., 2013). After 1 week, 2.5 mg DNBS in 100 μL of 50% ethanol were administered into rectum of overnight-fasted and anaesthetized (1 g/kg isoflurane) mice. Control (SHAM) mice were similarly treated with vehicle (50% ethanol solution). Mice were daily monitored, by assessing changes in body weight, general clinical status and the development of diarrhea and/or hematochezia. One week after DNBS rectal administration, animals were sacrificed by cervical dislocation without anesthesia, to avoid any anesthesia-induced side effects on intestinal motor responses which are the basis of our investigation (Faggin et al., 2025). All the treatments and experimental procedures were blindly conducted. The extent of small intestine damage was evaluated using the SIDS (small intestine damage score) scoring system, as previously described (Faggin et al., 2025). In particular, we determined: (i) ulceration (0: normal; 1: ulceration with inflammation at one area; 2: ulceration/inflammation at two or more areas; 3: major damage throughout the small intestine); (ii) adhesions (0: no adhesions; 1: minor adhesions (intestine can be separated from other tissue with effort); 2: major adhesions); (iii) hyperemia (0: no hyperemia; 1: focal hyperemia (at one site); 2: hyperemia multiple areas; 3: hyperemia throughout small intestine) and (iv) bleeding (0: no bleeding; 1: blood at one site; 2: blood in multiple areas; 3: blood throughout the small intestine). 2.3 Histopathology Histological analysis was performed on ileal full-thickness cross sections, to evaluate inflammation and villous atrophy. Briefly, after small intestine dissection, ileal samples were excised 5-10 cm from ileocecal valve and carefully washed in phosphate-buffered saline (PBS), fixed in 10% buffered formalin and paraffin embedded. Sections (5 μm in thickness; 6–8 sections/animal) were cut and stained with hematoxylin and eosin (H&E) (Cerantola et al., 2022). A minimum of 10 independent fields per animal was examined in a blinded manner at low (x10) and high (x40) magnification with a Leica D4500B microscope (Leica Microsystems, Wetzlar, Germany) connected to a Leica DC 200 high‐resolution digital camera (Leica Microsystems). 2.4 Gastrointestinal transit analysis Mice were orally gavaged with non-absorbable FITC‐labelled dextran (FITC‐dextran; 70 kDa; 25 mg/kg in 0.9% saline solution), as previously described (Caputi, Marsilio, Filpa, et al., 2017; Faggin et al., 2025). After 30 minutes mice were sacrificed and the whole GI tract was collected. The stomach and caecum were analyzed separately while the small intestine and the colon were divided into 10 and 3 segments of equal length, respectively. Tissue and luminal contents from each segment were homogenized and cleared by centrifugation (10 000 × g for 10 min at 4°C). The fluorescence intensity of FITC-dextran was measured in the supernatants at 460/530 nm using a Victor plate reader (Victor, PerkinElmer; Wallac Instruments, Turku, Finland). The transit of FITC‐dextran along the GI tract was determined by calculating the percentage fluorescence for each segment and expressed as geometric center (GC) using the following equation: GC = Σ (% of total fluorescence signal x segment * segment number)/100 (Caputi, Marsilio, Filpa, et al., 2017). 2.5 Fecal pellet frequency and fecal water content Fecal pellet output was examined in all experimental groups and before euthanasia. Animals were individually placed in a novel environment (clean cage) and observed for one hour. The number of pellets produced in one hour by each mouse was recorded and the pellets were weighed (wet weight). Subsequently, pellets were dried overnight at 65°C and weighed again to obtain the dry weight. Fecal water content was measured as the difference between wet and dry weights and expressed as percentage (Cerantola et al., 2022). 2.6 Ex vivo contractility studies Intestinal contractility was assessed ex vivo by measuring tension changes in ileal samples using isolated organ bath technique as previously described (Cerantola et al., 2022; Marsilio et al., 2021). Briefly, distal ileum segments (1 cm in length) were isolated and mounted in organ baths containing 10 ml of oxygenated (95% O 2 /5% CO 2 ) Krebs solution (NaCl 118 mM, KCl 4.7 mM, CaCl 2 ∙2H 2 O 2.5 mM, MgSO 4 ∙7H 2 O 1.2 mM, K 2 HPO 4 1.2 mM, NaHCO 3 25 mM, C 6 H 12 O 6 11 mM) maintained at 37°C. Variations in smooth muscle tension were recorded by isometric transducers (World Precision Instruments, Berlin, Germany) connected to a PowerLab 4/30 data acquisition system using LabChart8 software (ADInstruments, Besozzo, VA, Italy). Ileal preparations were subjected to an initial tension of 0.5 g and an equilibration period for at least 45 min to allow the development of spontaneous rhythmic contractions. At the end of the equilibration period, preparations were challenged with 1 μM carbachol (CCh) until stable responses were obtained (Faggin et al., 2025). To study cholinergic-mediated responses ileal preparations were exposed to increasing concentrations of CCh (0.001–100 μM) to obtain cumulative concentration–effect curves. Neuronal-mediated contractions were evaluated by electrical field stimulation (EFS, 0–40 Hz; 1-ms pulse duration; 10-s pulse trains, 40 V) using platinum electrodes connected to an S88 stimulator (Grass Instrument) (Faggin et al., 2025). In order to evaluate the influence of tachykininergic neurotransmission on ileal contraction, the 10 Hz EFS-mediated tachykininergic contraction was assessed under non-adrenergic non-cholinergic (NANC) conditions obtained after 20-minute incubation with 1 µM atropine + 1 µM guanethidine. Ileal relaxation was analyzed following 10 Hz-EFS in NANC conditions. To evaluate the involvement of the nitrergic inhibitory neurotransmission, 10 Hz‐EFS‐induced relaxations in NANC conditions were evaluated following the addition of 10 μM 1400 W (a selective inducible NOS (iNOS) inhibitor) or 100 µM Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, a non-selective nitric oxide synthase (NOS) inhibitor) (Cerantola et al., 2020). Concentration-response curves to 5-HT (0.3–100 μM) were obtained in a non-cumulative manner (Forcen et al., 2015; Marsilio et al., 2021; B R Tuladhar, Womack, & Naylor, 2000). To determine the neuronal-mediated 5-HTergic response, 10 Hz-EFS was performed after a 20-minute preincubation with ondansetron (0.1 μM, 5-HT3R antagonist), ketanserin (1 μM, 5-HT2AR antagonist) or GR113808 (0.1 µM, 5-HT4R antagonist)(Marsilio et al., 2021). The antagonist concentrations used were based on the pKi previously described in the literature (Faggin et al., 2025). Contractile responses were expressed as gram tension/gram dry tissue weight of ileal segments, and ileal relaxation was calculated as area under curve (AUC)/gram dry tissue weight (Cerantola et al., 2021). 2.7 Immunohistochemistry on ileal whole mount preparations Isolated distal ileum segments (10 cm in length) were rinsed with PBS and fixed in 4% paraformaldehyde in PBS solution for 2 h at room temperature. After washing in PBS (3 × 15 min), ileal segments were cut in 0.5 cm-pieces, opened along the mesenteric border and placed on Sylgard-coated dishes, with the mucosal side down. Longitudinal muscle-myenteric plexus (LMMP) whole-mount preparations were isolated using a dissecting microscope, ss previously described (Cerantola et al., 2021). LMMP preparations were pinned down on the bottom of Sylgard-coated dishes and washed in PBT (PBS with 0.3% Triton X-100) for 45 min with gentle shaking. After blocking nonspecific-binding sites with 5% bovine serum albumin (BSA) in PBT for 1.5 h at room temperature, LMMP preparations were incubated overnight at room temperature with primary antibodies ( Table S1 ) diluted in PBT and 5% BSA. LMMP preparations were then washed in PBT (3 × 15 min) and incubated at room temperature for 2 h with appropriate secondary antibodies ( Table S1 ) diluted in PBT and 5% BSA. LMMP preparations were mounted on glass slides using a Mowiol mounting medium (Citifluor™ Mountant Solution AF1) and stored in the dark at -20 °C until analysis. Negative controls were obtained by incubating sections with isotype-matched control antibodies at the same concentration as primary antibody and/or pre-incubating each antibody with the corresponding control peptide (final concentration as indicated by manufacturer’s instructions). The immuno-related procedures used comply with the recommendations made by the British Pharmacological Society (Alexander et al., 2018). 2.8 Confocal image acquisition and analysis Images were acquired with a Zeiss LSM 800 confocal imaging system (Oberkochen, Germany) equipped with an oil-immersion 63× objectives (NA 1.4) used for the LMMP preparation. Z-series images (25 planes) of 512 × 512 pixels were captured and processed as maximum intensity projections for LMMP whole mount preparations. All microscope settings were kept constant for all images. The analysis of the total neuron population in the myenteric ganglia was performed by counting HuC/D + cells in 10 randomly chosen images per mouse. The total number of HuC/D + neurons or SOX10 + or VIP + cells was measured in each image and normalized per myenteric ganglion area, as previously described (Caputi, Marsilio, Filpa, et al., 2017; Cerantola et al., 2020). Changes in protein immunoreactivity were determined in LMMP whole mount preparation (20 captured images per mouse) (Cerantola et al., 2020; Marsilio et al., 2021) and expressed as fluorescence intensity (density index) normalized per myenteric ganglion area. All analyses were performed using Image J (Fiji) v.1.54i. 2.10 RNA isolation and quantitative RT-PCR Total RNA was extracted from mice mucosa-deprived small intestine samples with TRIzol (Invitrogen, Carlsbad, CA, USA) and treated with DNase I (DNase Free, Ambion) to remove possible traces of contaminating DNA. Two μg of total RNA was retrotranscribed using the High-Capacity cDNA synthesis kit (Applied Biosystems, Life Technologies, Grand Island, NY, USA). Quantitative RT-PCR was performed on the QuantStudio 3 Real-Time PCR Systems (Thermo Fisher Scientific, Carlsbad, California) with Power Sybr Green Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) following manufacturer’s instructions. Primers were designed to have a similar amplicon size and similar amplification efficiency as required for the utilization of the 2-ΔΔCt method to compare gene expression (Cerantola et al., 2022), using Primer Express software (Applied Biosystems, Foster City, CA, USA) based on available sequences deposited in public database. Primer sequences are reported in Table S2. For quantitative RT-PCR, a final concentration of 500 nm for each primer was used (in particular, 200 nM for 5HT 3 R) and experiments were performed in at least seven different biological samples for each experimental group (n = 5), as previously described (Bistoletti et al., 2019). 2 -ΔΔCt values obtained from the comparison between normalized Ct values of DNBS-treated samples with those obtained from control were used to evaluate the effect of DNBS-induced ileitis on the expression of pro-inflammatory cytokines or 5-HT receptors in the small intestine. 2.11 Statistical analysis All data are expressed as mean ± SEM except for the geometric center, which is presented as median and range (minimum-maximum). All the results were analyzed by investigators blinded to the treatments, using GraphPad Prism software v. 8.4 (San Diego, California, USA). Animals were randomly assigned to experimental groups. The distribution of data was tested with the Shapiro–Wilk normality test. Statistical significance was calculated with paired or unpaired Student’s t-test for two sample comparisons, two-way ANOVA followed by Bonferroni post hoc test for multiple comparison, or the non-parametric Mann–Whitney’s U-test for independent variables. The differences between groups were considered significant at P < 0.05; “N” values indicated the number of animals/group. Post-hoc tests were run only if F achieved P < 0.05, and there was no significant variance in homogeneity. Data and statistical analysis complied with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2022). 2.12 Materials Chemicals were of the highest commercially available analytical grade and purchased from Sigma-Aldrich. Paraformaldehyde (PFA) was purchased from Electron Microscopy Sciences (Società Italiana Chimici, Rome, Italy), and Triton X-100 was from AppliChem (Milan, Italy). All drugs for in vitro contractility studies were dissolved in milliQ water. The source of the antibodies used can found supplementary Table 1. Details of other materials and suppliers were provided in the specific sections. 2.13 Nomenclature of targets and ligands Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2023/24 (AlexanderChristopoulos, et al., 2023; Alexander, Fabbro, et al., 2023; Alexander, Kelly, et al., 2023). 3. RESULTS 3.1 TLR4 deficiency reduces the severity of DBNS-induced ileitis During the 7-day experimental procedure, the body weight of both WT and TLR4 -/- SHAM mice increased gradually ( Figure 1A,B ). In contrast, 7 days after DNBS rectal instillation, both genotypes exhibited a significant body weight loss ( Figure 1A,B ). Notably, the changes in body weights of WT treated mice occurred as early as 2 days after DNBS administration, whereas in TLR4 -/- mice they arose on day 4. The extent of macroscopic damage in GI tract was confirmed by a significant increase in the SIDS of both genotypes, with a much greater effect observed in WT than TLR4 -/- mice (7.4- and 3.3-fold, respectively, compared to related SHAM animals; Figure 1C ). In WT mucosa-deprived ileum, DNBS treatment caused 4.0-fold and 2.9-fold increase of the pro-inflammatory cytokines IL-6 ( Figure 1D ) and TNFα ( Figure 1E ) mRNA levels, respectively. In SHAM conditions, TLR4 deficiency determines a low-grade inflammation in the gut (Caputi, Marsilio, Cerantola, et al., 2017; Faggin et al., 2025), which was confirmed by a marked increase of IL-6 and TNFα levels compared to WT animals ( Figure 1D,E ). Surprisingly, DNBS treatment lowered the levels of these pro-inflammatory markers in the ileal mucosa-deprived tissue of TLR4 -/- mice ( Figure 1D,E ), implying an involvement of TLR4 signaling in DNBS-induced inflammation. Histopathological analyses of the ileal segments showed no evident differences among the experimental groups, although slight oedema was observed in WT DNBS mice ( Figure S2A ). However, there was no evidence of villi and crypts disruption, epithelial alteration, mucosa compromission and/or inflammation-foci. TLR4 -/- DNBS ileal sections were not associated with inflammation-related morpho-structural changes and microscopically nearly comparable to that of TLR4 -/- SHAM specimens ( Figure S2A ). No changes in small intestine length were found in both genotypes ( Figure S2B ). Intriguingly, only in WT mice, DNBS treatment caused a significant reduction of colon length ( Figure S2C ), along with marked alterations in the weight of stomach and caecum ( Figure S2D,E ). The development of DNBS-induced inflammatory process was further confirmed by spleen enlargement and weight gain in DNBS-treated WT mice compared to SHAM ( Figure S2F ). 3.2 Gastrointestinal motility is compromised in DNBS‐treated mice Considering that altered bowel habits are observed in CD patients, we analyzed the in vivo GI transit and fecal water content to assess the presence of altered intestinal motility and/or diarrhea, respectively. WT DNBS‐treated mice showed a slower gut motility compared to WT SHAM as demonstrated by higher FITC-dextran fluorescence in the GI upper part ( Figure 2A ), and a marked decrease of the GC (GC WT DNBS =6.1 vs GC WT SHAM =7.1; Figure 2C ). In addition, reduced gastric emptying ( Figure 2D) , lower fecal pellet frequency ( Figure 2E ) and increased stool water content ( Figure 2F ) were also observed in WT DNBS compared to WT SHAM mice. In TLR4 -/- mice, DNBS treatment significantly accelerated the distribution of FITC-dextran compared to SHAM mice (GC TLR4-/- DNBS =7.2 vs GC TLR4-/- SHAM =5.9; Figure 2B,C ), and increased gastric emptying, fecal pellet frequency and water content ( Figure 2D-F), suggesting the involvement of TLR4 signaling in ileitis-induced dysmotility. 3.3 DNBS-induced ileitis impacts the myenteric neuroglial network in a TLR4-dependent manner Considering the morpho-functional changes following ileitis, we sought to determine the influence of DNBS treatment on ENS architecture by immunohistochemical analysis of the glial markers S100β, GFAP and SOX10 together with the neuronal markers HuC/D and α-syn with confocal microscopy ( Figure 3 and Figure S3 ). In WT mice, DNBS-induced inflammation changed the distribution of these ENS markers ( Figure 3 ), further evidenced by a significant increase in S100β density index ( Figure S3A ), GFAP ( Figure S3B ) and α-syn ( Figure S3C ), and in the total number of SOX10 + cells ( Figure 3D ). These phenomena were associated to neurodegeneration, confirmed by the significant reduction of the total number of HuC/D + neurons ( Figure 3F) . Ileal myenteric plexus of TLR4 −/− SHAM mice showed the presence of reactive gliosis ( Figure 3 and Figure S3 ), as previously reported (Caputi, Marsilio, Cerantola, et al., 2017; Cerantola et al., 2020; Faggin et al., 2025), whereas DNBS treatment caused reduced S100β density index ( Figure S3A ), higher α-syn immunoreactivity ( Figure S3C ) and increased the total number of SOX10 + cells ( Figure 3D ). These findings, associated with no changes in GFAP immunostaining ( Figure S3B) and HuC/D + cells count ( Figure 3F) , indicate an involvement of TLR4 signaling in the regulation of the enteric glial cells’ phenotype and neurodegenerative processes during inflammation. 3.4 TLR4 signaling influences enteric excitatory neurotransmission during DNBS-induced ileitis In WT mice, DNBS-induced inflammation determined a significant reduction of CCh-mediated contraction compared to SHAM animals, as shown by the significant downward shift of the concentration-response curve to CCh and lower Emax value ( Figure 4A ). In contrast, TLR4 -/- DNBS mice showed a marked increase in the maximum response to CCh compared to corresponding SHAM mice ( Figure 4A ), reaching values comparable to those observed in the concentration-response curve of WT SHAM mice. We then assessed the presence of alterations in ENS activity by stimulating with EFS the release of endogenous enteric neurotransmitters from ileal segments. DNBS treatment determined again an opposite neuronal excitatory contractile response in the two genotypes. WT DNBS mice showed a significant downward shift of the frequency-response curve to EFS, as shown by the reduction of the maximum response at 40 Hz ( Figure 4B ). Conversely, DNBS-induced ileitis caused a significant increase of EFS-induced contraction in TLR4 -/- mice ( Figure 4B ), determining Emax values comparable with those observed in WT SHAM mice. Considering the neuronal contribution of cholinergic neurotransmission in 10 Hz-EFS-elicited contractions (Caputi, Marsilio, Cerantola, et al., 2017; Caputi, Marsilio, Filpa, et al., 2017), DNBS-induced ileitis triggered opposite 10 Hz-EFS-mediated responses in the two genotypes compared with related control mice ( Figure 4B ), supporting TLR4 contribution in tuning enteric cholinergic neurotransmission during intestinal inflammation. To confirm these results obtained from functional studies, we assessed the status of the myenteric cholinergic network in LMMP preparations from WT and TLR4 -/- mice, by analyzing the immunofluorescence distribution of the enzyme ChAT, involved in ACh synthesis and marker of cholinergic neurons ( Figure 4C ). DNBS-induced ileitis caused opposite effect in the number of ChAT + myenteric neurons of both genotypes compared to related SHAM animals ( Figure 4D ). We next evaluated the participation of tachykininergic excitatory tone in ileitis-induced GI dysmotility, analyzing its contribution to 10 Hz-EFS-induced response under NANC conditions ( Figure 5A ) along with the myenteric distribution of SP + neurons in ileal whole-mount preparations by confocal immunohistochemistry ( Figure 5B,C ). In WT mice, DNBS treatment determined a marked increase in tachykininergic excitatory response (1.9-fold; Figure 5A ) and in the number of myenteric SP + neurons (2.1-fold; Figure 5B,C ). Conversely, ileal preparations of TLR4 -/- SHAM mice showed a higher physiological tachykininergic response ( Figure 5A ) and a 1.7-fold increase in the number of myenteric SP + neurons compared to WT SHAM ( Figure 5B,C ). In TLR4 -/- DNBS mice, ileitis impaired tachykininergic-mediated contraction ( Figure 5A ) with no further changes in the total number of myenteric SP + neurons ( Figure 5B,C ). In WT mice, DNBS-induced ileitis determined an altered distribution of IBA1, a macrophage marker, suggestive of a current immune-mediated process, closely juxtaposed around myenteric plexus and enteric SP + neurons ( Figure 5D ). In the myenteric plexus of TLR4 −/− SHAM mice, IBA1 immunostaining appears much higher around neuroglia network compared to those observed in WT SHAM mice and decreased during ileitis, suggesting a TLR4-dependent macrophage recruitment in the neuromuscular layers. These findings further support an involvement of TLR4-mediated neuroimmune crosstalk in the altered efficiency of the excitatory pathways in the different experimental groups during DNBS-mediated inflammation. 3.5 DNBS-induced ileitis affects enteric inhibitory motor function Considering the alterations in the excitatory neurotransmission, we analyzed the impact of DNBS-induced ileitis on enteric inhibitory neuromuscular responses by evaluating 10-Hz-EFS-mediated relaxation in NANC conditions in ileal segments isolated from all the experimental groups. No changes in the NANC inhibitory response were found in WT mice following DNBS treatment compared to controls ( Figure 6A ). However, the addition of 1400W, a selective inhibitor of iNOS, significantly reduced 10 Hz-EFS-evoked relaxation ( Figure 6A ), which was further reduced after the pretreatment with pan-NOS inhibitor L-NAME ( Figure 6A ). As previously demonstrated by our group (Caputi, Marsilio, Cerantola, et al., 2017; Cerantola et al., 2020) and confirmed in this study, TLR4 -/- mice showed a 1.9-fold increase in NANC ileal relaxation with respect to WT SHAM mice ( Figure 6B ), which was roughly halved following DNBS treatment ( Figure 6B). In TLR4 -/- DNBS mice, the incubation with 1400W significantly lowered 10 Hz-EFS-evoked response, which was further reduced in presence of L-NAME ( Figure 6B) . Overall, these results suggest that DNBS treatment affects inhibitory tone of both genotypes, partially mediated by iNOS-produced NO sustaining ileal relaxation. In WT myenteric ganglia, DNBS treatment determined an iNOS overexpression, as confirmed by 1.8-fold increase of iNOS immunoreactivity ( Figure 6C,D ) and 3.5-fold rise of iNOS mRNA levels ( Figure 6E ). Slight alterations in iNOS immunoreactivity and transcript levels were found in TLR4 -/- mice following ileitis ( Figure 6C-E ), confirming our functional data on inhibitory neuromuscular response ( Figure 6B ). The immunohistochemical analysis of the myenteric distribution of nitrergic neurons revealed that DNBS treatment was associated with a marked increase of nNOS + neurons in WT DNBS mice with no changes in TLR4 -/- DNBS animals ( Figure 7A,B ). Given the alterations in EFS-induced response observed in WT mice following DNBS, we hypothesized an involvement of VIPergic inhibitory neurotransmission besides the nitrergic one. Immunofluorescence analysis revealed a 2.4-fold increase in the total number of myenteric VIP + neurons in WT DNBS mice ( Figure 7C,D ). In control conditions, TLR4 -/- mice are characterized by a 2-fold increase of VIP + neurons compared to WT SHAM mice, that was roughly halved following DNBS treatment ( Figure 7C,D ). 3.6 TLR4 signaling influences 5-HTergic neurotransmission during DNBS-induced ileitis Considering that intestinal inflammation is associated to changes in 5-HT signaling and content (Coates, Tekin, Vrana, & Mawe, 2017; Faggin et al., 2025), we evaluated the presence of DNBS-induced changes in intestinal motor response to 5-HT, analyzing the ileal contractility evoked by the non-cumulative addition of endogenous 5-HT. In WT DNBS mice, we found a significant upward shift of the concentration-response curve to 5-HT associated with a marked increase in the maximum contraction compared to SHAM group ( Figure 8A ). Transgenic mice in SHAM conditions showed an enhanced 5-HT-evoked contraction compared to WT mice ( Figure 8A ), which was reduced following DNBS treatment ( Figure 8A ), reaching values comparable to those obtained in WT SHAM. Concentration-dependent relaxations following the non-cumulative addition of 5-HT were significantly increased in small intestine preparations isolated from WT DNBS mice ( Figure 8B ). In contrast, a significant decrease in the response to exogenous 5-HT was observed in TLR4 -/- DNBS mice, with values returning to levels comparable to those observed in WT SHAM ( Figure 8B ). Considering that both intestinal inflammation and TLR4 signaling have been associated with alterations in the pattern of 5-HTRs expression as well as in the motor response mediated by 5-HT (Coates et al., 2017; Faggin et al., 2025), we assessed the effect of ketanserin (5-HT 2A R antagonist, Figure 9A ) ondansetron (5-HT 3 R antagonist, Figure 9B ), or GR113808 (5-HT 4 R antagonist, Figure 9C ) on EFS-elicited contractile response. In WT SHAM mice, ondansetron or GR113808 caused opposite effects in EFS-induced contractions ( Figure 9B,C ), with no alterations following the incubation with ketanserin ( Figure 9A ). Following DNBS-induced ileitis, EFS-induced contractions were markedly reduced in presence of either 5-HT 2A R or 5-HT 3 R antagonists ( Figure 9A,B ), whereas isolated preparations pretreated with the 5-HT 4 R inhibitor resulted to be insensitive to 5-HT 4 R inhibition ( Figure 9C ). Interestingly, TLR4 -/- SHAM ileal preparations showed a significant increase in EFS-mediated contraction after 5-HT 2A R, 5-HT 3 R or 5-HT 4 R inhibition ( Figure 9A-C ). In contrast, all three antagonists significantly reduced, in a comparable manner, the EFS-induced response of TLR4 -/- segments following DNBS treatment ( Figure 9A-C) , to indicate a clear impact of TLR4 deficiency on the 5-HTergic neurotransmission in presence of ileitis. In agreement, the analysis of 5-HTRs mRNA levels ( Figure 9D-F ) in mucosa-deprived ileal preparations highlighted a significant increase of 5-HT 4 R in both genotypes following DNBS treatment ( Figure 9F ). As previously observed (Faggin et al., 2025), 5-HT 2A R and 5-HT 3 R mRNA transcripts were found significantly increased in mucosa-deprived segments of TLR4 -/- SHAM and markedly reduced after DNBS-induced ileitis ( Figure 9D,E ). 4. DISCUSSION Sensory-motor alterations are hallmark features of gut dysfunction commonly reported by CD patients, that further corroborate the detrimental effect of inflammation on ENS. Indeed, hypertrophic and/or hyperplastic nerve bundles and glia have been observed in CD ileal and colonic specimens, mostly associated with the extent of inflammatory infiltrate (Lakhan & Kirchgessner, 2010). Considering our recent findings on the critical involvement of TLR4 in gut dysmotility and ENS plasticity in DSS-induced ileitis (Faggin et al., 2025), in this study we evaluated the impact of TLR4 signaling on enteric neurodysfunction in DNBS-mediated ileitis which resulted in distinctive ENS morpho-functional changes compared to those revealed in DSS model. We show for the first time that, TLR4 deficiency mostly mitigates DNBS-induced alterations in ENS circuitries, gut motility, excitatory neuromuscular responses and 5-HTergic pathways. Specifically, our findings reveal that DNBS ileitis leads to the following changes associated to TLR4 signaling: (i) slower GI transit; (ii) reactive gliosis and neurodegeneration, associated with abnormal distribution of α-syn protein and increased IBA1 + macrophages infiltrate in myenteric plexus; (iii) depressed excitatory neuromuscular responses, mainly mediated by cholinergic and tachykinergic pathways; (iv) evident iNOS-derived NO-dependent inhibitory neurotransmission; (v) increased exogenous 5-HT-mediated neuromuscular responses, involving an altered 5-HT 4 R activity. Although DNBS-mediated effects on gut motility and ENS function have been well documented in colon (Antoniou et al., 2016; Palenca et al., 2022), only few studies have explored the impact on small intestine (Barada et al., 2006; Barada et al., 2007). The temporal (e.g., 3, 7, 21 days) changes in the expression of pro-inflammatory cytokines in rat duodenum, jejunum, ileum and colon, have been characterized following TNBS-induced colitis (Barada et al., 2006). A marked increase in TNF-α and IL-6 levels occurred at 3 h, peaked at 3–7 days, and began to subside 21 days after colitis induction in apparently non-inflamed small intestine as well as in inflamed colon. In our study, similar ileal inflammatory and histopathological changes were observed after 8-day DNBS administration and were partially preserved by TLR4 deficiency, paralleling our previous results in DSS-induced ileitis (Faggin et al., 2025). TLR4 signaling is essential for maintaining intestinal homeostasis and regulating susceptibility to DSS (Faggin et al., 2025), DNBS (Wu et al., 2019) and bacteria clearance (Fukata et al., 2005). In physiological conditions, TLR4 deficiency elicit low-grade inflammation, characterized by dysmotility and neurogliopathy (Anitha, Vijay-Kumar, Sitaraman, Gewirtz, & Srinivasan, 2012; Caputi, Marsilio, Cerantola, et al., 2017). However, during ileitis, TLR4 knockout downregulates IBA1 expression, index of lower macrophage infiltrate (Anitha et al., 2012; Fukata et al., 2005; Rakoff-Nahoum et al., 2004). These findings highlight a TLR4-mediated dual role in protecting gut homeostasis and influencing inflammatory processes (Wu et al., 2019; Zeng et al., 2025), further contributing to individual susceptibility to CD (Feki et al., 2017). In our study, DNBS treatment in WT mice reduced HuC/D + neurons and myenteric ganglia size, associated with increased glial GFAP and S100β immunoreactivity and higher content of SOX10 + cells, suggesting an influence of inflammatory process on EGCs transcriptomes, possibly affecting neural progenitors, as previously noted in DSS ileitis (Faggin et al., 2025; Rosenbaum et al., 2016). The myenteric GFAP-expressing glial subpopulation are susceptible to inflammation by secreting different inflammatory cytokines (von Boyen et al., 2004). Differential expression patterns of several EGCs markers (e.g., SOX10, S100β, GFAP) account for glial heterogeneity and plasticity to ensure a multipotent state primed for neurogenesis as well as ENS resilience to luminal stimuli (Boesmans, Lasrado, Vanden Berghe, & Pachnis, 2015). Neurodegeneration and neurodysfunction have been shown in CD biopsies together with higher expression of neuronal α-syn and glial S100β (Casini et al., 2025). Increased α-syn levels in patients with either acute or chronic gut inflammation likely result from decreased protein degradation and higher mobilization within ENS, as an immune defense mechanism (Prigent et al., 2019; Suman, 2024; Swaminathan, Fung, Finkelstein, Bornstein, & Foong, 2019). Conversely, S100β expression and secretion have been related to inflammation severity (Cirillo et al., 2011). Interestingly, α-syn and S100β can interact with TLR4 expressed on neurons and EGCs, leading to increased gut permeability and inflammatory response, contributing to dysmotility and dysregulated ENS functions (Casini et al., 2025). Accordingly, here we observed that TLR4 deficiency counteracted neuroglial reactivity during DNBS-mediated ileitis. Comparable findings were obtained in DSS-treated TLR4 -/- mice (Faggin et al., 2025) and in EGCs deriving from DSS-treated mice or IBD patients incubated with palmitoylethanolamide, an inhibitor of S100β-TLR4 axis (Esposito et al., 2014). Recent results have highlighted the key role of TLR4 activation/inhibition in modulating excitatory (e.g., cholinergic) and inhibitory (e.g., nitrergic) neurotransmissions to ensure gut homeostasis (Anitha et al., 2012; Caputi, Marsilio, Cerantola, et al., 2017; Layunta, Forcen, & Grasa, 2022). Dysmotility is a common characteristics of intestinal inflammation in human and drug- or infection-induced animal models (Cheon, Cui, Yeon, Kwon, & Park, 2012). These changes, observed in CD patients, may result from anomalies in neuromuscular tone and/or activity of gut extrinsic (e.g., sympathetic and parasympathetic) and intrinsic (e.g., nitrergic) innervation (Hosie et al., 2024). Considering the neuropathy observed in CD biopsies, we here add new evidence that, during ileitis, TLR4 signaling is highly implicated in coordinating bidirectional neuron-glial communication, likely tuning excitatory (e.g., cholinergic and tachykinergic) and inhibitory (e.g., nitrergic and VIPergic) neurotransmission, contributing to their effects on motility. Prior studies have shown that 10 Hz-EFS generally elicits a maximal cholinergic contraction (Brun et al., 2013) whereas 10 Hz-EFS in NANC conditions uncovers tachykinergic-evoked rebound contractions (Caputi, Marsilio, Cerantola, et al., 2017; Zizzo, Mule, & Serio, 2005). DSS ileitis determines evident changes such as enhanced EFS-elicited neurogenic contractions/relaxations (Faggin et al., 2025). Conversely, DNBS ileitis depressed EFS-elicited neurogenic contractions, with no effects on relaxations. Despite the observed reduction of NANC-mediated relaxation after L-NAME incubation, no proportional differences were found between control and DNBS groups, whereas a significant decrement appeared only after iNOS inhibition, reinforcing the involvement of iNOS-derived NO in ileal dysmotility during inflammation. In humans and animal models, both increased and decreased neuromuscular contractility has been observed during colitis, due to the altered mixture of inflammatory factors, known to influence neuromuscular activity by acting either on calcium channels or cholinergic receptors (Akiho, Ihara, Motomura, & Nakamura, 2011; Khan & Collins, 2006; Ohama, Hori, & Ozaki, 2007). Intriguingly, we provide new evidence that TLR4 signaling contributes to cholinergic and tachykinergic dysfunctions in ileitis. Reduced muscarinic response and enhanced acetylcholine degradation through abnormal acetylcholinesterase activity has been observed in colitis-induced immune infiltrate in the ileum and colon (Gomez-Bris et al., 2025), further highlighting the impact of gut neuroimmune crosstalk during inflammation. Enteric tachykinergic signaling is prominent in motor disorders associated with inflammation (i.e., IBD, diverticulitis). Higher SP release has been detected from both ENS neurons and immune cells of intestinal lamina propria (e.g., macrophages), suggesting the involvement of the tachykinergic system in the pathogenesis of motor dysfunctions (Pellegrini et al., 2016). Accordingly, TLR4 deficiency determined opposite results on excitatory-mediated contraction, counteracting DNBS-dependent neuroimmune effects and related inflammatory pathways, as evidenced by reduced myenteric infiltrate of IBA1 + macrophages, cytokines milieu and α-syn distribution. Therefore, our results implicate a myenteric tachykinergic/TLR4 axis in curbing DNBS-induced neuro-immunological changes. Intriguingly, we and others have reported TLR4 contribution in tuning 5-HT control of gut motility and ENS homeostasis (Forcen et al., 2015; Marsilio et al., 2021). However, unlike the DSS model (Faggin et al., 2025), DNBS-induced ileitis affected ileal maximum response evoked by non-cumulative addition of exogenous 5-HT, probably due to compensatory neuroplastic changes in myenteric ganglia as well as 5-HT 4 R activity. Following DNBS treatment, TLR4 deficiency markedly tuned 5-HTergic-mediated contraction and relaxation together with transcript levels of 5-HT 2A R, 5-HT 3 R and 5-HT 4 R. Exogenous 5-HT-mediated regulation of intestinal contraction and relaxation is well documented (B. R. Tuladhar, Costall, & Naylor, 1996; B R Tuladhar et al., 2000). Considering the existing species-specific differences in 5-HTR activity, the agonist/antagonist profiles of 5-HTR subtypes in ileum may differ from those observed in other animal tissues (B R Tuladhar et al., 2000). In mouse ileum, neuronal 5-HT 3 R, 5-HT 2A R and facilitate neuromuscular contraction primarily through cholinergic pathways (Kelley et al., 2014; Mashhadi, Naylor, & Javid, 2014; B R Tuladhar et al., 2000). In our study, the different modulation of EFS-induced response with 5-HTRs antagonists may result from changes in 5-HTRs activity during DNBS-induced inflammation. Indeed, in vivo treatment with specific 5‐HT 2A R or 5‐HT 3 R antagonists or 5‐HT 4 R agonist has been shown to determine a sustained benefit in DSS/TNBS-induced colitis by promoting GI motility, attenuating histological damage and reducing visceral hypersensitivity (Raouf et al., 2024; Spohn et al., 2016; Utsumi, Matsumoto, Amagase, Horie, & Kato, 2016; Xiao et al., 2016). Additionally, 5‐HT 4 R-dependent mechanisms appear to favor neurogenesis during DSS through EGCs differentiation into neurons (Belkind-Gerson et al., 2015). In conclusion, these findings further corroborate the existence of a downstream 5-HTergic-TLR4 axis involved in the regulation of neuroimmune responses that could potentially attenuate some of the dysmotility symptoms of IBD, as previously highlighted in DSS model (Faggin et al., 2025). However, the precise mechanisms by which cholinergic-5-HTergic-TLR4 axis may reverse IBD dysmotility need to be clarified and may involve a positive relationship between serotonin content, excitatory neurotransmission and intestinal susceptibility to inflammation (Raouf et al., 2024). Acknowledgments This study was supported by grants from University of Padova to M.C.G (San Camillo Hospital Grant, Treviso (Italy); UNIPD-DSF-PRID-2023; AlfaWassermann spa VC2016SC2019AW), to S.F. (MUR/University of Padova PhD Fellowship 2020; Department of Pharmaceutical and Pharmacological Sciences PostDoc Fellowship ARD-B 2023; Department of Pharmaceutical and Pharmacological Sciences PostDoc Fellowship ARD-A 2024), to S.C. (Department of Pharmaceutical and Pharmacological Sciences PostDoc Fellowship ARD-B 2020; ECCO Grant 2022), and to G.C. (MUR-PNRR/University of Padova PhD Fellowship 2023). The funders had no role in study design, data collection and analysis, or the preparation of or decision to publish the manuscript. The authors would like to thank Francesca Patrese, DMV, and Ludovico Scenna, DMV (University of Padova, Padova, Italy), for veterinary assistance, Alessia Forgiarini, Andrea Pagetta, Carla Argentini and Massimo Rizza (University of Padova, Padova, Italy) for technical assistance in animal handling and experimental procedures. CRediT authorship contribution statement Sofia Faggin : conceptualization, data curation, formal analysis, investigation, methodology, writing—original draft preparation, writing—review and editing; Silvia Cerantola : conceptualization, data curation, formal analysis, investigation, methodology, writing—review and editing; Gloria Carrossa : data curation, formal analysis, investigation, methodology; Annalisa Bosi : investigation, formal analysis, methodology; Alessandra Ponti : investigation, formal analysis; Eleonora Napoli : investigation, methodology, writing—review and editing; Elena Stocco : formal analysis, methodology, writing—original draft preparation; Andrea Porzionato : conceptualization, formal analysis, methodology, writing—original draft preparation, writing—review and editing; Edoardo V. Savarino : conceptualization, funding acquisition, supervision, writing—original draft preparation, writing—review and editing; Valentina Caputi : conceptualization, formal analysis, methodology, supervision, writing—original draft preparation, writing—review and editing; Cristina Giaroni : conceptualization, funding acquisition, methodology, project administration, supervision, writing—original draft preparation, writing—review and editing; Maria Cecilia Giron : conceptualization, data curation, formal analysis, funding acquisition, methodology, project administration, supervision, writing—original draft preparation, writing—review and editing. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest EVS has served as speaker for Abbvie, Abivax, Agave, AGPharma, Alfasigma, CaDiGroup, Celltrion, Dr Falk, EG Stada Group, Fenix Pharma, Galapagos, Johnson&Johnson, JB Pharmaceuticals, Innovamedica/Adacyte, Eli Lilly, Malesci, Mayoly Biohealth, Omega Pharma, Pfizer, Reckitt Benckiser, Sandoz, SILA, Sofar, Takeda, Tillots, Unifarco; has served as consultant for Abbvie, Agave, Alfasigma, Biogen, Bristol-Myers Squibb, Celltrion, Dr. Falk, Eli Lilly, Fenix Pharma, Johnson&Johnson, JB Pharmaceuticals, Merck & Co, Nestlè, Pfizer, Reckitt Benckiser, Regeneron, Sanofi, SILA, Sofar, Takeda, Unifarco; he received research support from Bonollo, Difass, Pfizer, Reckitt Benckiser, SILA, Sofar, Unifarco, Zeta Farmaceutici. The Authors have no affiliations with organizations that have direct or indirect financial interests in the topic or materials covered in the manuscript. REFERENCES https://doi.org/10.1038/sj.bjp.0703747 Akiho, H., Ihara, E., Motomura, Y., & Nakamura, K. 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TLR4 deficiency alleviates dinitrobenzene sulfonic acid (DNBS)-induced ileitis in mice. (A,B) Changes in percentage of body weight during 7-day DNBS treatment in both WT (A) and TLR4 -/- (B) mice; N = 30. (C) Small intestine damage score (SIDS) assessment of DNBS-induced ileitis; N = 20. (D,E) qRT-PCR quantification of IL-6 (D) and TNF-α (E) mRNA levels in the small intestine of WT and TLR4 -/- mice in absence or presence of DNBS treatment; N = 5. Data are reported as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus related SHAM genotype; °°°P < 0.001 versus WT SHAM mice; ### P < 0.001 versus WT DNBS mice. FIGURE 2. Dinitrobenzene sulfonic acid (DNBS) treatment affects GI motility in WT and TLR4 -/- mice . (A,B) Distribution of the non-absorbable FITC dextran 70 kDa, reported as % fluorescence ± SEM in the individual segments of the digestive tract (Cec, caecum; Col1–Col3, colon segments; Sb1–Sb10, small bowel segments; Sto, stomach) in WT (A) and TLR4 -/- (B) mice in absence or presence of DNBS treatment; N = 10. (C) Geometric center calculated following the analysis of the GI transit, reported as median with its percentile; N = 10. (D) Percentage of gastric emptying in WT and TLR4 -/- mice in absence or presence of DNBS treatment; N = 10. (E,F) Fecal pellet frequency per hour and percentage of fecal water content in WT and TLR4 -/- mice in absence or presence of DNBS treatment; N = 20. Data are reported as mean ± SEM. **P < 0.01, ***P < 0.001 versus related SHAM genotype; °P < 0.05, °°P < 0.01, °°°P < 0.001 versus WT SHAM mice; ### P < 0.001 versus WT DNBS mice. FIGURE 3. Dinitrobenzene sulfonic acid (DNBS) treatment produces TLR4-dependent neuroplastic changes in the myenteric neuroglial network of WT mice. (A,B,C,E) Representative confocal microphotographs showing the distribution of HuC/D + (red) neurons, S100β + (green, A ), glial fibrillary acidic protein (GFAP) + (green, B ), SOX10 + (yellow, C ) glial cells, and α-synuclein (cyan, E ) in longitudinal muscle–myenteric plexus (LMMP) preparations of WT and TLR4 -/- mice in the absence or presence of DNBS treatment (bars = 22 μm). (D,F) Analysis of the total number of SOX10 + glial cells (D) or HuC/D + neurons (F) in LMMP preparations of WT and TLR4 -/- mice, in the absence or presence of DNBS treatment; N = 5. Data are reported as mean ± SEM. **P < 0.01, ***P < 0.001 versus related SHAM genotype; °°°P < 0.001 versus WT SHAM mice. FIGURE 4. TLR4 signaling influences ileal excitatory cholinergic pathway upon dinitrobenzene sulfonic acid (DNBS) treatment. (A) Concentration-response curves to carbachol (CCh; 0.001–100 μM) in isolated ileal preparations of WT and TLR4 -/- mice in the absence or presence of DNBS treatment; N = 10. (B) Electric field stimulation (EFS; 0–40 Hz)-induced contraction in ileal preparations isolated from WT and TLR4 -/- mice in the absence or presence of DNBS treatment; N = 10. (C) Representative confocal microphotographs showing the distribution of HuC/D + (green) and ChAT + (red) neurons in longitudinal muscle–myenteric plexus (LMMP) preparations of WT and TLR4 -/- mice in absence or presence of DNBS treatment (bars = 22 μm). (D) Analysis of total number of ChAT + neurons in LMMP preparations of WT and TLR4 -/- mice in the absence or presence of DNBS treatment; N = 5. Data are reported as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus related SHAM genotype; °P < 0.05, °°P < 0.01, °°°P < 0.001 versus WT SHAM mice; # P < 0.05, ## P < 0.01, ### P < 0.001 versus WT DNBS mice. FIGURE 5. TLR4 signaling impacts myenteric tachikininergic neurochemical coding upon dinitrobenzene sulfonic acid (DNBS) treatment. (A) Tachykininergic nerve-evoked contractions induced by 10 Hz-EFS in NANC conditions, in isolated ileal preparations of WT and TLR4 -/- mice in the absence or presence of DNBS treatment; N = 10. (B) Representative confocal microphotographs showing the distribution of HuC/D + (red) and SP + (green) neurons in longitudinal muscle–myenteric plexus (LMMP) preparations of WT and TLR4 -/- mice in absence or presence of DNBS treatment (bars = 22 μm). (C) Analysis of total number of SP + neurons in LMMP preparations of WT and TLR4 -/- mice in the absence or presence of DNBS treatment; N = 5. (D) Representative confocal microphotographs showing the distribution of substance P + (SP; magenta) neuronal fibers and IBA1 + (yellow) macrophages in LMMP preparations of WT and TLR4 -/- mice in the absence or presence of DNBS treatment (bars = 22 μm). Data are reported as mean ± SEM. ***P < 0.001 versus related SHAM genotype; °°P < 0.01 and °°°P < 0.001 versus WT SHAM mice; ### P < 0.001 versus WT DNBS mice. FIGURE 6. Dinitrobenzene sulfonic acid (DNBS) treatment affects enteric nitrergic-mediated relaxation . (A,B) 10-Hz electric field stimulation (EFS)-evoked NANC relaxation with or without 1 μM 1400W (iNOS inhibitor) or 100 μM L-NAME (pan-NOS inhibitor) in isolated ileal preparations of WT (A) and TLR4 -/- (B) mice in the absence or presence of DNBS treatment; N = 10. (C) Representative confocal microphotographs showing the distribution of HuC/D + (red) neurons and iNOS (yellow) in longitudinal muscle–myenteric plexus (LMMP) preparations of WT and TLR4 -/- mice in the absence or presence of DNBS treatment (bars = 22 μm). (D,E) Analysis of iNOS fluorescence intensity (D) or RT-PCR quantification of iNOS transcripts (E) in LMMP preparations of WT and TLR4 -/- mice in the absence or presence of DNBS treatment; N = 5. Data are reported as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus related SHAM genotype; °P < 0.05, °°P < 0.01, °°°P < 0.001 versus WT SHAM mice; ## P < 0.01, ### P < 0.001 versus WT DNBS mice. FIGURE 7. TLR4 signaling influences myenteric neurochemical coding upon dinitrobenzene sulfonic acid (DNBS) treatment. (A,C) Representative confocal microphotographs showing the distribution of HuC/D + (red, A,C ), nNOS + (green, A ), or VIP + (green, C ) neurons in longitudinal muscle–myenteric plexus (LMMP) preparations of WT and TLR4 -/- mice in absence or presence of DNBS treatment (bars = 22 μm). (B,D) Analysis of total number of nNOS + (B) , or VIP + (D) neurons in LMMP preparations of WT and TLR4 -/- mice in the absence or presence of DNBS treatment; N = 5. Data are reported as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus related SHAM genotype; °P < 0.05 versus WT SHAM mice; ## P < 0.01, ### P < 0.001 versus WT DNBS mice. FIGURE 8. TLR4 signaling alters 5-HT-mediated ileal responses following dinitrobenzene sulfonic acid (DNBS) treatment. Non-cumulative concentration-response curves to 5-HT (0.3–100 μM) inducing ileal contraction (A) and rebound relaxation (B) in isolated ileal preparations from WT and TLR4 -/- mice in the absence or presence of DNBS treatment; N =10. Data are reported as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus related sham genotype; °P < 0.05, °°P < 0.01 versus WT SHAM mice; ### P < 0.001 versus WT DNBS mice. FIGURE 9. TLR4 signaling influences 5-HT 3 R, 5-HT 2A R and 5-HT 4 R activity and expression upon dinitrobenzene sulfonic acid (DNBS) treatment. (A-C) 10-Hz electric field stimulation (EFS)-evoked contraction with or without 1 μM ketanserin (A) , or 0.1 μM ondansetron (B) , or 0.1 µM GR113808 (C) in isolated ileal segments of WT and TLR4 -/- mice in absence or presence of DNBS treatment; N = 10. Data are reported as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 versus respective control in absence of antagonists. (D-F) RT-PCR quantification of 5-HT 2A receptor (D) , 5-HT 3 receptor (E) , and 5-HT 4 receptor (F) mRNA levels in the ileum of WT and TLR4 -/- mice in absence or presence of DNBS treatment; N = 5. Data are reported as mean ± SEM. **P < 0.01, ***P < 0.001 versus related SHAM genotype; °°°P < 0.001 versus WT SHAM mice; ### P < 0.001 versus WT DNBS mice. SUPPORTING INFORMATION FIGURE S1. Experimental design and treatment strategy. FIGURE S2. Dinitrobenzene sulfonic acid (DNBS) treatment alters GI morphology only in WT mice. (A) Representative images showing ileal cross-sections stained with hematoxylin and eosin (H&E) stained ileal sections from WT and TLR4 -/- mice in absence or presence of DNBS treatment (bars = 200 μm). (B-F) Analysis of small intestine (B) and colon (C) length, and stomach (D) , caecum (E) , and spleen (F) weight in WT and TLR4 -/- mice in absence or presence of DNBS treatment; N=30. Data are reported as mean ± SEM. **P < 0.01, ***P < 0.001 versus related SHAM genotype; °P < 0.05 versus WT SHAM mice; # P < 0.05, ## P < 0.01, ### P < 0.001 versus WT DNBS mice. FIGURE S3. Dinitrobenzene sulfonic acid (DNBS) treatment alters ENS phenotype only in WT mice. (A-C) Analysis of S100β (A) , GFAP (B) and α-synuclein (C) fluorescence intensity index in LMMP preparations of WT and TLR4 -/- mice, in the absence or presence of DNBS treatment; N = 5. Data are reported as mean ± SEM. **P < 0.01, ***P < 0.001 versus related SHAM genotype; °°°P < 0.001 versus WT SHAM mice; ## P < 0.01, ### P < 0.001 versus WT DNBS mice. 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Authors Affiliations Sofia Faggin 0000-0003-4813-2259 Universita degli Studi di Padova Dipartimento di Scienze del Farmaco View all articles by this author Silvia Cerantola Universita degli Studi di Padova Dipartimento di Scienze del Farmaco View all articles by this author Gloria Carrossa Universita degli Studi di Padova Dipartimento di Scienze del Farmaco View all articles by this author Annalisa Bosi Universita degli Studi dell'Insubria Dipartimento di Medicina e Chirurgia View all articles by this author Alessandra Ponti Universita degli Studi dell'Insubria Dipartimento di Medicina e Chirurgia View all articles by this author Eleonora Napoli University of California Davis Department of Neurology View all articles by this author Elena Stocco Universita degli Studi di Padova Dipartimento di Neuroscienze View all articles by this author Andrea Porzionato Universita degli Studi di Padova Dipartimento di Neuroscienze View all articles by this author Edoardo Savarino 0000-0002-3187-2894 University of Padova View all articles by this author Valentina Caputi University of Arkansas Department of Poultry Science View all articles by this author Cristina Giaroni Universita degli Studi dell'Insubria Dipartimento di Medicina e Chirurgia View all articles by this author Maria Cecilia Giron 0000-0002-0825-7965 [email protected] Universita degli Studi di Padova Dipartimento di Scienze del Farmaco View all articles by this author Metrics & Citations Metrics Article Usage 268 views 186 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Sofia Faggin, Silvia Cerantola, Gloria Carrossa, et al. 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