We evaluated in a multimethodological approach the anti-inflammatory and anti-oxidant properties of hydroalcoholic extracts obtained from husks, grains, flour and pasta derived from durum wheat belonging to the ancient cultivar “Senatore Cappelli”, assessing their potential as bioactive compound sources in terms of phytochemical, antioxidant, and anti-inflammatory properties

Colonic goblet cells by secreting Muc2 mucin and specific proteins is critical for physically entrapping and expelling invading enteropathogens. Thus, is not surprising that Muc2-/- littermates exhibit increased susceptibility to attaching/effacing C. rodentium colonization. The colonic epithelium also secretes small cathelicidin peptide, which potentially interacts intimately with goblet cells and were presumed to accumulate within the sterile inner mucus layer as a simple antimicrobial peptide defense. In this study, we aimed to determine the effects of cathelicidin on mucin secretion in goblet cells during C. rodentium-induced colitis and the impact on the mucus barrier defense. For this, we used cathelicidin-deficient (Camp-/-) mice, mouse colonoids and human colonic LS174T goblet cells to elucidate the mechanisms by which cathelicidin regulates goblet cell secretions. Our results showed Camp-/- littermates infected with C. rodentium displayed increased fecal shedding and epithelial colonization. Camp-/- littermates at the peak of C. rodentium infection (7 dpi) had a deficient mucin layer with fewer Alcian blue/PAS filled goblet cells and a reduction in fucose and N-acetylglucosamine (WGA+) glycoproteins. By transmission electron microscopy (TEM), goblet cells in Camp-/- colons were swollen and retained a large number of mucus granules during C. rodentium infection. C. rodentium infected Camp-/- littermates showed impaired reactive oxygen species (ROS) production and a transcriptomic profiling associated with decreased ROS biosynthesis and an increase in ROS negative regulators. In mucin producing LS174T colonic epithelial cells, human cathelicidin LL-37 promptly induced the secretion of goblet cell-associated TFF3 and RELMβ, via a ROS-dependent mechanism. These findings revealed that mice lacking cathelicidin (Camp-/-) were more susceptible to C. rodentium colonization caused by defective goblet cell mucus and mucin-associated protein secretion via a ROS-dependent mechanism. Importantly, cathelicidin regulated mucus secretion revealing a non microbicidal actions of this peptide with homeostatic properties on the colonic mucus barrier, critical in excluding luminal microbiota away from the epithelia to clear bacterial infections and restore gut homeostasis.

Inflammation is associated with reactive nitrogen species generation (e.g., peroxynitrite) and the subsequent nitration of the constituent tyrosine residues within proteins, generating 3-nitrotyrosine residues. To assess if endogenous nitrated proteins can be measured in human blood, it is important to select the correct blood processing method. Processing to provide serum is a popular choice in both research and routine biochemical analyses, where coagulation results in the removal of fibrinogen. In the present study, 3-nitrotyrosine-containing proteins were detected and identified in healthy human plasma and serum samples by both native and reducing SDS PAGE with western blotting. Western blots using an anti-nitrotyrosine antibody, following native PAGE, revealed that most of the nitrated protein was in a single band, present in plasma but barely detectable in serum. This protein band was identified as fibrinogen using an anti-fibrinogen antibody. Further investigations using reducing SDS PAGE followed by western blotting, revealed that commercially sourced fibrinogen, from human plasma, contained endogenous 3-nitrotyrosine residues. Under reducing conditions, the β subunit of fibrinogen (52 kDa) was seen to be the most susceptible to nitration of the three fibrinogen subunits, in both the commercially sourced fibrinogen and in freshly sourced plasma samples. Nitrated albumin represented only a minor constituent of the plasma nitroproteome. In conclusion, nitrated fibrinogen was observed as a major constituent of the nitroproteome in human blood plasma. We propose that analyses of non-erythrocytic blood protein nitration are optimally conducted using plasma.

Interleukin-6 signaling activates STAT3, leading to matrix metalloproteinase (MMP)-3 production. The hip joints with rapidly destructive coxopathy (RDC) show rapid chondrolysis, probably by MMP-3 increased in the synovial fluid in the affected hip joint. Currently, no information is available on STAT3 activation in the RDC synovial tissues. This study aimed to investigate STAT3 activation in the synovial tissues with joint destruction in the early stage of RDC. This study also investigated the effect of tofacitinib on STAT3 activation in the synovial tissues from the hip joint with RDC. Synovial tissues within 7 months from the disease onset were obtained from four RDC patients with femoral head destruction and high serum levels of MMP-3. The tissues were incubated with or without tofacitinib. Immunohistochemical examination was performed to detect STAT3 phosphorylation with anti-phospho-STAT3 antibody. RDC synovial tissues demonstrated the synovial lining hyperplasia with an increase of CD68-positive macrophages and CD3-positive T lymphocytes. STAT3 activation was found in the synovial tissues. The majority of phospho-STAT3-positive cells were the synovial lining cells and exhibited negative expression of macrophage or T cell marker. Treatment with tofacitinib resulted in a decrease in phospho-STAT3-positive cells, especially with high intensity, indicating effective suppression of STAT3 activation in RDC synovial tissues. Inhibitory effect of tofacitinib could work through the Janus Kinase/STAT3 axis in the synovial tissues in the early stage of RDC. Thus, STAT3 may be a potential therapeutic target for prevention of joint structural damage in RDC.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterised by the degeneration of upper and lower motoneurons. Onset and progression of the disease are determined by both cell-autonomous neuronal dysfunctions and non-cell-autonomous factors, mainly due to activation of glial cells. Activated astrocytes posses both neurotoxic and neuroinflammatory properties in ALS, and the study of the molecular pathways underlying their activation may reveal new therapeutical targets to counter their toxicity. The Extracellular Matrix Metalloproteinases INducer (EMMPRIN), a glycoprotein expressed by various cell types including neurons, is the major activator of matrix metalloproteinases (MMP) synthesis and release. EMMPRIN activation can be induced by peptidyl-prolyl isomerase A (PPIA), a chaperone protein with cis/trans isomerase activity, that exhibits cytokine- and chemokine-like behaviour. Interestingly, PPIA is highly released in the cerebrospinal fluid (CSF) of ALS patients and animal models where, by activating EMMPRIN on motoneurons, induces MMP-9 expression and neuronal death. Here we show that EMMPRIN is expressed also by astrocytes where it is upregulated under inflammatory conditions or in presence of the SOD1G93A mutation that characterises familial ALS cases. PPIA-mediated stimulation of EMMPRIN in healthy astrocytes induces NF-kB activation and a proinflammatory phenotype. Interestingly, SOD1G93A astrocytes release high levels of PPIA and present the same activated phenotype induced by PPIA in healthy astrocytes. Finally, treatment of SOD1G93A astrocytes with an anti-EMMPRIN antibody, known to block PPIA/EMMPRIN interaction, reverses the toxic phenotype and suggests an autocrine mode in the astrocytic activation of EMMPRIN. In conclusion, here we demonstrate that EMMPRIN activation in ALS occurs also in astrocytes where it exacerbates their neuroinflammatory and toxic phenotype. Furthermore, we suggest the potential use of an anti-EMMPRIN antibody to reduce astrocytic activation during the disease.

Systemic Sclerosis (SSc) is a rare systemic autoimmune disease. Hypertrophic scars (Hsc) occur after deep skin injuries. Their common hallmark is excessive pathological skin fibrosis due to extracellular matrix (ECM) accumulation. Current treatments didn’t delay or stop fibrosis progression, highlighting the urgent need for novel therapeutic strategies. In this context, we dug into the role of the Placental Growth Factor (PlGF) in skin fibrosis development. Fibroblasts were isolated from skin biopsies of healthy individuals, SSc or Hsc patients, and cultured in monolayer or three-dimensional (3D) to form a reconstructed dermis. PlGF expression was visualized in skin biopsies and 3D reconstructed dermis using immunofluorescence. PlGF effect on the ECM was assessed using fluoroenzymatic and enzyme-linked immunosorbent assay in fibroblast monolayers or reconstructed dermis following treatments with PlGF-1 or -2, VEGF or TGF-β. Finally, the dermal thickness of the 3D reconstructed dermis, with or without treatments, was measured with a laser telemeter. PlGF was absent in healthy skin but expressed in Hsc skin and 3D reconstructed dermis from Hsc or SSc fibroblasts. PlGF treatment stimulated collagen I production in healthy fibroblasts cultured in monolayer and 3D reconstructed dermis from healthy and Hsc fibroblasts. Moreover, PlGF treatment increased dermal thickness in 3D reconstructed dermis from healthy and SSc fibroblasts. PlGF may directly contribute to fibrosis development by promoting ECM production. Targeting PlGF could represent a new therapeutic approach in skin fibrosis associated with SSc or Hsc, with potential applications in other fibrosis-related diseases.

OBJECTIVE: We and others have found that intestinal epithelial cells express Nucleotide (P2) receptors that modulate inflammation. In inflammatory conditions, cellular damage occurs due to bacterial invasion, leading to the release of nucleotides into the extracellular environment. ATP and UTP, released by stressed intestinal epithelial cells (IECs), microbiota and other cells, activate P2Y2 receptors, triggering the release of PGE2 by IECs. In this study, we evaluated the role of the P2Y2 receptor in the DSS colitis model and its effect also on maintaining IEC homeostasis by regulating its integrity. METHODS: P2Y2-deficient (P2ry2-/-) and WT mice were treated with 3% DSS for 7 days. The disease score was assessed daily. On day 7, the colon was harvested for assessment of inflammatory markers. In addition, IECs were isolated from mouse colons and cultured. IECs were stimulated by TNF plus a TLR4 receptor ligand in the presence or absence of P2 receptor antagonists. The permeability and expression of tight junction proteins were evaluated by FITC-dextran and qPCR, respectively. RESULTS: Mice deficient in the P2Y2 receptor showed an increased disease activity index (DAI), indicating increased severity of inflammation in the DSS colitis model. Intriguingly, despite this proinflammatory phenotype, both in vitro and in vivo experiments demonstrated a decrease in the permeability of IECs in the absence of P2Y2 signaling. Accordingly, analysis of stimulated IECs in the presence of a P2Y2 antagonist revealed a significant increase in the RNA expression level of junctional adhesion molecule (JAM), suggesting a potential role of P2Y2 in regulating epithelial barrier integrity. CONCLUSION: Although the global absence of P2Y2 in mice exacerbated intestinal inflammation in the DSS model of colitis, our data also point out the involvement of P2Y2 in intestinal integrity. Preliminary analysis with IEC cultures provides further insights highlighting the role of P2Y2 in enhancing epithelial barrier integrity by affecting adhesion molecules. As P2Y2 is expressed on both leukocytes and IECs further experiments will be needed to discriminate these effects. Therefore, our data suggest the involvement of P2Y2 signaling in intestinal inflammation which warrants future investigations.

Among many functions, the intestinal epithelium prevents the intrusion of microorganisms from the intestinal microbiota into the systemic circulation. However, in inflammatory bowel disease, increased permeability of intestinal epithelial cells (IECs) allows these microorganisms to entry into the lamina propria where they activate several cells via their microbe-associated molecular patterns (MAMPs) triggering proinflammatory responses. Activated, stressed, or injured cells release endogenous nucleotides such as ATP, ADP, UTP and UDP into the extracellular environment activating cell surface bound proinflammatory P2 receptors. In the lamina propria of the intestine, fibroblasts are among the first cells to respond to MAMPs as they reside beneath the IECs. In this work, we show a role for nucleotide signaling related to innate immune responses of fibroblasts. Fibroblasts isolated from healthy human colon and the mouse fibroblast cell line NIH/3T3 express innate immune toll-like receptors (TLRs) -3 and -4 that, when activated by polyinosinic-polycytidylic acid (Poly(I:C)) or lipopolysaccharide (LPS), respectively, trigger the release of the neutrophils chemoattractant CXCL1/KC (mouse) or CXCL8/IL-8 (human). Activation of TLR-3 and -4 in the presence of the general P2 receptor blockers suramine, RB2 and apyrase, reduces the release of CXCL1/KC and CXCL8/IL-8. We determined by qPCR that human primary colonic fibroblasts and NIH/3T3 express P2Y1, P2Y12 and P2Y13 receptors that all respond to ADP. We showed by luciferin-luciferase that NIH/3T3 cells release ATP following the activation of TLR-4 and that ATPase activity is significantly higher than ADPase activity suggesting an accumulation of ADP at the cell surface upon TLRs activation. In agreement, we found that ADP enhances CXCL1/KC release by LPS-stimulated NIH/3T3 cells. In human primary colonic fibroblasts, blockade of P2Y13 receptor with the specific antagonist MRS 2211 following the activation of TLR-3 with Poly(I:C) significantly reduced the release of CXCL8/IL-8 when compared to controls thus supporting the effect of ADP in NIH/3T3 cells. Taken together, our preliminary results suggest a role for ADP in promoting the innate immune responses of fibroblasts.

Asthma patients with airway neutrophilia tend to have more severe clinical disease compared to the eosinophilic asthma endotype. Despite the gap in effective therapeutics for neutrophilic asthma, much of the research on novel asthma therapies has focused on targets relevant to eosinophilic asthma. This highlights the compelling need for innovative anti-inflammatory strategies for neutrophilic asthma. Withaferin A (WFA), derived from the Withania somnifera plant, is a promising drug candidate for neutrophilic asthma. WFA has therapeutic effects in murine models of acute lung injury, pulmonary fibrosis, and other neutrophil-mediated diseases. Our published in vitro data demonstrate that WFA directly inhibits key inflammatory neutrophil functions and promotes timely apoptosis of primed neutrophils. The objectives of this study were to 1) evaluate the safety of pulmonary WFA delivery (via oropharyngeal aspiration, o.p.) in healthy mice and 2) investigate the effect of WFA o.p. on pulmonary neutrophil recruitment in an established murine model of neutrophilic asthma. Repeated administration of WFA via oropharyngeal aspiration (0.25-1 mg/kg on Days 0, 2, 4, 7, 9, 11, 14-16) was well-tolerated in healthy mice, with no evidence that local delivery of WFA into the lungs caused systemic disease or airway inflammation or cytotoxicity. Neutrophilic asthma was induced in separate cohorts of mice via OVA/LPS (50 µg/100 ng o.p.) sensitization on Days 0 and 7, followed by OVA challenge (50 µg o.p.) on Days 14-16. On Day 17, neutrophilic asthma mice treated with 1 mg/kg WFA o.p. had significantly less neutrophilic airway inflammation compared to vehicle-treated neutrophilic asthma mice. This blunting of airway neutrophilia by WFA occurred under both prophylactic (Days 0, 2, 4, 7, 9, 11, 14-16) and therapeutic (Days 14-16) WFA dosing schedules and was replicated across independent experiments. These data support the safety and efficacy of inhaled WFA as a novel therapy for neutrophilic asthma. Our ongoing investigations of WFA treatment for neutrophilic asthma includes refinement of inhaled delivery methods, optimization of dose, assessment of additional clinically relevant outcome measures, and evaluation in more chronic neutrophilic asthma models.

Systemic lupus erythematosus (lupus) is an autoimmune disease mainly affecting women of reproductive age. Its unpredictable manifestations can affect almost any part of the body. In lupus, neutrophils are held to drive overt inflammation through interactions with autoimmune complexes by producing reactive oxygen species and by releasing proteases that can cause tissue damage. Neutrophils recognize immune complexes via their Fc gamma receptors (FcγRs). In the present study, we observed that stimulation of whole blood from healthy volunteers with immune complexes specifically up-regulated FcγRI on leukocytes in circulation. This increase was particularly marked on neutrophils when compared to monocytes and lymphocytes. Immunofluorescence microscopy confirmed the specific up-regulation of FcγRI on neutrophils and further revealed a clustered distribution pattern for FcγRI. Exposure of isolated neutrophils to immune complexes resulted in the production of reactive oxygen species, which was prevented by blocking FcγRI. Because levels of autoimmune complexes can be higher in patients with lupus, we measured the surface expression of FcγRs on neutrophils in a cohort of patients, by flow cytometry. We observed that FcγRI was specifically elevated in lupus compared with healthy volunteers, although less pronounced in patients taking hydroxychloroquine, the first line of treatment for lupus. Preincubation of isolated neutrophils from healthy volunteers with hydroxychloroquine also diminished reactive oxygen species generation in response to immune complexes. This study identifies neutrophil FcγRI as a new and potentially relevant factor in the inflammatory component of lupus. Studies are in progress to delineate its precise functions for therapy purposes. This project is funded by the Arthritis Society (Canada), grant no. 21-0000000121. SH is the recipient of a Frederick Banting & Charles Best studentship from the Canadian Institutes of Health Research (CIHR).

In our lower respiratory tract, the alveolar spaces are devided from the bloodstream and the external environment by only a few microns of interstitial tissue. Alveolar macrophages (AMs) defend this delicate mucosal surface from invading infections by regularly patrolling the site. Using intravital microscopy we have uncovered that AMs display three behaviour modalities to achieve the goal of proper survelience of the alveolar space: (i) extending cell protrusions to sample surrounding areas, (ii) squeezing their whole cell body between alveoli, and (iii) patrolling by moving their cell body around each alveolus. In this study we found Rho GTPase, cell division control protein 42 (Cdc42) expression significantly decreased specifically after berry-flavoured e-cigarette (e-cig) exposure. This resulted in a shift in AM behaviour from squeezing to probing. Alterations is AM behaviour resulted in decreased clearance of inhaled bacteria, Pseudomonas aeruginosa. Together these data unravel important pathways in AM migration and show that flavored e-cig vapes have deleterious effects on immune cell function.

Red blood cells (RBCs) are well known for their pivotal function in oxygen and carbon dioxide transport, however recently they are also recognoized for their major role in regulation of inflammation, including systemic inflammation. In sepsis, RBCs bind pathogens and store cytokines, but their plasticity is compromised and they release extracellularly hemoglobin/heme. Here we identify a modulatory effect of RBCs on murine neutrophils leading to their quiescence via the sialic acid-Siglec-E receptor pathway. We report that ex vivo murine RBCs increase neutrophil viability and release of IL-1beta but diminish lipopolysaccharide-induced Siglec E-dependent release of neutrophil extracellular traps (NETs). To further elucidate the impact of the interactions, we aimed at establishing a method of in vivo RBC imaging (anti-Ter-119 antibody) in the vasculature of live mice with intravital microscopy (IVM) at different stages of bacterial sepsis/endotoxemia. In vivo RBC-leukocyte interactions were clearly observed in the liver vasculature during sepsis (leading to either phagocytosis of RBCs or were transient in nature) and they exceeded those observed in healthy animals. Moreover, we applied phenylhydrazine-induced anemia model and blocked Siglec-E receptor in order to test whether these manipulations affect NET formation during sepsis. Prolonged neutrophil-RBC interactions, downregulation of NET formation and enhanced phagocytosis were observed as a result of RBC depletion. In the Siglec-E blocking approach, despite the lack of cell interactions, more NETs were formed both during systemic inflammation and in the accompanying hemolytic anemia. Our studies confirm the RBC-neutrophil crosstalk via sialic acids-Siglec-E axis but further studies are required to verify if these intrerations are beneficial or pathological for the sepsis outcome. The study was supported by National Science Centre of Poland, OPUS 22, 2021/43/B/NZ6/00782.

Macrophages, either resident or derived from blood monocytes, are key players in the wound healing process, with their differentiation influenced by the time elapsed since injury and the pathological or physiological state of the wound. While blood monocyte-derived macrophages have been extensively studied, skin-resident macrophages remain unexplored. In addition, extracellular vesicles (EVs), known for their involvement in intercellular communication and transport of bioactive elements, have a largely unknown effect on immune cells involved in wound healing. The content of EVs produced by myofibroblasts from healthy wounds has been previously highlighted, revealing the presence of pro- and anti-inflammatory cytokines. The aim of this study was to evaluate the role of EVs in the differentiation of skin-resident macrophages in normal and hypertrophic wound healing. Dermal cells were isolated by a series of enzymatic steps and characterized by flow cytometry. The resulting cell mixture was cultured to induce macrophage differentiation into different subtypes: pro-inflammatory macrophages (M1) and anti-inflammatory macrophages (M2). Macrophages were then characterized by flow cytometry using specific markers. Fluorescent EVs were isolated from myofibroblasts of normal and hypertrophic scars. In the dermal fraction, resident macrophages expressed the markers CD45 and CD163low. A protocol for differentiation of resident macrophages was developed and the differentiation state of macrophages was characterized by TNF-alpha expression for M1 and CD163high marker expression for M2. EVs will be added to undifferentiated macrophages and their differentiation state will be analyzed. We expect significant differences in macrophage behavior depending on the origin of the EVs used in co-culture, with a tendency for EVs from hypertrophic scars to promote differentiation into M2. These findings will deepen our understanding of the role of EVs in the immune response associated with wound healing, opening new perspectives in the medical field.

Systemic lupus erythematosus (SLE) is characterised by excessive production autoantibodies (Abs), yet the antigen specificities remain elusive for most of these Abs. We performed a broad explorative screen of IgG and IgA antibodies against autoantigen specificities to gain insight into SLE pathogenesis and identify novel Abs that could enhance diagnostics. We analysed plasma samples from 289 patients with SLE and 196 age-/sex-matched healthy controls (HC) from the PRECISESADS project (NTC02890121). Samples were screened for IgG and IgA seroreactivity against a panel of >1,600 protein autoantigens using KREX-based i-Ome arrays. Differentially abundant Ab (DAAb) analysis revealed 14 IgG and 2 IgA DAAbs that were elevated in patients with SLE vs HC. Of those IgG DAAbs, anti-LIN28A (sen; spe; AUC: 0.77; 0.69; 0.80), anti-HBGB2 (0.65; 0.81; 0.79), anti-HMG20B (0.75; 0.71; 0.70), and anti-NRF1 (0.63; 0.80; 0.77) demonstrated the best ability to discriminate between SLE patients and HC. Of the IgA DAAbs, anti-LIN28A (0.65; 0.83; 0.80) and anti-SSB (0.64; 0.82; 0.78) demonstrated best metrics. Functional analysis of IgG DAAbs revealed 2 GO-term enrichments at the cellular component level: extracellular and cell surface, suggesting that autoantigens eliciting IgG are accessible at the cell surface or released by cell lysis. Functional analysis of IgA DAAbs revealed 17 GO-term enrichments related to DNA-binding transcription repressor activity, chromatin binding, transcription factor binding, and regulation of transcription by RNA polymerase II, suggesting that autoantigens eliciting IgA are enriched for nucleic acid binding. This study corroborated previously reported specificities (anti-HBGB2, anti-HMG20B) and revealed novel IgG and IgA (anti-LIN28A, anti-NRF1, anti-TGIF2, anti-SUB1, anti-PSIP1, anti-CCNB1) Abs described for the first time in SLE, with robust accuracy in distinguishing SLE from HC.

Low-grade "chronic" inflammation (LGI) during the process of reconditioning after COVID-19 undoubtedly plays an important role. One of the main problems of LGI, which poses a risk to human health, is a significant increase in the risk of cardiovascular complications in the postcovid period, which include death from cardiovascular disease (CVD). Aim: To evaluate the efficacy of the effect of the drug rebamipide on C-reactive protein (CRP) and major lipopolysaccharide binding systems (lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) in postcovid patients with severe arthralgias. Methods. A prospective single-center study included 62 patients aged 46±5.6 years with a history of COVID-19, taking non-steroidal anti-inflammatory drugs (NSAIDs) for joint pain on the background of postcovid syndrome. The patients were divided into two groups. The first group (n=34) received rebamipide at a dose of 100 mg 3 times a day and omeprazole 20 mg daily for 28 days. Group 2 - 28 patients, received only omeprazole 20 mg daily for 28 days. All patients underwent clinical examination, anamnestic data collection and blood tests (ELISA) for levels of CRP, LBP and BPI. Results. Significantly higher levels of CRP and LBP were registered in the studied groups before the treatment compared to the control group (p<0,05). On the contrary, BPI index was significantly lower in the groups of patients with postcovid syndrome (p<0,05). In peripheral blood in group No. 1, which took rebamipide in addition to PPI, a significant decrease in CRP level (mg/L) was found to 3.75 [2.82; 4.21] and post-drug 2.05 [1.85; 2.62] (p<0.05), respectively (p<0.05). In the second group (omeprazole monotherapy), no significant changes were found before the drug 3.4 [2.56; 4.0] and after PPI course 3.52 [2.68; 3.9] (p>0.05). Conclusion. Rebamipide can potentially become a therapeutic tool for combating LGI driven by LPS in individuals who have undergone a new coronavirus infection and have signs of postcovid syndrome accompanied by an increase in peripheral blood CRP and LBP as part of LPS-driven LGI. This work was supported by the Russian Science Foundation under grant no. 23-15-20021, https://rscf.ru/project/23-15-20021/.

Some endocrine-disrupting chemicals (EDCs), such as the plasticizers bisphenol A (BPA) and bisphenol S (BPS), can affect not only endocrine functions but also the immune responses of various cells and tissues. Eastman TritanTM copolyester, a novel plastic used as a replacement for BPA and BPS and manufactured utilizing three monomers, 1,4-cyclohexanedimethanol (CHDM), 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD) and dimethyl terephthalate (DMTP), can leach from products and could exert estrogenic activity. In this sense, various phthalates have been shown to increase inflammation and induce cellular metabolic changes. Since neutrophils are inflammatory cells that express hormone receptors and display a glycolysis-focused metabolism to fuel their antimicrobial functions, we hypothesized that TritanTM compounds could modulate the metabolism and functions of neutrophils. We collected neutrophils purified from healthy women and men and treated them with TritanTM compounds in the presence of pro-inflammatory cytokines to mimic inflammatory conditions. We assessed neutrophil viability by annexin V/propidium iodide staining, their metabolic activity with MTS assays and an extracellular flux analyzer, and their functions by evaluating reactive oxygen species production and cytokine secretion. We observed that TritanTM compounds did not compromise neutrophil viability and glycolytic response. However, DMTP was found to reduce the respiratory burst induced by TNF and IL-1β, and all three TritanTM monomers increased CXCL8/IL-8 secretion induced by these pro-inflammatory cytokines. Overall, these results suggest that TritanTM compounds can modulate specific neutrophil functions. A better understanding of how TritanTM compounds can impact the inflammatory functions of neutrophils will help determine whether Eastman TritanTM copolyester may have potential adverse effects on our health or the health of future generations and if it is genuinely a safe replacement for other plasticizers such as BPA and BPS.

Endometrial cells play a key role in the control of the inflammatory response at postpartum, in addition to their physiological reproductive function. Endometrial cells express receptors for free fatty acids (FFA) such as FFA1 and FFA4 receptors. Both FFA1 and FFA4 receptors bind long chain fatty acids, such as linoleic acid (LA) and docosahexaenoic acid (DHA), respectively. In cows, linoleic acid increases in the blood during early postpartum period, however, its role on the inflammatory response in endometrial cells is unclear. On the contrary, DHA is added to the feed of postpartum cows, because DHA has been shown to have an anti-inflammatory effects on various cells. The aim of this study was to determine the inflammatory response-associated effects of LA and DHA in bovine endometrial cells. Bovine endometrial (BEND) cells were cultured with linoleic acid (LA) or docosahexaenoic acid (DHA) for 30 min, then lipopolysaccharide (LPS) was added and incubated for 24 h. Interleukin-6 (IL-6), IL-8 and prostaglandin E2 (PGE2) were assessed by ELISA assay. The extracellular adenosine triphosphate (ATP) production was measured with a luminescent assay in supernatants of BEND cells incubated with LA for 15 s. Also, a possible effect of LA and DHA on changes in metabolic routes was assessed through metabolomic assay. The results showed that LA reduced the IL-6 and IL-8 production induced by LPS, but did not reduce PGE2. LA increased the extracellular ATP levels at 15 s of incubation. DHA reduced the IL-6 and PGE2 production induced by LPS, but did not affect IL-8 production. The metabolomic assay showed that LA and DHA induced changes in metabolites such as adenosine, arachidonic acid, stearic acid, and others. In conclusion, both LA and DHA showed an anti-inflammatory effect on LPS-activated endometrial cells. Furthermore, LA and DHA induced metabolomic changes, which will be studied in detail to establish a link between immunity and metabolism. Understanding the interplay between immunity and metabolism will be useful to propose strategies based on fatty acids for adequate control of inflammatory processes in the endometrium. (Funded by Fondecyt No. 1200905)

Neutrophils are no longer viewed as a homogenous population of short-lived cells. In this context, we identified a subset of inflammatory neutrophils from patients with autoimmune diseases that produce the peptidase inhibitor elafin. Such neutrophils are similar to a reprogrammed human population of long-lived (LL) neutrophils generated by a combination of cytokines associated with inflammatory diseases, e.g., granulocytemacrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), and interleukin (IL)-4. These LL neutrophils exhibit characteristics of neutrophils and dendritic cells and produce elafin. Here, we characterized, using an extracellular flux analyzer, the metabolic changes in neutrophils exposed to IL-4, TNF, and GM-CSF, alone or in combination. Under inflammatory concentrations, TNF and GM-CSF, but not IL-4, increased the glycolytic response of neutrophils, while only TNF induced transient oxygen consumption (known as the respiratory burst). Reprogramming with TNF and IL-4 led to an increased glycolytic response without any effect on respiration. After 48h of reprogramming in culture, LL neutrophils exhibited an increased metabolic phenotype characterized by augmented glycolysis and respiration, both in resting state and following their activation with pro-inflammatory cytokines. To better understand the molecular mechanisms by which metabolic processes influence neutrophil functions, we went back to the transcriptomics of LL neutrophils (doi: 10.4049/jimmunol.2000852) and found an important modulation of mitochondria-associated genes. We detected higher transcript levels of the mitochondria-associated genes SLC25A27, P2RX7, and PLPP3/PPA2B, confirming LL neutrophils’ distinctive metabolic features. These results suggest that LL neutrophils display an energized metabolic phenotype and could possess functional mitochondria capable of respiration to generate energy and sustain their functions. A better characterization of the metabolic features of these inflammatory neutrophils with proresolutive properties could represent new ways for therapeutic intervention in chronic inflammatory diseases.

The COVID-19 pandemic due to the coronavirus SARS-CoV-2 has been responsible for early 7 million deaths globally since 2020. In spite of vaccines, the virus continues to propagate, with the immunosuppressed, aged, and non-vaccinated populations remaining vulnerable. An unmet therapeutic challenge caused by COVID-19 is acute respiratory distress syndrome (ARDS), which develops typically from pulmonary hyper-inflammatory response degenerating into the so-called cytokine storm. The cluster of differentiation 36 receptor (CD36) is expressed in macrophages and associated with the inflammatory response. Ligands of the CD36 receptor have been shown to reduce inflammation in murine models. Hypothesizing the potential to curb ARDS in COVID-19, the CD36 ligand hexarelin, an analog of the growth hormone-releasing peptide (GHRP) family, was examined for the ability to attenuate hyper-inflammation of pulmonary macrophages. Dose-response studies for determining the optimal dose of hexarelin were performed in a non infectious lung inflammation model. Transgenic mice expressing the human ACE2 protein under regulation of the human cytokeratin 18 promoter in epithelial cells (K18- hACE2) were treated by a subcutaneous injection of hexarelin (10 µmol/kg) or 0.9% NaCl vehicle 30 minutes before intranasal instillation with SARS-CoV-2 (250 TCID50), and then treated daily with hexarelin or vehicle over the next 9 days. Survival and body weight were documented daily. Cytokine levels were assayed in lung homogenates. Hexarelin increased survival, decreased body weight loss and reduced pro-inflammatory pulmonary cytokine and chemokine levels in lung homogenates. Modulation of CD36 offers potential as a novel therapeutic approach for mitigating virus-induced hyper-inflammatory response of lung macrophages in SARS-CoV-2 infection. Supported by the Réseau québécois de recherche sur le médicament (RQRM) and the Faculty of Pharmacy, Université de Montréal

Early-onset colorectal cancer (EOCRC) is increasing at an alarming rate worldwide. Presenting in young individuals (18-49y) EOCRC is being detected at advanced stages of disease resulting in more unfavourable features as compared to late-onset CRC (>50y). Unlike late-onset CRC, EOCRC is highly prevalent in the distal/rectal parts of the colon. Sidedness, the location within the colon, has become an important prognostic and predictive factor in CRC patients. Also, oxidative stress has been associated with inflammatory bowel disease (IBD) and CRC. In earlier studies, we found that the loss of NFE2L3 in mice with colitis induced CRC reduced the numbers of distal/rectal tumors of the colon. Recently, our RNA-seq data from an acute inflammation mouse model showed that variance of genes regulated following was more prominent in the distal and mid colon and that the effect of knockdown of NFE2L3 was most defined in the distal colon as compared to the proximal colon. Our results revealed a role for NFE2l3 in oxidative stress signaling and in the inflammatory response in mice. We found that NFE2L3 stringently regulates a specific set of oxidative stress target genes including STAT1 and SLC7A11, which have been linked to IBD and CRC previously. We are currently validating the role of regulatory proteins in IBD and CRC by analysis of human tissue specimens obtained from IBD and CRC patients. Overall, our project aims to gain insights into the molecular mechanisms involved in colon inflammation and CRC by elucidating the role NFE2L3 is playing in colitis and colon carcinogenesis.

We have developed a Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) based method for monitoring the concentrations of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulators: elexacaftor (VX-445), tezacaftor (VX-661), and ivacaftor (VX-770) in biological fluids from individuals with cystic fibrosis. This innovative methodology facilitates swift and sensitive quantification of CFTR modulators from minimal samples, offering significant benefits in practical applications.

Inflammatory bowel disease (IBD), which affects millions of people world-wide, is a chronic inflammatory condition of the gut caused by microbiome dysbiosis as well as environmental and genetic factors. IBD can be broadly classified as ulcerative colitis (UC) and Crohn’s disease (CD). Among the various animal models that are used to mimic human IBD, the dextran sulfate sodium (DSS)-induced colitis model is more widely used due to its simplicity and recapitulation of several features observed in human ulcerative colitis. In the present study, we compared the efficacy of two anti-inflammatory therapies in the DSS colitis model in mice by examining their effects on clinical signs and pro-inflammatory cytokines release in colon tissues. First, induction of colitis was evaluated using two different concentrations of DSS (2% and 3%). DSS was provided ad libitum in drinking water continuously from Day 1 to Day 10. Disease-related clinical signs such as weight loss score, stool consistency score and fecal blood score were measured daily, and a disease activity index (DAI) score was calculated by summation of the aforementioned three scores. A DSS concentration-dependent induction of colitis was observed in the mice as evidenced by relatively higher clinical scores with 3% DSS compared with 2% DSS. Based on relatively higher and optimal level of colitis induction with 3% DSS, we selected this concentration to examine effects of the two therapies. Effects of 5-amino salicylic acid and cyclosporine A, both of which are used clinically as well as in preclinical studies as positive controls, were evaluated. Both drugs were administered orally once daily from Day 1 to Day 10. While cyclosporine A (80 mg/kg) attenuated the colitis scores in the DSS-treated mice, 5-amino salicylic acid (75 mg/kg) was unable to exert any effects. Furthermore, at necropsy, colon tissues were collected from the mice to examine the effects of these two drugs on the pro-inflammatory cytokine levels. Similar to the in-life results, only cyclosporine A was able to block the release of pro-inflammatory cytokines in the colon tissues. In conclusion, these results suggest that cyclosporine A can be used as a positive control tool drug in the DSS colitis model in mice

Damage-associated molecular pattern proteins (DAMPs) are innate immunity sentinels that are actively or passively released from tissue cells. Intracellular nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis and extracellular NAMPT (eNAMPT) is a potent DAMP amplifying innate immunity-mediated signals via Toll-like receptor 4 (TLR4) activation. Plasma eNAMPT levels are directly linked to disease severity in multiple acute lung and systemic inflammatory disorders. We utilized human THP-1 monocytic cells to mechanistically examine canonical NLRP3 inflammasome involvement in secretion/release of two DAMPs, eNAMPT and high mobility group box 1 protein (HMGB1). THP-1 cells exposed to Nigericin (triggering K+ efflux) showed rapid LDH release consistent with pyroptosis and NLRP3 inflammasome-dependent release of both DAMPs. eNAMPT and HMGB1 release was blocked by the NLRP3 inhibitor MCC950 and was abolished in caspase-1 deficient cells. Gasdermin D (GSDMD) cleavage is critical to GSDMD pore formation and induction of pyroptosis. Nigericin-induced release of eNAMPT and HMGB1 was abolished in GSDMD KO THP-1 cells. Lipopolysaccharide (LPS)/TLR4 signaling promoted GSDMD-dependent eNAMPT and HMGB1 secretion (abolished in GSDMD KO cells) but in the absence of LDH leakage and pyroptotic cell death. The pore-forming N-terminal GSDMD cleavage fragment did not appear in LPS-treated cells indicating eNAMPT/HMGB1 secretion/release is independent of GSDMD pore formation. We conclude that the NLRP3 inflammasome regulates LPS/TLR4-induced eNAMPT and HMGB1 secretion/release through lytic and non-lytic GSDMD functionality independently of pyroptosis involving an undefined mechanism.

We examined the role of NADPH oxidase 2 (NOX2) and Endothelin (ET) pathways in the contractile dysfunction of popliteal lymphatic vessels draining inflamed ankles of TNFΔARE/+mice. Popliteal lymphatic vessels (pLVs) were isolated from female TNFΔARE/+ and wild-type (WT) mice and mounted on a pressure myograph. Systolic (SD) and diastolic diameter (DD), amplitude, ejection fraction (EF), fractional pump flow (FPF), and contraction frequency (CF) were then measured in response to a stepwise increase in transmural pressure or increase in the concentration of acetylcholine (ACh). Effects of NOX2 inhibitor VAS2870 (10 µM), free radical scavenger tempol (10 µM), and ET receptor antagonist bosentan (10 µM) were evaluated. Protein expression of phosphorylated (p-) and total (t-) eNOS in the vessels were determined by western blot. Differences were assessed by multiple t-tests and one-way ANOVA followed by Sidak’s posthoc test. Significant increase in the pLV CF (min-1, 11.7±2.5 vs 4.9±0.4, P<0.05, n:10), SD (µm, 94.1±3.6 vs 77.5±6.2, P<0.05, n:10), and FPF (µm/min-1, 3.9±1.4 vs 2.3±0.2, P<0.05, n:10) was measured in TNFΔARE/+ mice compared to WT. In the presence of VAS2870, tempol, or bosentan, differences in CF (min-1, 8.0±1.3 vs 5.8±2.1; 11.3±2.1 vs 9.6±1.1; 9.3±1.0 vs 9.4±1.1, respectively) and SD (µm, VAS: 80.2±7.1 vs 71.8±5.6; tempol: 77.7±5.3 vs 76.8±6.8) were abolished. Additionally, administration of a concentration of ACh inducing a maximal response in WT vessels was not sufficient to inhibit lymphatic pumping in TNFΔARE/+ mice (min-1, 4.7±1.7 vs 0.5±0.5, P<0.05). A significant increase in the protein level of p-eNOS, but not t-eNOS was observed in the TNFΔARE/+ mice (%, 450.4±143.8 vs 99.9±29.3, P<0.05, n: 5) compared to WT. These results indicate a correlation between increased activity of lymphatic vessels and activation of the endothelin pathway, together with increased production of eNOS-NO. Consequently, this leads to an increase in free radicals in arthritic TNFΔARE/+ mice. It is likely that such changes impact the effective drainage of inflammatory content from the joints to the pLN, thus influencing the immune response.

During the resolution of inflammation, Ly-6C+F4/80- monocytes differentiate to Ly-6C-F4/80+ macrophages that exert efferocytic properties and consequently convert to IFN-β-producing macrophages. Here, we report that exposure to IFN-β, or TGF-β, or a deficiency in the pro-apoptotic protein ARTS, results in the conversion of mature macrophages to a rejuvenated Ly-6C+F4/80+CCR2+ phenotype. This phenotype appeared exclusively in peritoneal resolution phase macrophages and not their unchallenged peritoneal, splenic or bone marrow counterparts. Moreover, IFN-β-triggered rejuvenated macrophages were hyper-efferocytic and expressed higher levels of the efferocytic receptor CD36. Inhibition of CD36 ligation resulted in complete abrogation of efferocytosis ex vivo in both mature and rejuvenated macrophages. Altogether, our findings indicate an unprecedented phenomenon in which IFN-β promotes macrophage rejuvenation and efferocytosis that are limited by ARTS-mediated apoptosis during the resolution of inflammation.

Juvenile dermatomyositis (JDM) is a polygenic autoimmune interferonopathy with predominant involvement of the adaptive system. It is the most common inflammatory myopathy in childhood. The objective of our study is to review the clinical-analytical characteristics of patients referred to the transition consultation and assess their evolution. 14 patients were included (50% men) with a mean age (SD) of 6.53 (3.59) years at the onset of the disease and 6.92 (3.81) years at diagnosis. The median (Q1;Q3) follow-up of the patients is 18.68 (3.95;13.92) years. At diagnosis, 9(64%) patients presented positive ANAs, all had a speckled nuclear pattern. Regarding the specific antibodies, three AntiMi2, two AntiTIF1, two antiMDA5, one antiRO52 and one AntiSAE2. Eight patients met definitive disease criteria according to Bohan and Peter. In the remaining six, the diagnosis was probable. All patients had CK levels above the laboratory's upper reference value at the time of diagnosis. Currently, five patients have elevated CKs levels. Most patients have been treated with a combination of immunosuppressive drugs; Tacrolimus and methotrexate are the most used immunosuppressants with earlier initiation compared to other series published in the literature. At the last visit, 85% of patients are in remission and 40% are untreated.

Polyphenols and type 1 angiotensin II blockers can be used to help reduce the chronic inflammation and oxidative damage caused by metabolic syndrome. Polyphenols have antioxidant, hypoglycemic effects and nephroprotective properties. Azilsartan is a lipophilic molecule that can penetrate cells and stimulate the PPARy pathway, which improves carbohydrate and lipid metabolism. The metabolic syndrome was modeling in 158 male Wistar rats, based on a fructose-feeding model. Group 1 received drug "Fenokor". Group 2 received grape seed concentrate. Group 3 received Azilsartan. The 4th group - MetS without correction. Different group 5 was the control without MetS. Experimental modeling of MetS is associated with characteristic signs, such as abdominal obesity, hyperglycemia and dyslipidemia. Polyphenols' drugs have a diverse positive effect on blood glucose levels and lipids, as well as inflammatory and anti-inflammatory indicators. Our data suggest the potential of using these preparations to correct metabolic syndrome, including when used in conjunction with Azilsartan. However, more research is needed to confirm these findings.

Megakaryocytes, the parental cells of platelets, are diverse cells with immune cell characteristics and functional changes may affect the platelets they produce. They can express Toll-like receptors and the IgG-receptor FcγRIIa, which may enable the recognition of immune complexes by MKs, but this has never been shown. Here, we investigated if Megakaryocytes express functional FcγRIIa and if this facilitates the interaction with immune complexes and cellular responses. MKs were obtained by 4-day differentiation of progenitor cells, isolated from bone marrow of healthy 7-15 weeks old C57BL6/J mice (FcγRIIatgn or FcγRIIanull) and stimulated with microbial ligands and immune complexes (ICs). Extracellular Vesicles (EVs) and cytokines in supernatants and IC-uptake were analyzed by flow cytometry, ELISA and immunofluorescence microscopy. MKs released various cytokines in response to TLR2 and TLR4 ligands. Only the cytokine MIP-2 was significantly increased in response to FcγRIIa stimulation with heat-aggregated IgG (ha-IgG). CD41+ and CD9+ MK-EV release was constitutive and refractory to TLR ligand stimulation. However, FcγRIIatgn MKs, but not FcγRIIanull MKs, were found to bind and internalize ha-IgG, which induced cytokine and EV release. FcγRIIatgn MKs internalized mitochondrial ICs and coronavirus SARS-CoV-2 ICs, but only the latter induced MIP-2 and EV-release. These effects were not seen if MKs were incubated with SARS-CoV-2 in absence of antibodies or in presence of non-SARS-CoV-2 reactive IgG. MKs may participate in adaptive immune responses through FcγRIIa expression. In particular, FcγRIIa activation can induce morphological changes, IC internalization and the release of EVs and cytokines by MKs. The nature of the response may depend on the type of IC as SARS-CoV-2 ICs modulated cytokine and EV-release by MKs in contrast to mitochondrial ICs.

The protease plasma kallikrein (PKal) cleaves high molecular weight kininogen which leads to the creation of the pro-inflammatory autocrine bradykinin. Bradykinin has long been implicated in a number of inflammation and edema associated disorders such as diabetic macular edema, hereditary angioedema, vasculopathy, neuropathy and cancer. Bradykinin, signaling through the B1 and B2 receptors, induces vasodilation, edema, sensitization of afferent nerves involved in pain, as well as pro-inflammatory signaling (NETs, IL-1β, TNFα). Rezolute has created a novel suite of orally available small molecule plasma kallikrein inhibitors (PKIs). Our in vitro and in vivo preclinical evaluation funnel has been used to characterize the feasibility of these novel compounds to treat inflammatory disorders. These compounds exhibit IC50 values in the picomolar to single digit nanomolar range, and preliminary in vitro profiling of the lead candidates suggests an excellent pharmacology and drugability profile. One of the Rezolute compounds, RZLT – B, exhibits promising properties and is largely representative of the PKIs in development at Rezolute. RZLT – B has shown excellent plasma kallikrein inhibition both in purified PKal and human plasma PKal potency assays. Additionally, preliminary in vitro toxicity screens have highlighted a defined and understood safety profile. Pharmacokinetic studies have elucidated the consistent plasma exposure after oral dosing and ex vivo plasma potency studies have been used to characterize the in vivo inhibition of plasma kallikrein as a function of oral dosing. To better understand the use of RZLT – B in treating inflammation, a rat hind paw inflammation model has been used, with results comparable to the non-selective COX inhibitor indomethacin. Though an established target for HAE, Plasma Kallikrein has only recently become a therapeutic target for inflammatory and leakage conditions due to the advent of small molecule inhibitors. The suite of novel plasma kallikrein inhibitors at Rezolute were created using a combination of rational design and computational chemistry approaches with a focus on a derisked safety profile and once daily oral dosing. Future studies with these compounds will seek to determine their application in Bradykinin mediated disorders.

In this study we develope a new preclinical model of vulvodynia disease by the constrictions of the pudendal nerve.

Despite representing 50% of the heart failure (HF) population, clinical outcomes and quality of life of individuals with heart failure with mildly reduced (HFmrEF) or preserved (HFpEF) ejection fraction remain poor. Inflammation is central to the pathophysiological process of HF in many of these patients and is mainly driven by their numerous comorbidities. Many mechanisms have been postulated for this inflammatory milieu, amongst which myocardial damage due to coronary microvascular dysfunction (CMD) seems fundamental, reinforcing the importance of targeting inflammation as a potential treatment for patients with HFmrEF or HFpEF. Colchicine is an anti-inflammatory agent that has recently shown benefits in patients with coronary artery disease. In patients with HFmrEF or HFpEF, colchicine may improve CMD and circulating biomarkers of inflammation and fibrosis. In a single-center, randomized, double-blinded, placebo-controlled study, we aim to evaluate the effects of colchicine on CMD in patients with either HFmrEF or HFpEF and inflammation. We will enroll 70 participants with HF and left ventricular ejection fraction > 40% and elevated high-sensitivity C-reactive protein levels (hs-CRP, ≥2 mg/L). Patients will be randomly assigned in a 1:1 ratio to receive a 6-month course of colchicine at a dose of 0.5 mg twice daily or placebo. We will compare the change between baseline and six months in coronary flow reserve, a marker of CMD, assessed using adenosine-based positron emission tomography imaging. In addition, a broad set of circulating inflammation (including MPO, IL-1b, IL-6, TNF-a, sICAM-1) and myocardial fibrosis (including sST2, MMP-2/-9) biomarkers will be measured at baseline and six months. HFmrEF and HFpEF can be considered systemic inflammatory states induced by their frequent associated co-morbidities. While inflammation seems central to disease development and progression, whether a potent anti-inflammatory agent such as colchicine may improve CMD related to inflammation and circulating biomarkers of myocardial fibrosis is unknown. This mechanistic study aims to validate the role of inflammation as a potentially modifiable promoter of myocardial damage associated with CMD in patients with HFmrEF or HFpEF.

Cognitive impairment is one of the most widely reported neurological symptoms of the post-acute sequalae of SARS-CoV-2 infection. There are no FDA-approved drugs to treat this condition. The SARS-CoV-2 spike protein attaches to angiotensin converting enzyme 2 (ACE2), initiating ACE2 internalization for infection, decreasing membrane-bound ACE2 and shifting the renin angiotensin system towards Ang II production. By activating the Ang II type 1 receptor (AT1R), Ang II can induce neuroinflammation and oxidative stress that contribute to cognitive impairment in several animal models (e.g., heart failure, AD). Therapeutics targeting Ang II/AT1R are protective against cognitive impairment in human and animal studies. Ang II also alters sphingolipid metabolism by activating enzymes involved in the biosynthesis of sphingosine-1-phosphate (S1P), a potent inflammatory sphingolipid which we have implicated in the development of cognitive impairment in a model of neurotoxicity. Noteworthy, studies of COVID-19 patient serum have shown increased levels of S1P predicting severity. We developed a mouse model that blocks ACE2 with chronic delivery of the ACE2 inhibitor MLN 4760 (ACE2i model) which results in impairment in the spatial memory and recognition memory mimicking the cognitive deficits associated with SARS-CoV-2. Our data revealed increased S1P levels in the hippocampus and prefrontal cortex (PFC), key centers of cognition, where we found S1PR1 expression. This was associated with markers of oxidative stress and neuroinflammation (two proposed mechanisms thought to drive cognitive deficits in various animal models). Systemic administration of the orally bioavailable functional S1PR1 antagonist ozanimod prevented oxidative stress, neuroinflammation and cognitive impairment in the ACE2i model in the same behavioral tests without affecting locomotor activity. Our results suggest targeting the S1PR1 receptor with ozanimod can prevent the cognitive impairment that occurs from SARS-CoV-2 infection.

In mouse models, NX-13 is an orally active, gut-restricted NLRX1 agonist that reduces colitis in multiple UC models through a novel immunometabolic, bimodal mechanism. Most preclinical UC studies rely upon rodent models, though the human gastrointestinal physiology, microbiome, and immune system bear a greater similarity to that of pigs. Here, we validate our preclinical murine NX-13 results using the dextran sulfate sodium (DSS) pig model of colitis and confirm the immunometabolic mechanism of action. Pigs were randomized by weight (n=6 per group) and were administered tablets of placebo, NX-13 10 mg, NX-13 50 mg, or NX-13 100mg once daily. Simultaneously, pigs were challenged with 1% DSS in drinking water for 6 days and monitored daily. Feces were collected for fecal calprotectin (FCP) quantification. Necropsy was conducted on day 7, and colonic tissue was macroscopically scored and collected for analysis by flow cytometry, histopathology and gene expression. Oral NX-13 treatment protected against weight loss and reduced colitis development with differences as early as day 2, becoming significant between days 4 and 6 (Fig1A). NX-13 yielded a dose-dependent improvement in macroscopic lesion severity and microscopic immune cell infiltration. Specifically, Th1 cells (Fig1B) and TNF producing myeloid cells in the colonic were reduced by NX-13, as well as FCP levels in stool (Fig1C) and mRNA expression in the colon. Oral NX-13 treatment reduced inflammatory cytokines and chemokines. Additionally, NX-13 treatment had MOA specific effects, namely upregulation of NLRX1 (Fig1D) and mitochondrial metabolism gene COX3, and reduction of NFkB and NLRP3. NX-13 demonstrated fast and clinically meaningful improvement in disease activity and impact on key inflammatory markers. These results can be used to guide translational studies in the ongoing NEXUS phase 2 clinical trial.

Iron plays a critical role in lung infections due to its function in the inflammatory immune response but also as an important factor for bacterial growth. Iron chelation represents a potential pharmacological approach to inhibit bacterial growth and pathologically increased pro-inflammatory mediator production. The present study was designed to investigate the impact of the novel iron chelator, DIBI, in murine lung infection induced by intratracheal Pseudomonas aeruginosa (strain PA14) administration. DIBI (80mg/kg) was given by intraperitoneal injection either as a single dose immediately after PA14 administration or a double dose (second dose 4 h after PA14 administration). The results showed that lung NF-κB p65 levels, as well as levels of various inflammatory cytokines (IL-6, TNF-α, IL-1β) both in lung tissue and bronchial alveolar lavage fluid (BALF), were significantly increased 24 h after PA14 administration. Single-dose DIBI did not reduce the inflammatory response and bacterial load in the lungs or BALF. However, two doses of DIBI significantly attenuated levels of NF-κB p65, reduced levels of inflammatory cytokines, and decreased bacterial load. Our findings support the conclusion that the iron chelator, DIBI, can reduce bacterial growth and exhibits anti-inflammatory effects in experimental lung infection induced by P. aeruginosa.

The approval of vaccines against the SARS-CoV-2 virus placed much hope in the attenuation of the global cost of the pandemic. As the innate immune response is an important first checkpoint in the evolution of an infection, we sought to determine the impact of vaccination on neutrophil activation by a viral analog acting on TLR 7/8 ' R848. To this end, a cohort of 304 food and retail workers from Quebec City have given blood samples for three visits at 12-week intervals. Neutrophils were isolated at the first and third visits and were directly stimulated with R848 to assess the innate immune response. After 24h, supernatants were collected to measure IL-8 production by ELISA. In view of the study's scope, from Spring 2021 to Spring 2022, we compared neutrophil response throughout the seasons. The results showed that IL-8 production after stimulation was significantly different in our cohort depending on the season of the visit. This difference was observed in vaccinated and unvaccinated participants. We also found that the participants’ response changed between visits, and that there was an increase in IL-8 production after vaccination. More research is needed to understand the connection between vaccination and season on the neutrophil. Another finding was that subjects who received the Moderna vaccine produced more IL-8 at resting than the unvaccinated and Pfizer groups, and that resting levels of IL-8 were lower for subjects who received their last vaccine dose more than 4 months prior to the visit. This study highlights that neutrophil response could be affected by the season, and by vaccination. It also shows the importance of taking into consideration the period of sampling when studying neutrophils and innate immunity.

INTRODUCTION. An increasing body of evidence indicates that gut microbiota has a significant impact on human health. Indeed, the gut microbiota is the source of several bioactive metabolites. Its taxonomic composition can be dysregulated (dysbiosis) in diseases, notably metabolic syndrome. Some bacteria from the genus Bacteroides, a genus that is increased in obesity, can biosynthesize commendamide, which is an acylated glycine (N-3-hydroxy-oxohexadecyl-glycine). Thus, commendamide shares structural similarities with N-oleoyl-glycine, an N-acyl-amine that is part of the endocannabinoidome. Commendamide could thus represent a potential pharmacological target in dysregulated states linked to the endocannabinoidome, such as obesity and inflammation. In order to fully understand the role(s) of commendamide, we first need to develop an analytical method allowing its quantification in numerous biological matrices, notably feces. OBJECTIVE. To develop an analytical method to quantify commendamide and its congeners by tandem mass spectrometry. METHOD. Commendamide and and commendamide-d2 were syntesized as described previously by Villano et al. Oleoyl-commendamide, palmitoyl-commendamide and their deuterated counterparts were synthesized using a similar strategy. We optimized the detection of the componds and developed a liquid chromatography method allowing the analysis of the compounds. Three extraction methods were also compared allowed us to analyze biological matrices, notably mouse feces and samples from a Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). RESULTS. Commendamide and its congeners were better detected in the negative mode (M-H-) rather than the positive mode (M+H+). Compound extraction was best achieved using a liquid-liquid extraction with acidified methanol and chloroform. Reproducibility experiments will be presented. Finally, commendamide and some of its derivatives were found in both feces and SHIME® effluents. CONCLUSIONS. Our analytical method allows the detection and quantification of commendamide and its congeners. We are now focusing at establishing if there is a link between commendamide levels and Bacteroides species in selected samples.

INTRODUCTION. The endocannabinoids 2-arachidonoyl-glycerol (2-AG) and N arachidonoyl-ethanolamine (AEA) are lipid mediators regulating many physiological processes, notably inflammation. 2-AG and AEA are respectively part of the monoacyl-glycerol (MAG) and N-acyl-ethanolamine (NAE) families. Thus, MAGs and NAEs are considered as part of the endocannabinoidome. Endocannabinoid hydrolysis inhibitors are being investigated as potential treatment in numerous conditions. This strategy will not only increase the levels of 2-AG and/or AEA, but also those of other MAGs and/or NAEs. Increasing MAG and/or NAE levels will likely increase the levels of their metabolites. Herein we investigated whether MAGs and NAEs were substrates for the 15-lipoxygenase pathway, which is strongly involved in asthma and its severity. We thus assessed if human eosinophils and neutrophils biosynthesized the 15-lipoxygenase metabolites of MAGs and NAEs derived from linoleic acid (LA), eicosapentaenoic acid (EPA), docosapentaenoic acid n-3 (DPA) and docosahexaenoic acid (DHA). METHODS. We chemoenzymatically synthesized some putative 15-lipoxygenase metabolites of MAGs and NAEs containing LA, EPA, DPA and DHA and optimized their detection by LC-MS/MS. Human eosinophils and neutrophils were isolated from the blood of healthy volunteers and incubated with MAGs and NAEs at different concentrations and times. RESULTS. Eosinophils, which express the 15-lipoxygenase-1, metabolized all the MAGs and NAEs to the expected 15-lipoxygenase metabolites. Human neutrophils, which might express the 15-lipoxygenase-2, also metabolized most of the MAGs and NAEs, but to a much lower extent than eosinophils. Importantly, some of the new 15-lipoxygenase metabolites we disclose were found in tissues from humans and mice. CONCLUSIONS. We successfully showed that human eosinophils and neutrophils transform MAGs and NAEs into novel 15-lipoxygenase metabolites. How these new metabolites modulate the inflammatory cascade is now being explored as they could participate in the effects of endocannabinoid hydrolysis inhibitors in vivo.

Environmental factors alter human physiology through two main factors: temperature (Tm) and photoperiod variance (Ppv). Tm directly increases the number of mitochondria along with endocannabinoid components in the metabolic systems such as liver, white, and brown adipose tissue. The endocannabinoid (eCB) system consists of enzymes and fatty acids with regulatory effects, namely on adipose and gut tissue/microbiome to brain and behavior. Exposure to constant light disrupts circadian rhythms, leading to alterations in gene expression and endocannabinoid molecule levels. In this study, we investigated the impact of disrupted circadian rhythms on gene expression and endocannabinoid levels in mice liver tissue. Our results revealed significant downregulation of Phospholipase C Beta 1 (PLCB1) and GPR110 in mice liver tissue following circadian disruption. To further elucidate the role of circadian rhythm in gene regulation, we generated a knockout cell line targeting the BMAL1 gene. Gene expression analysis in the knockout cell line showed consistent downregulation of PLCB1 and GPR110. PLCB1 plays a crucial role in calcium signaling by catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). This enzymatic activity leads to the release of calcium ions from intracellular stores, such as the endoplasmic reticulum, into the cytoplasm, thus modulating various cellular processes, including gene expression, cell proliferation, and neurotransmitter release. Our results show an increased calcium level in mitochondria of Bmal1 KO cells. Also, Treatment with docosahexaenoylethanolamine (DHEA), an agonist of GPR110, resulted in decreased calcium levels in mitochondria, suggesting a potential link between circadian rhythm, endocannabinoid signaling, and mitochondrial function. Overall, our findings suggest a complex interplay between circadian rhythm, endocannabinoid signaling, and mitochondrial function, with implications for lipid metabolism and immunological processes.

Annexin A1 (AnxA1) is a calcium-dependent phospholipid-binding protein that plays an important role in regulating neuroinflammation and innate immunity. However, the role of AnxA1 in Parkinson's Disease (PD) was poorly explored. This study presents the role of AnxA1 in a PD mouse model induced by 6-hydroxydopamine (6-OHDA). Male C57BL/6 AnxA1+/+ and AnxA1-/- mice were distributed in two groups: PD+Saline and PD+Ac2-26. Both groups underwent stereotactic surgery for intracerebral injection of 1 μL of neurotoxin 6-OHDA (5 μg/μl) directly into right striatum (ipsilateral) and 1 μL of vehicle on the left (control; contralateral). The PD+Ac2-26 group was treated with Ac2-26 (100 μg/animal) intraperitoneally after the 6-OHDA infusion for 7 days; PD+Saline received only saline. Cylinder test was performed to assess sensory-motor function of mice on 0-, 7- and 14-days post-lesion. All saline-treated mice showed significant decreased contralateral forelimb use compared to ipsilateral on days 7 and 14. Ac2-26 treatment produced a reversal of forelimb use asymmetry for both genotypes and no differences were detected between use contralateral and ipsilateral on days 7 and 14. Western blotting corroborated these findings. A significant reduction in tyrosine hydroxylase (TH) levels was detected in the right striatum and substantia nigra (SN) from both genotypes compared to the left side (control). Ac2-26 administration reversed this effect and no differences were detected for TH levels in right and left sides of the striatum and SN. Lack of endogenous AnxA1 was associated with reduced levels of the IL-4 on both sides analyzed (R, L) of striatum and SN, regardless of systemic treatment (saline or Ac2-26). In AnxA1-/- SN, the addition of Ac2-26 produces a broad anti-inflammatory effect with a reduction in the levels of IL-1β, IL-6, IL-17 and TNF-α, especially on the right side (6-OHDA), compared to AnxA1+/+ SN. Taken together, our results show that in the PD model, the endogenous lack of AnxA1 does not worsen the development of the disease. On the other hand, Ac2-26 therapy shows neuroprotective and anti-inflammatory effects on SN in both genotypes, highlighting its potential use in neurodegenerative diseases. Funding: FAPESP, CAPES, CNPq.

Acute lung injury (ALI) is the most frequent distant organ dysfunction in acute pancreatitis (AP), with more than a quarter of patients admitted for AP eventually developing ALI. It has a high mortality rate and is the main cause of death in AP patients. We have previously shown that a disintegrin and metalloprotease 17 (ADAM17) plays a role in promoting AP using the well-documented cerulein-induced experimental AP model. This is via a form of ADAM17 mediated signaling known as interleukin-6 (IL-6) trans-signaling whereby ADAM17 cleaves membrane bound IL-6 receptor to produce its soluble receptor (sIL-6R). However, the role of ADAM17 in ALI is undetermined. To assess the role that ADAM17 plays in ALI, we have initially employed a single day experimental model, with mice receiving 7 intraperitoneal injections of cerulein (50ug/kg in 50ul PBS). We found that this model showcases early molecular changes of ALI in the lung, such as increases in mRNA expression of inflammatory mediators and protein expression of signaling pathways known to be involved in ALI and linked to ADAM17. In particular, IL-6 trans-signaling components, interleukin-6 (IL-6) and signal transducer and activator of transcription 3 (STAT3) was augmented in cerulein-treated wild-type mice. Moreover, cerulein treated Adam17ex/ex mice (>90% reduction in global ADAM17 expression) showed a reduction in proinflammatory mediators and signaling pathways implicated in ALI, suggesting a role for ADAM17 in ALI pathology. Taken together, the single day cerulein model is useful for preliminary studies in determining whether ADAM17 plays a role in ALI and in characterizing its early changes. Studies using a longer model is currently underway to further explore the changes seen.

INTRODUCTION: The autoimmune character of multiple sclerosis (MS) is well defined and the source of many treatments targeting immune cells of people living with the disease. Most immune-modifying drugs used for MS slow down the disease course, but generate an increased vulnerability to infections without curing MS. Using the experimental autoimmune encephalomyelitis (EAE) mouse model, we and others have demonstrated that interleukin-1β (IL-1β) is a key inflammatory mediator in the pathophysiology of EAE, as IL-1β-knockout mice are protected from the disease. To exert its proinflammatory role, IL-1β requires two different signals. A first one induces the transcription of the immature form of the cytokine, pro-IL-1β, and a second one activates the canonical inflammasome pathway, a large multiprotein complex responsible for the cleavage of pro-IL-1β into its bioactive form. We hypothesize that blocking all mechanisms of IL-1β maturation, whether inflammasome-dependent or not, will alter EAE progression and alleviate the symptoms. As a first step towards addressing this hypothesis, we aim to identify the proteases responsible for the maturation of IL-1β during inflammatory conditions using proximity-dependent biotinylation. METHODS: Proximity-dependent biotinylation takes advantage of a biotin ligase to attach a biotin molecule to every protein within proximity of a protein of interest. These proteins are then captured with streptavidin beads and identified using mass spectrometry. Here, we generated a plasmid coding for a doxycycline-inducible biotin ligase coupled to pro-IL-1β (TurboID-HA-pro-IL-1β) and expressed it in a RAW264.7 murine macrophage cell line using lentiviruses. RAW264.7 macrophages transduced with a plasmid coding for a doxycycline-inducible TurboID-HA-eGFP were used as a negative control. RESULTS: Immunoblotting analysis revealed a doxycycline-dependent expression of the construct in transduced RAW264.7 macrophages, and that the biotinylation capacity of TurboID increases over time, as anticipated. The expression and cellular localization of the construct were confirmed by immunofluorescence. Hence, we expect that this technique could help elucidate the IL-1β interactome through mass spectrometry. These findings could unveil novel therapeutic targets and pave the way for future research avenues aimed at discovering effective and safe treatments for MS patients.

Severe dengue (DG) is marked by intense inflammation, causing cytokine storm, resulting in vascular leakage, hemorrhage, and shock. Innate immune cells and products are vital in these processes. No licensed antiviral drugs or host-targeting therapies exist for dengue, underscoring the urgent need for new treatments. Our research has identified Annexin A1 (AnxA1) as a critical mediator of the resolution response in dengue. AnxA1 acts through its FPR2/ALX receptor expressed on various cell types, including leukocytes, mast cells and endothelial cells. We observed reduced circulating levels of AnxA1 in dengue patients, particularly in those with severe disease, as well as in DENV-infected mice. AnxA1 knockout (AnxA1KO) mice displayed increased susceptibility and delayed disease resolution, emphasizing the importance of AnxA1 in dengue pathogenesis. Conversely, treatment of mice with the AnxA1 mimetic peptide (Ac2-26) improved disease outcomes without compromising the host's ability to deal with the infection. However, systemic administration of proteins like AnxA1 is hindered by instability and rapid degradation. To address this, we created a formulation using cyclodextrins (CDX), known for stability and enhanced bioavailability. We utilized Hydroxypropyl-β-cyclodextrin (HP-BCD) to improve the stability and in vivo effects of Ac2-26. Experiments were conducted on A129 mice infected with DENV-2, treated with pure or formulated Ac2-26 via intraperitoneal (i.p.) or oral routes of administration, starting 36 hours post-infection. Euthanasia occurred at days 3 or 5 post-infection, representing the peak of viral replication and disease manifestation, respectively. Results showed that both pure and formulated Ac2-26 treatments reversed clinical scores and thrombocytopenia induced by DENV infection. Moreover, formulated Ac2-26 exhibited potentiated anti-inflammatory effects by reducing mast cell degranulation and plasma levels of MCPT-1, as well as, reducing levels of several cytokines in the spleen and liver damage induced by infection. Notably, the formulation containing Ac2-26 did not impact DENV titers, indicating its selective anti-inflammatory and pro-resolving properties. Finally, the association of formulated Ac2-26 with and antiviral drug (a nucleotide analogue), fully prevented mice from DENV-induced disease and lethality.These findings underscore the importance of developing combined therapeutic approaches that aim for synergistic effects, leading to more effective strategies with reduced side effects on the host.

The production of specialized pro-resolving mediators (SPMs) during the resolution phase in the inflammatory milieu is key to orchestrating the complete resolution of the acute inflammatory response. Resolvin D2 (RvD2) exhibits potent pro-resolving functions in sepsis via activation of the GPR18 receptor (Nature, 2009, PMID: 19865173; JEM, 2015, PMID: 26195725). Here we uncovered a new resolvin in fresh human saliva. We identified it as epimeric to RvD2 after carrying out the first total organic synthesis of this new resolvin. The epi-RvD2 was produced by M2-like macrophages, M1-like macrophages and monocytes incubated with docosahexaenoic acid (5 ug) and zymosan (100 ng/ml) 45 minutes at 37oC. The physical properties, namely the chromatography and tandem mass spectrometry fragmentation pattern, of the biologically produced epi-RvD2 matched those of the newly synthesized material. We provide evidence that this new synthetic epi-RvD2 binds and activates the GPR18 receptor, in an equipotent manner as RvD2 (1-100 nM). In addition, the synthetic epi-RvD2 was shown to exhibit pro-resolving functions. Flow cytometric analyses revealed that topical application of epi-RvD2 (1 ug) 15 minutes prior to the addition of LTB4 (1 ug) and PGE2 (1 ug) significantly decreased the number of neutrophils in the mouse ear compared with the application of LTB4 and PGE2 alone. The synthetic epi-RvD2 increased M2 makers CD206 and CD163 on human monocyte-derived macrophages and enhanced efferocytosis of senescent red blood cells by M2-like macrophages. Also, increasing concentration of epi-RvD2 (0.1-10 nM) significantly increased phagocytosis of E. coli by human neutrophils in a time- and dose-dependent manner compared to vehicle alone. Together, these results establish this novel hydroxy epimer of resolvin D2 for its structure and potent pro-resolving functions. The authors acknowledge support by US NIH grants P01GM095467 and R35GM139430

Alzheimer's disease (AD), an age-associated neurodegenerative disorder, is characterized by progressive neuronal loss and the accumulation of misfolded proteins like amyloid-β and tau. Neuroinflammation, mediated by microglia and brain-resident macrophages, plays a pivotal role in AD pathogenesis, yet the intricate interactions among age, genes, and other risk factors remain elusive. Somatic mutations, known to accumulate with age, instigate clonal expansion across diverse cell types, impacting both cancer and non-cancerous conditions. Utilizing molecular-barcoded deep panel sequencing on 311 prefrontal cortex samples of AD patients and matched controls, our study unveiled an elevated occurrence of somatic mutations within cancer driver genes in AD brains. Recurrent somatic mutations, often multiple, were observed in genes associated with clonal hematopoiesis (CH). Remarkably, these somatic cancer driver mutations were specifically enriched in CSF1R+ microglia of AD brains and exhibited signs of positive selection, suggesting mutation-driven microglial clonal expansion (MiCE). Single-nucleus RNA sequencing of the temporal neocortex samples from additional 62 AD patients and matched controls revealed a nominal increase in subchromosome-level somatic mutations associated with CH in AD microglia, with mutant microglia exhibiting upregulated pro-inflammatory and AD-associated genes. We further created three lines of induced pluripotent stem cell (iPSC)-derived microglia, each carrying driver mutations in one of the most mutated genes in AD brain. Through single-cell RNA sequencing, we verified that these mutant microglia consistently displayed an amplified inflammatory and disease-associated transcriptional profile compared to their wild-type counterparts. Our findings indicate that somatic cancer driver mutations in microglia are prevalent in normal aging but further enriched in AD, driving MiCE and promoting inflammatory, disease-related microglial signatures. This study provides crucial insights into microglial clonal dynamics in AD, potentially paving the way for novel approaches to AD diagnosis and therapy.

Evaluation of the role of the hydroxyl carboxylic acid receptor type 2 (HCAR2) in microglia pathophysiology and experimental model of neurophatic and nociplastic-like pain.

G-protein coupled estrogen receptor (GPER) is recognized as a novel therapeutic target in various diseases including cancer, cerebrovascular, metabolic, neurodegenerative, and chronic inflammatory diseases like arthritis, and inflammatory bowel disease. The anticancer effect of G1, a GPER agonist, has been investigated alone or in combination with pembrolizumab in patients with metastatic uveal melanoma. However, there are conflicting reports that the anticancer effect of G1 appears to be due to cytoskeleton destruction independent of GPER. In this study, we investigated the cytotoxic mechanism of GPER agonist G1 and GPER antagonist G15 in cancer cells and macrophages. Our findings revealed that both G1 and G15 induced concentration-dependent necroptosis in cancer cells, such as HT-29 human colon cancer and PC-3 human prostate cancer, and macrophages (TPA-differentiated THP-1 cells). The necroptosis induction was comparable to that observed with TNFα treatment, as demonstrated by FACS analysis with annexin V antibody/propidium iodide staining and the phosphorylation of RIP1, RIP3, and MLKL. G1- and G15-induced necroptosis and activities of p-RIP1, p-RIP3, and p-MLKL remained unaffected by GPER knockdown. Rather, G1- and G15-induced RIP1 phosphorylation was inhibited by the RIP1 inhibitor necrostatin-1 in a concentration-dependent manner. Additionally, the necroptosis triggered by G1 and G15 was effectively inhibited by taurodeoxycholic acid, an ER stress blocker, and vitamin E. On the other hand, G1 demonstrated the ability to attenuate the disruptive effect of TNFα on the intestinal epithelial barrier formation, while G15 did not. Taken together, GPER ligands, G1 and G15, induce cytotoxicity irrespective of cell type, and this effect is not mediated through GPER action. The current findings suggest that it is important to consider cytotoxicity issues when using GPER ligands G1 and G15 for GPER-dependent therapeutic effects.

INTRODUCTION: Pain affects 20% of adults worldwide. In patients with inflammatory autoimmune diseases such as multiple sclerosis (MS), this prevalence is over 50%. Painful information is transmitted from the periphery to the spinal cord and then the brain through the dorsal root ganglia (DRGs), where sensory neurons called nociceptors reside. Interleukin-1β (IL-1β) is a pro-inflammatory cytokine capable of triggering both inflammation and pain. OBJECTIVE: To identify the mechanisms mediating inflammatory and painful responses induced by IL-1β. METHODS: IL-1β injections were performed in the cerebellomedullary cistern of wild-type mice, mice globally knockout for the Il1r1 gene, and mice in which Il1r1 gene expression was specifically knockdown or restored in TRPV1+ nociceptors. We then characterized neurons expressing IL-1R1, studied the presence of transcription starting sites (TSS) from total RNA extracted from DRGs using 5’RACE-PCR, and performed immunoblotting to verify the presence of IL-1R1 isoforms. We also studied the expression of the two isoforms of the IL-1R1 coreceptor, IL-1RAcP and IL-1RAcPb, by in situ hybridization. RESULTS: IL-1R1 is expressed exclusively in a subtype of nociceptors expressing TRPV1. Invalidation of the Il1r1 gene in these nociceptors prevented tactile pain (allodynia) in mice with experimental autoimmune encephalomyelitis, a model of MS, without affecting other clinical signs. Restoration of IL-1R1 resulted in the return of painful behaviors. We also identified a TSS encoding a novel Il1r1 mRNA and confirmed the existence of a novel truncated isoform that we named τIL-1R1. Finally, we detected the presence of IL-1RAcP and IL-1RAcPb in DRG neurons. CONCLUSION: IL-1β triggers inflammation and pain through independent mechanisms. Painful response is likely to be mediated by TRPV1+ nociceptors via the τIL-1R1 isoform.