Mucosal healing, the primary goal for IBD treatment, plays an important role in the re-establishment of the intestinal epithelial barrier function. Emerging evidence shows that key players of the inflammation initiation is also involved in the mucosal repair program. Our previous data showed that rhamnogalacturonan (RGal), a pectic polysaccharide, enhances the intestinal epithelial barrier function through TLR4 and PKC activation, and accelerates wound healing in Caco-2 cells and colonoids. RNAseq data and pathway analysis have indicated the involvement of the canonical nuclear factor B (NF-B) signaling pathway. We hypothesize that RGal drives an inflammatory gene expression profile to promote resolution of inflammatory phase and increase wound healing. Confluent Caco-2 and colonoids monolayers were apically treated with vehicle (media) or RGal (1000 g/ml) for 6, 12 and 24 h. RNA was isolated and RNAseq was performed. Selected proteins were evaluated through electrochemiluminescence. For the neutrophil migration assay, Caco-2 monolayers were seeded on 24-well plates and treated with vehicle (media), TNF (10 ng/ml) or RGal (1000 g/ml). Neutrophils (2 x 105) were added to 8 m porous filter transwells and incubated for 4 h at 37 C. Female and male mice with DSS-induced colitis were orally treated with RGal (10 mg/kg) for 7 days during the recovery phase. RGal treatment at different time-points induced changes in gene expression, upregulating the expression of inflammatory response genes responsible for neutrophil production and chemotaxis (CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, G-CSF). RGal also increased CXCL8 family chemokines, confirming the gene expression data, and neutrophil migration by 98%. RGal treatment of male mice reduced the weight loss at later days, fecal lipocalin-2 levels and restored the colon length. In addition, RGal reduced the number of neutrophils when compared to DSS group but increased it compared to naïve group. These data suggest that RGal upregulates inflammatory genes and proteins downstream NF-B activation. The RGal effect is mainly on neutrophil-dependent pathway since we observed increased neutrophil migration in vitro and in vivo. Our findings show that RGal can modulate the inflammation/repair program of the intestinal epithelium and this could lead to the resolution of intestinal inflammation and mucosal healing.

The normal immune response to infection requires a well-regulated mechanism of leukocyte activation and recruitment, a process generally termed inflammation. Dysregulation of this inflammatory response is a major problem in modern medicine and is associated with several disease conditions including autoimmune diseases, cancer and even COVID-19. Food-derived peptides with anti-inflammatory properties have gained popularity due to their availability in the daily diet and limited side effects. Previous reports have shown that the potato protein patatin has anti-inflammatory property. However, the sequence of amino acids in patatin that is critical for anti-inflammatory activity is not well established. In this work, we investigated the ability of the potato protein-derived peptide DIKTNKPVIF to down-regulate the inflammatory response of monocyte-derived macrophages (MDMs) activated with lipopolysaccharide (LPS). We found that DIKTNKPVIF inhibited the inflammatory response of MDMs to LPS as evidenced by decreased production of pro-inflammatory cytokines. Importantly, using computational and experimental screening techniques, we identified TNKPVI as the bioaccessible and biostable pharmacophore necessary for the activity of DIKTNKPVIF. Our results highlight the potential of potato-derived bioactive peptides to regulate the inflammatory response.

B lymphocyte stimulator factor (BLyS) is produced by wide range of cells of the immune system, and has proven to be a key factor in the selection and survival of B cells. BLyS is an important factor in the pathology of Systemic Lupus Erythematosus; elevated serum levels of soluble BlyS are at increased risk of flare. Objectives: We aimed to analyze the association between the BLyS serological levels and clinical manifestations, as well as with disease activity in SLE patients. Methods: A cross-sectional, observational study in SLE patients (SLICC 2012 criteria) and healthy controls (HC) was performed. In SLE patients complete laboratory test, clinical evaluation and SLEDAI score was carried out. Serum concentration of BLyS was analyzed by colorimetric methods. Lupus patients were dichotomized as high and normaL BLyS levels based on BLyS levels above 2 SD of the mean in healthy controls. Results: 166 SLE patients (86.7% female) participated in the study, with a mean age at diagnosis of 34 (14) years and a mean time of disease evolution of 16 (11) years, and 87 HC (67% female) with a mean age of 45 (14) years. The 32.5% of patients showed SLEDAI>6. The 57.8% were under glucocorticoid treatment, 47% under immunosupressants or biological therapy and 58.43% under antimalarials. BLyS levels were significantly higher in SLE patients (3133.8ng/mL) than in HC (2618.02ng/mL) (P=0.036), and 4600.72ng/mL was the cut-off point. The 12% of patients and the 3.4% of HC displayed increased BLyS levels. Higher BLys levels showed a significant association to elevated SLEDAI score (P=0.03) and were significantly associated to anti-dsDNA positivity (P=0,01), but showed no association with hypocomplementemia. The statistical analysis yield differences in belimumab treatment (P=0.002) and corticoid therapy (P=0.01) between patients with lower and higher BLyS levels. No influence of age at diagnosis, time of evolution and tobacco use in BLyS levels was observed. Conclusion: In our series we observed a 12% of patients with high levels of BLyS, and BLyS high levels showed a tendency to be associated to high SLEDAI score. BLyS levels are influenced by anti-dsDNA positivity, as well as to corticoid therapy and belimumab treatment.

Dietary cadmium (Cd2+) intake is implicated in the pathogenesis of hypertension and anaemia, but there is a paucity of information regarding haematological changes in hypertensive conditions. This study, therefore, evaluated the effects of Cd2+ on blood pressure (BP) and haematological indices in Sprague-Dawley rat model. Three cohorts (n=10 each) of control and Cd2+-fed male Sprague-Dawley rats were selected. Cd2+-exposed rats received 2.5 or 5 mg/kg b.w. cadmium chloride via gavage thrice-weekly for eight weeks, while control animals received tap water. BP and blood flow were measured non-invasively from rat tails twice-weekly using a CODA machine, while weights were measured thrice-weekly. Haematological indices were assessed using the Cell-Dyn Emerald Haematology Analyzer. Data were reported as mean ± SEM, and statistically analyzed using One-Way Analysis of Variance. Bonferroni post hoc test was used for multiple comparisons. Cd2+-exposure induced hypertension by significantly (p<0.05) elevating systolic, diastolic, and mean arterial BPs, pulse pressure, heart rate, and blood flow. Mean cell haemoglobin and mean cell volume were significantly (p<0.05) reduced, and red cell distribution width significantly (p<0.01) increased by exposure to 5 mg/kg b.w. Cd2+. Mean cell haemoglobin concentration, haematocrit, haemoglobin, red blood cell, platelet, mean platelet volume, and white blood cell counts were unaffected by Cd2+-exposure. Cd2+ induced hypertension with microcytosis, possible hypochromicity, and anisocytosis without anaemia, which may be precursor to microcytic anaemia and coronary artery disease. This study is important in Cd2+-exposed environments and warrants further investigations into possible association between Cd2+ exposure and hypertension management and evaluating hypochromicity using blood smears.

Traumatic Brain Injury (TBI) represents a significant global health concern, contributing to long term disability and cognitive impairments. TBI induces a robust inflammatory response with levels of cytokines such as TNF and IL-1, playing pivotal roles in the secondary injury cascade. Synthesized molecules will be discussed and if they have a potential neuroprotective effect on inhibiting TNF and IL-1 and thus reducing neuronal cell death, preserving synaptic integrity and enhancing neuroregeneration.

G protein-coupled receptors (GPCRs), the largest family of receptors in humans, activate effector proteins to rapidly generate a multitude of second messengers such as cyclic adenosine monophosphate (cAMP), diacylglycerol (DAG), phosphatidic acid (PA), inositol 1,4,5-trisphosphate (IP3), calcium, nitric oxide (NO), cyclic guanosine monophosphate (cGMP) and reactive species of oxygen (ROS). Our research group has previously demonstrated the essential role of the ubiquitin ligase TRAF6 in the very rapid activation of the protein kinases TGFβ-Activated Kinase 1 (TAK1) and inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), leading to an inflammatory response in vascular smooth muscle cells upon activation of the angiotensin II (Ang II) AT1 receptor. Following the covalent attachment of lysine 63-linked polyubiquitin (polyUbiK63) to substrates such as NEMO (IKKγ) or TAB2, TRAF6 controls the activation of IKKβ and TAK1 respectively. In vitro evidences also suggest that TRAF6 produces free unattached polyUbiK63 chains. Since TRAF6 is responsible for the very rapid activation of the inflammatory kinases TAK1 and IKKβ, we propose that following the activation of GPCRs belonging to the Gq/G11 and Gi families, there will be rapid and transient production of polyUbiK63 free chains, which will be found in immunocomplexes comprising the activated TAK1 and IKKβ kinases and leading to an inflammatory response. Using wild-type mouse embryonic fibroblasts and TRAF6-deficient cells, we show that the absence of TRAF6 reduces the anti-phospho signals of several effectors controlling the TAK1 and IKKb signaling cascades in response to lysophosphatidic acid (LPA) and thrombin (Thr). Furthermore, cells overexpressing domain that specifically binds free/unanchored polyubiquitin chains also show deficient signaling events in response to LPA stimulation. Our results demonstrate the pivotal role of TRAF6-mediated polyUbiK63 free chains in orchestrating the downstream inflammatory responses triggered by GPCR activation. These findings shed light on potential therapeutic targets for modulating the NF-κB inflammatory signaling pathways.

Bone tissue is continuously remodeled through the concerted actions of bone cells, including osteoclasts and osteoblasts. An optimal balance between bone resorption by osteoclasts and bone formation by osteoblasts is required for bone homeostasis. The TNF family cytokine RANKL is an essential cytokine for osteoclast differentiaion. RANKL, which are produced by the supporting mesenchymal cells including osteoblast lineage cells binds to its receptor RANK on the precursor cells of monocyte/macrophage lineage. Pathologically, excess RANKL signal leads to abnormal osteoclast activation and bone loss in rheumatoid arthritis, osteoporosis, and bone metastasis. On the other hand, RANKL also plays crucial roles in the immune system, including lymph node development, thymic epithelial cell differentiation and M cell differentiation in the gut. Thus, RANKL exactly acts as a multifunctional cytokine that influences the skeletal and immune systems. RANKL is synthesized as a membrane-bound molecule, which is cleaved into the soluble form by proteases. By generating mice selectively lacking soluble RANKL, we have revealed that soluble RANKL is dispensable for physiological regulation of bone and immune systems. In addition, we found that soluble RANKL made no contribution to inflammation-induced bone desctrutsion. These findings indicated the importance of local regulation of the RANKL/RANK system. Since RANKL is a mulfuncaional cytokine produced in a wide variety of tissues, assessing the dynamics of the producing cells under both physiological and pathological conditions is important for the elucidation of the pathogenesis of various bone loss disorders. We established the strategies to analyze the spaito-temporal expression of RANKL, which would contribute to the understanding of the mechanisms underlying the local regulation of the RANKL/ RANK system and the development of the therapeutic intervention for various bone loss.

The production and release of interleukin-8 (IL-8/CXCL8) by endothelial cells play a critical role in recruiting immune cells to the site of infection. In agreement, various stimuli such as pathogen-associated molecular patterns (PAMPs) and ligands for Toll-like receptors (TLRs) induce the release of IL-8, along with the release of various danger molecules such as nucleotides. Several studies, including the work of our research team, suggest the involvement of endogenously released nucleotides in inflammatory processes through the activation of their P2 receptors. In this research work, we investigated the role of extracellular nucleotides in PAMP-induced IL-8 release by human umbilical vein endothelial cells (HUVECs) expressing P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 receptors. ELISA analysis of supernatants collected after stimulating HUVECs with TLR-specific PAMPs for 18 hours revealed significant IL-8 production in response to TLR3 (poly(I:C)) and TLR4 (LPS) ligands but not to TLR1/2 (Pam3CSK4) and TLR5 (flagellin) ligands. This response was inhibited in the presence of apyrase, an enzyme that hydrolyzes extracellular nucleotides, non-selective inhibitors of nucleotide receptors (suramin and RB2) as well as the specific antagonist of the P2Y2 receptor (AR-C118925XX). Partial knock-down of the P2Y2 receptor using specific small hairpin RNAs also resulted in a significant reduction in IL-8 release by HUVECs in response to poly(I:C) and LPS. Our results suggest that there is a release of nucleotides following the activation of TLR3 and TLR4. These nucleotides, in turn, activate the P2Y2 receptor on the cell surface, leading to substantial IL-8 secretion. This highlights the critical role of the P2Y2 receptor signaling in the modulation of poly(I:C)- and LPS-induced IL-8 secretion in HUVECs. Blocking P2Y2 receptor could potentially prevent excessive inflammation and endothelial dysfunction.

INTRODUCTION: Most viable cells generate EVs, which originate from their endosomal compartment (exosomes, 50-100 nm in size) and/or from plasma membrane blebbing and budding (ectosomes, 100-1000 nm). Neutrophil-derived ectosomes (NDEs) are produced in response to several stimuli and are involved in a myriad of physiological and pathological processes (1). Flow cytometry (FCM) is often used to study NDEs and other EVs. However, due to the small size of EVs, their measure is hindered by the unavoidable presence of non-vesicular nanoparticles in the samples, buffers, sheath fluid and other reagents, which can yield high background or even EV swarming by nonspecific events (2,3). These problems are fairly common and have been the subject of specialized publications (2-4). METHODS: Neutrophils were isolated from healthy donors and stimulated with fMLP to produce NDEs. These were isolated through serial centrifugations and analyzed by flow cytometry (Beckman Coulter Cytoflex) using the blue (SSC-488) and violet side scattering settings (SSC-405). The primary threshold was set on FSC (size) or calcein blue fluorescence, to compare the background signal. NDEs were characterized as events positive for Calcein Blue (closed vesicles), Annexin V (exposed phosphatidylserine) and expressing the neutrophil membrane markers CD15 or CD66b . This cytometry strategy was then applied to NDE quantitation in human plasma from healthy and septic non-fasting subjects. RESULTS: The use of SSC-405 increased the detection of EVs and other nanoparticles. However, event swarming and abort rate rose from <4% to 25-40%. The swarming of non-vesicular events could be mostly eliminated by changing the setting of the detection threshold from FSC (size) to calcein fluorescence. This reduced abort rates to <5% and yielded consistent results along a wide range of NDE concentrations. In plasma samples, NDEs were detected in low concentrations in healthy donors and were 10-fold more abundant in sepsis patients, without interference by lipoproteins or other analytes. CONCLUSION: We set up a highly sensitive, calcein-based cytometry method that eliminates most of the background noise by focusing on the acquisition of vesicular events without the interference of non-vesicular nanoparticles for NDE analysis in complex samples. Our method also dispenses with commonly used dilution and/or filtration steps during sample preparation.

The paper focuses on studying specific mutations in BRCA1, BRCA2, PALB2 and CHEK2 genes characterized by high occurance rate among women in Crimea as well as their role in ovarian cancer development

During early stages of systemic inflammation neutrophil extracellular traps (NETs) released by neutrophils are engaged in pathogen trapping and immobilization. However, as the inflammatory response progresses, NETs persist in vasculature even if not needed any more. This is leading to bystander tissue damage and organ injury. Hardly anything is known about mechanisms and kinetics of NET removal in vivo therefore we aimed at investigating it. The process was studied during endotoxemia and NETs composed of neutrophil elastase (NE), histones and extracellular DNA (extDNA) were followed for 365 days in liver sinusoids with intravital microscopy (IVM). Protein NET components, unlike extDNA, were not detached from endothelium for months. Liver macrophages (Kupffer cells), but not monocytes, were mostly engaged in their removal but also neutrophils themselves participated in the process. Multiple receptors were engaged in this process, including TLR2 and TLR4 but also scavenger receptors. Although lipopolysaccharide (LPS) was used to induce inflammation, to avoid pathogen replication and survival in leukocytes, we detected self-renewal of NETs. The second wave of NETs was initiated by histones which triggered an inflammatory milieu, activated platelets and coagulation-related events, including factor VII-activating protease (FSAP) activity. Our study shows that complete removal of NETs in vivo is a very long process leading to a vicious cycle of NET formation. This finding explains detection of NET components in inflammatory disorders at their various stages and thus has a therapeutic potential. The study was funded by the National Science Centre of Poland (grant 2021/43/B/NZ6/00782).

Upon prolonged eye closure at night, over hundreds of thousands of leukocytes can be collected from the ocular surface upon awakening, with polymorphonuclear neutrophils (PMNs) representing the main population. While leukocytes have been previously observed in tears of individuals suffering from ocular allergies, there is limited knowledge on tear PMNs and their potential role in ocular allergy. This study aimed to characterize the phenotype of tear PMNs collected from participants suffering from ocular allergy. Participants with and without ocular allergies were recruited to collect tear leukocytes after a full-night of sleep (closed-eye tear PMNs) and at the end of day (open-eye tear PMNs) using a gentle eyewash. Cells were counted and characterized by flow cytometry before and after stimulation with fMLP. Significantly more closed-eye tear leukocytes were collected from ocular allergy participants compared to healthy participants, while there was no difference in the number of open-eye tear leukocytes. Furthermore, closed-eye tear PMNs from ocular allergy participants exhibited a less activated phenotype but a higher activation potential in response to fMLP. There was no significant difference in the production of ROS, suggesting that oxidative stress may not be a key contributor to ocular discomfort associated with ocular allergy. Further research is needed to characterize the potential contribution of tear PMNs to the development or progression of symptoms of ocular allergy.

Systemic sclerosis (SSc) is a fibrosing disorder, characterized by a vasculopathy, an exacerbated inflammation and an autoimmunity, partly due to the aberrant production of IFNα by the plasmacytoid dendritic cells (pDCs) and to defective central B cell tolerance. Indeed, pDCs have been documented to produce large amounts of IFNα via the TLR7 and TLR9 signaling. Moreover, deficiency of MYD88, which mediates the function of both TLRs, results in a failure to silence autoreactive B cells. CXCL4 is one of the inflammatory molecules found increased in the serum of SSc patients and its level correlated with skin and pulmonary disease. Our objective was to investigate whether CXCL4 regulates TLR9 signaling in pDCs and B cells. pDCs and B cells were isolated from the blood of healthy donors (HDs) or SSc patients using BDCA4 and CD19-magnetic beads respectively. pDCs or B cells were then cultured with medium, TLR9 ligand (TLR9-L) alone or with CXCL4. Cytokines gene expression and secretion were analyzed. Delivery of TLR9-L was analyzed by Amnis and confocal microscopy. We demonstrated that pDCs from SSc patients spontaneously secreted CXCL4, which subsequently potentiated the production of IFNα from TLR9-induced pDCs. We demonstrated that the CXCL4 formed nanoparticles with DNA, which promoted its uptake and skewed its delivery from the late endosomes to the early endosomes. This is concordant with our previous data that show that the intracellular trafficking of the TLR9 ligand in human pDCs is important. Indeed, IFN was produced from pDCs when the TLR9-L was localized in the early endosomal compartment where IRF7 is engaged. Surprisingly, we observed that CXCL4 abrogated TLR9 response in human B cells. CXCL4 prevented the delivery of TLR9-L to the late endosomes, where the signaling occurs in B cells. CXCL4 in vivo expression led to defective TLR9 responses and increased the number of polyreactive B cells, hence impeding central B cell tolerance. Our data provide evidence for a novel mechanism by which CXCL4 superinduces the interferon production by TLR9-induced pDCs. CXCL4 also impairs central B cell tolerance by altering the intracellular trafficking of TLR9 ligands, hence inhibiting TLR9 response which is required for the removal of developing autoreactive B cells. Taken together, our data shows a pathogenic role of CXCL4 in both pDCs and B cells.

[Objective] Rheumatoid arthritis is a prototypical inflammatory disease characterized by persistent chronic inflammation. Our study focused on immunophenotypic stratification, aiming to optimize molecular targeted therapies for rheumatoid arthritis. [Methods] We stratified 533 bio-naive patients diagnosed with rheumatoid arthritis through immunophenotyping of immunocompetent cells (T cells, B cells, NK cells, monocytes, and dendritic cells) in peripheral blood using flow cytometry. We analyzed the treatment response using molecular targeted drugs, ensuring matching of patient backgrounds through inverse probability of treatment weighting (IPTW). [Results] Cluster analysis stratified 533 RA patients into 5 clusters. 2 of these showed distinctive RA phenotypes differing HC, marked by significant increases in CD4 effector memory T cells re-expressing CD45RA (TEMRA). We assessed the clinical efficacy of each molecular targeted therapy, including TNF inhibitor, IL-6 inhibitor, CTLA4-Ig, and JAK inhibitor, within each group and observed significant differences in their effectiveness. Subsequently, within each cluster, the utilization of b/tsDMARDs with high efficacy was designated as the Preferred group, while the use of alternative drugs was categorized as the Non-preferred group. In the Preferred group, both the 24-week remission rate and the rate of achieving low disease activity were significantly higher compared to the Non-preferred group. To validate these findings, immunophenotyping was conducted in a new validation cohort comprising 183 cases. The subjects were then reassigned to the aforementioned clusters using the k-nearest neighbor method. Significantly, in the validation cohort, the remission rate of the Preferred group at 24 weeks exceeded that of the Non-preferred group, demonstrating an effectiveness more than twice as high. [Conclusion] Immunophenotypic stratification underscored the potential for treatment optimization.

Extracellular trap (ET) formation, initially recognized in neutrophils, has emerged as a phenomenon exhibited by various innate immune cells, including monocytes/macrophages. Monocyte/macrophage extracellular traps (METs) constitute a novel strategy in the cellular defense repertoire against pathogens. Despite their significance, the mechanistic insights into MET formation remain incomplete. In this study, we aimed to validate the formation of METs by bone marrow-derived macrophages (BMDMs) and to delineate the involvement of reactive nitrogen species (RNS) in this process. BMDMs were generated by differentiation of bone marrow cells from C57BL/6J male mice. Differentiation was facilitated using conditioned medium from the mouse fibroblast cell line L929, and functional activity was confirmed by flow cytometry, assessing the expression of F4/80+ and CD11a+ markers. Differentiated BMDMs were subjected to stimulation with a nitric oxide synthase (NOS) inhibitor (L-NAME), a nitric oxide donor (SNAP), or lipopolysaccharide (LPS). Visualization of METs was achieved through confocal microscopy, employing Sytox Green DNA stain and antibodies targeting MET components, including histones (H2A.X) and MMP-9. Our findings demonstrate that fully differentiated and functional macrophages, characterized by adherence, morphology, iNOS expression, and NO production, are proficient in forming METs upon stimulation. Notably, we present evidence for the pivotal role of RNS in MET release. L-NAME effectively inhibited MET formation by BMDMs in response to LPS, while SNAP induced METs. This study unveils the intricate interplay between macrophages, reactive nitrogen species, and MET formation, shedding light on a previously unexplored aspect of macrophage-mediated immune responses. The study was funded by the National Science Centre of Poland (grant 2021/43/B/NZ6/00782).

Connective tissue diseases (CTDs) including systemic lupus erythematosus (SLE), primary Sjögren’s syndrome (pSS) and systemic sclerosis (SSc) frequently share clinical and serological features, rendering differentiation challenging. Currently used autoantibodies (Abs) either lack specificity (e.g., ANA) or sensitivity (e.g., anti-dsDNA). We performed a broad explorative screen of IgG and IgA Abs in SLE vs pSS/SSc to unravel disease-specific Abs. We analysed plasma samples from SLE (n=289), pSS (n=208), and SSc (n=187) patients 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. Comparison between SLE and pSS revealed 10 IgG differentitally abadunt Abs (DAAbs; 8 elevated, 2 reduced) and 1 IgA DAAb (reduced). Of the elevated IgG DAAbs in SLE, anti-LIN28A (sen; spe; AUC: 0.71; 0.75; 0.78), anti-PCBP2 (0.56; 0.80; 0.74), anti-HMG20B (0.57; 0.81; 0.74), and anti-NRF1 (0.78; 0.64; 0.75) demonstrated best ability to distinguish SLE from pSS. Analysis of SLE vs SSc revealed 15 IgG (9 elevated, 6 reduced) and 4 elevated IgA DAAbs. Of the elevated IgG DAAbs in SLE vs SSc, anti-LIN28A (0.67; 0.84; 0.81), anti-HBGB2 (0.61; 0.85; 0.77), anti-HMG20B (0.66; 0.81; 0.79), and anti-NRF1 (0.64; 0.82; 0.77) demonstrated best metrics. Elevated IgA DAAbs in SLE vs SSc including anti-LIN28A (0.61; 0.91; 0.83), anti-HMG20B (0.78; 0.69; 0.80) and anti-NOL4 (0.68; 0.75; 0.76), showed good accuracy in differentiating between the two groups. This study corroborated traditional and previously described IgG Ab specificities (anti-LIN28A, anti-HBGB2, anti-HMG20B, anti-HNRNPA2B1) and identified novel IgG and IgA (anti-NRF1, anti-CCNB1, anti-LIN28A, anti-NOL4, anti-HMG20B) Abs described for the first time in SLE, with robust accuracy in distinguishing SLE from pSS or SSc.

Selective Estrogen Receptor Modulators (SERMs) are pharmacological drugs that are prescribed to postmenopausal women to treat symptoms of estrogen deficiency and to prevent osteoporosis and breast cancer. However, these medications, such as bazedoxifene, have shown secondary effects like increased inflammation in the body, but little is known about their impact on immune cells. Neutrophils are critical initiators of inflammation that play specific roles modulated by favoring their energetic metabolism towards glycolysis. It has been shown that neutrophils react to estrogen treatment and that it can modulate their metabolic activity. Similarly, SERMs can differentially impact certain functions in neutrophils, like NETosis. Hence, we hypothesize that SERMs could induce inflammation by upregulating glycolysis in neutrophils and thus activating them. We collected neutrophils from healthy women and men and treated them with physiological doses of bazedoxifene. We assessed neutrophil viability by Annexin V and propidium iodide staining, inflammatory functions by measuring the production of chemokines, like CCL3/MIP-1alpha and CXCL8/IL-8, and their metabolic activity with MTS assays and an extracellular flux analyzer (Agilent/Seahorse Bioscience). We further performed treatments with and without LPS to mimic infectious conditions. Physiological doses of bazedoxifene do not compromise neutrophil viability after 24 hours of treatment. However, higher doses of bazedoxifene alone can increase chemokine production and release, while lower concentrations can prime neutrophils to release more proinflammatory chemokines when challenged with LPS. Interestingly, neutrophil sensitivity to the drug is sex-dependent, with male neutrophils reacting at lower doses than female neutrophils. Furthermore, the response to bazedoxifene occurs rapidly, as observed by an acute dose-dependent effect on glycolysis, with different dose responses seen between men and women. Overall, we have seen that bazedoxifene can modulate neutrophil energetic profiles and affect their anti-microbial activity. Understanding how SERMs interact with neutrophils and impact their inflammatory functions will help improve drug regimens that will consider the potential side effects that patients could encounter while using these drugs.

Metabolism fuels all biological processes and accordingly, alterations in immuno-metabolic pathways lead to, or accompany, numerous pathological conditions such as systemic inflammation. Continuous activation of the immune response, cell dysregulation and formation of neutrophil extracellular traps (NETs) lead eventually to robust inflammation, resulting in damage to the host cells and organs. We are therefore targeting immunometabolism as a potential anti-inflammatory therapy. In particular, we have been investigating itaconic acid, an intermediate produced as a consequence of metabolic shifts during macrophage activation. Itaconate was shown previously to impact macrophage functioning. Interestingly, recent reports indicate that itaconate can be also produced by a subset of highly mature neutrophils. In our ex vivo approach murine neutrophils were treated with 4-octyl itaconate (4-OI; itaconate derivative) or inhibitors of various metabolic pathways in the presence or absence of lipopolysaccharide (LPS). 4-OI dramatically inhibited NET formation via inhibition of hypoxia-inducible factor-1α (Hif-1α) and induction of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase (HO-1) pathway. In the second approach, NETs were visualized in the liver sinusoids of living mice using intravital microscopy (IVM). IVM confirmed that 4-OI injected into endotoxemic mice reduces NETs without affecting neutrophil accumulation. Furthermore, we detected that itaconate reduced numbers of lymphocytes (CD3+) in the liver vasculature. Moreover we detected impact of itaconic acid on Kupffer cells (F40/80+; morphology), without affecting monocytes (Ly6C+). Our results indicate that itaconic acid can metabolically modulates inflammatory leukocytes and their responses during systemic inflammation. The study was supported by National Science Centre of Poland, OPUS 22, 2021/43/B/NZ6/00782.

Currently, the pathophysiologic mechanisms of acute organ and system damage as a result of coronavirus infection have been sufficiently widely investigated; however, the mechanisms underlying the clinical manifestations of long-COVID have not yet been accurately described. The mechanisms of persistence of a number of symptoms in COVID-19 patients and the role of markers of systemic inflammation and endotoxinemia in it remains an understudied aspect and a promising area for further study. Aim of the study: to evaluate markers of systemic inflammation, endotoxin-realizing systems and intestinal permeability, as well as endothelial dysfunction in patients with post-COVID-19 at the sanatorium-resort stage of treatment. Methods. Thirty-two patients who had undergone coronavirus infection and were on sanatorium-resort treatment in the pulmonology department of the State Budgetary Institution of the Republic of Crimea "Academic Research Institute of Physical Methods of Treatment, Medical Climatology and Rehabilitation named after I.M. Sechenov" were examined. A control group (n=20) was also selected. All patients were analyzed peripheral blood for the levels of markers of systemic inflammation and endotoxin-binding systems: C-reactive protein (CRP), lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI). Results. A statistically significant increase in the level of CRP (3.4 [2.56; 4.0] mg/L), LBP (18.46 [14.0; 25.5] ng/mL) and decreased BPI (1576 [276; 3588] pg/mL (p<0.05) was found in patients with postcovid syndrome, compared to the control group. Conclusion. Significant increase in markers of systemic inflammation and endotoxinemia in the group of patients with postcovid syndrome indicates an imbalance of endotoxin-binding and endotoxin-realizing systems in patients who have undergone coronavirus infection. It is necessary to further study the described markers to improve approaches to long-term personalized therapy of this category of patients. This work was supported by the Russian Science Foundation under grant no. 23-15-20021, https://rscf.ru/project/23-15-20021/.

Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) is a master regulator of innate immunity, playing a central role in both toll-like receptor (TLR) and interleukin-1 (IL-1) inflammation. IRAK4 regulates cytokine transcription important to the autoimmune pathophysiology, including atopic dermatitis (IL-1α/IL-1) and asthma (IL-33). Proteolysis targeting chimeras (PROTACs) are hetero-bifunctional molecules that degrade target proteins using the ubiquitin–proteasome system. PROTACs are a novel therapeutic strategy to target IRAK4, which has the efficacy advantage of removing both the scaffolding and kinase function of the protein compared to small molecule kinase activity inhibitors like PF-06650833/Zimlovisertib, which was discontinued in Phase 2. Recently, an IRAK4 PROTAC, KT-474, has entered Phase 2 clinical trials for the treatment of hidradenitis suppurativa and atopic dermatitis. This provided an opportunity to compare the in vitro to in vivo translation of an IRAK4 degrader with an activity inhibitor. Initially, we characterized the ability of KT-474 and PF-06650833 to degrade IRAK4 and inhibit downstream LPS-driven cytokine production using the THP-1 cell line, human peripheral blood monocytes (PBMCs), and mouse splenocytes. Furthermore, we have determined the PK of KT-474 and PF-06650833 prior to profiling both compounds in a mouse LPS model of acute inflammation. Our results demonstrate that KT-474 potently degrades IRAK4 (0.88 nM DC50, 101% Dmax) and inhibits LPS/R848-driven PBMC IL-6 production. Importantly, the inhibitory effect of KT-474 is maintained following its removal, unlike PF-06650833, demonstrating the longevity of this therapeutic modality over kinase activity inhibitors. In mouse PK, KT-474 reaches a Cmax after 2 hours, and there are measurable plasma levels up to 24 hours. This data has been used to design a mouse LPS PD model, in which IRAK4 degradation and inflammatory cytokines suppression will be used to evaluate PK PD relationships. To conclude, we have demonstrated that the degradation of IRAK4 by KT-474 is an effective therapeutic modality to inhibit cytokine generation with potential advantages over conventional kinase activity inhibitors.

Hypersensitivity pneumonitis involves antigen-induced inflammatory flares, however its etiology remains misunderstood. Consequently, the diagnosis is complex and evidence-based therapies are lacking, especially in the chronic fibrotic stages of the disease. B cell-derived mediators, including pro-inflammatory cytokines and antigen-specific antibodies, are central to the pathogenesis, and experimental models of hypersensitivity pneumonitis support the concept that B cell functions are modulated by the sphingosine-1-phosphate receptor 1. The aims of this exploratory study were to characterize lymphocyte populations in patients with chronic hypersensitivity pneumonitis and to determine if sphingosine-1-phosphate receptor 1 ligands directly interfere with B cell functions. In this study, venous blood of eleven chronic hypersensitivity pneumonitis patients and ten control subjects was processed for flow cytometric/functional cellular analyses and quantification of soluble mediators. Isotype-switched circulating memory B cells were reduced while naïve B cell frequencies were increased in patients compared to control. Contrarily to B cells, T cell subpopulations were similar in both groups. Plasmatic concentration of IL-21, TNF and B cell survival factor BAFF were increased in patients. Ex vivo, sphingosine-1-phosphate receptor 1 ligands modified surface expression of activation markers on B cells and the release of TNF and IL-6 in response to a T cell-independent antigen. We conclude that chronic hypersensitivity pneumonitis leads to changes in circulating B cell subpopulations and that sphingosine-1-phosphate receptor 1 modulators interfere ex vivo with the activation of circulating B cells in response to a T cell-independent antigen. The impact of sphingosine-1-phosphate receptor 1 ligands on T cell activation after CD3/CD28 stimulation is currently under investigation.

Psoriasis is an inflammatory skin disease characterized by thick red plaques and by important leucocyte infiltration, especially of T lymphocytes and dendritic cells. Lack of effectiveness and toxic side effects are the main concerns with conventional treatments, and research involving new antipsoriatic molecules is essential. Recently, the anti-inflammatory properties of polyphenols have attracted interest. A systemic administration of balsacone C has already induced improvement in a 3D psoriatic skin model; epidermal thickness and cell differentiation were more comparable to healthy reconstructed skin after the treatment. However, its anti-inflammatory properties have never yet been evaluated in a psoriatic model. Additionally, the anti-inflammatory activity of phloretin has been reported in the literature, but the compound has never been used on psoriatic keratinocytes to examine inflammation and cell proliferation. In this study, the main objective was to determine the anti-inflammatory and antiproliferative effects of two polyphenols, balsacone C and phloretin, in a coculture of T cells and psoriatic keratinocytes. The treatments were administrated for one week at their median inhibitory concentrations (125 μM balsacone C and 166 μM phloretin) and compared with methotrexate, a reference treatment for psoriasis. The coculture of psoriatic keratinocytes and T cells (N = 3 psoriatic keratinocyte donors, n = 3 cocultures/condition) were compared with healthy keratinocyte cultures (N = 3 healthy donors, n = 3 cultures). Phloretin and balsacone C reduced cell proliferation; the expression of Ki67 and PCNA was lower with phloretin and comparable to the healthy control, and balsacone C reduced Ki67 expression. Although the expression of IL-1α and IL-1β was reduced with balsacone C, phloretin reduced the expression of many more cytokines, including TNF-α, IL-17A, CCL2, MIP-1α, G-CSF, GM-CSF, IL-1α, IL-1β and IL-6, and also increased IL-2 secretion. The level of expression of CD45, a protein expressed on T cells, was closer to that of the healthy control after using phloretin.

Acute Kidney Injury (AKI) is a severe pathological condition characterized by rapid onset and high mortality rates. It is often associated with the release of inflammatory cytokines, instigating extensive tissue damage, particularly in vital organs such as the kidneys. Clinical investigations have revealed elevated protein levels of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, in the bloodstream of AKI patients. Lipopolysaccharide (LPS) can induce robust inflammatory reactions, leading to the release of multiple inflammatory cytokines. In this study, we examined the effects of Dexamethasone and TAK-242 on LPS-induced AKI in male C57BL6 mice. Our LPS mouse model involved a single intraperitoneal (IP) LPS administration (0.3 mg/kg), with samples collected at 4h post-LPS. IL-6, IL-1β, TNF-α, IFN-γ were assessed by MSD, while MCP-1, CRP-1, and NGAL were evaluated by ELISA. Gene expression of CCL5, CCL2, KIM-1, podocine, and IL-6, IL-1β, TNF-α, IFN-γ was evaluated by qPCR. Plasma creatinine and urea levels were measured by COBAS and drug levels were quantitated using LC-MS. LPS administration resulted in increased kidney levels of IL-6, IL-1β, TNF-α, IFN-γ, MCP-1, CRP-1, and NGAL compared to the control group. Both Dexamethasone and TAK-242 significantly reduced these levels (p<0.001), with NGAL being unaffected by Dexamethasone. TAK-242 reduced LPS-induced upregulation in IL-6, IL-1β, CCL2, CCL5, and KIM-1 gene expression, while Dexamethasone reduced IFN-γ, IL-6, KIM-1, and IL-1β gene expression. Plasma creatinine and urea levels, elevated in LPS-induced animals, remained unchanged with treatment. Dexamethasone level was twice higher in the kidney than in the plasma, whilst TAK-242 was undetectable in both at termination. In summary, LPS injection induces cytokine release, leading to a subsequent decline in kidney function. Understanding LPS-induced kidney responses may help to investigate potential therapeutic targets for conditions characterized by excessive inflammation. LPS-PK:PD models serve as valuable translational tools, guiding the development of therapies for inflammatory diseases and ultimately improving patient outcomes.

Neutrophils are key players in the immune system and play a crucial role in inflammation. Neutrophils release pro-inflammatory cytokines and NETs (Neutrophil Extracellular Traps), which play an active role in the process of vascular thrombosis. The interaction of neutrophils with platelets is a major component of the thrombo-inflammatory phenomenon. This interaction may be more pronounced in certain pathologies with a pro-inflammatory profile, such as heart failure (HF). Recently, it was discovered that neutrophils in lung cancer patients can express the platelet protein complex CD41/CD61 (GPIIb/IIIa). The CD41/CD61 complex is known for its role in platelet adhesion and aggregation. The aim of our study was to demonstrate the expression of the CD41/CD61 complex on neutrophils and biological activity in healthy volunteers and HF patients. Neutrophils were isolated by density gradient and analyzed by flow cytometry and confocal microscopy to determine the localization and expression of the CD41/CD61 complex. Our preliminary data indicate the intracellular presence of this complex in 80%-90% of neutrophils, while it is only expressed between 8% and 13% on their extracellular membrane. Our results also demonstrate that the CD41/CD61 complex plays a role in neutrophil adhesion to the extracellular matrix, since its blockade with a CD41/CD61 monoclonal antibody decreases HV neutrophil adhesion induced by IL-8 (100 nM) by up to 76%. Meanwhile, the use of eptifibatide, an antagonist of the CD41/CD61 complex, at a concentration of 1.5 or 50 µg/mL reduced the IL-8-induced adhesion by up to 74% and 70% respectively. Our data suggest that the CD41/CD61 complex is not exclusive to platelets, and its expression on neutrophils may confer them pro-thrombotic properties. The inhibition of CD41/CD61 complex could lead to a treatment of neutrophil-regulated thrombosis in heart failure patients.

Brain inflammation occurs in Alzheimer’s disease (AD), and inflammation is often associated with reactive nitrogen species generation (RNS). 3-Nitrotyosine, a product of RNS generation was assessed in frontal lobe brain homogenates of AD or vascular dementia (VaD) patients and non-dementia (ND) control volunteers. 3-Nitrotyrosine-containing proteins detected by western blotting revealed a dominant 15 kDa nitrated protein band in both dementia (AD/VaD) and ND frontal lobe brain tissue which was identified by mass spectrometry as hemoglobin. The same band stained positively when western blotted using an anti-hemoglobin antibody. Image analysis of the fluorescence staining intensities of western blots indicated that there was a significantly increased median level of the extent of hemoglobin nitration in the frontal lobe brain tissue from both the AD (n=10) and VaD (n=10) groups compared to ND volunteers (n=11; Mann-Whitney U test: AD v ND, P < 0.0005; VaD v ND, P < 0.05). The median normalized staining intensity of the nitrated hemoglobin band was higher in advanced AD patients (Braak stages 5-6) compared with early-stage AD (Braak stage 0), (P < 0.005). In parallel, nitrite (NO2¯) and nitrate (NO3¯) were measured by ozone-based chemiluminescence and the median NO2¯ levels (nmol/mg protein) were significantly higher in AD samples than in ND controls (P < 0.05). There were no statistically significant differences between the three groups when comparing median NO3¯ concentrations. Image analysis of western blots of lysates from peripheral blood erythrocytes suggested that hemoglobin nitration was increased in AD compared to ND (n=4 in each group; P < 0.05). Total protein-associated 3-nitrotyrosine was measured by an electrochemiluminescence-based immunosorbent assay (ECLISA), but showed no statistically significant differences between AD, VaD and ND. Females showed larger increases in hemoglobin nitration, total nitration and NO2¯ levels between disease and control groups compared to males, however, the group sizes in these sub-analyses were small. In conclusion, the extent of hemoglobin nitration was increased in AD and VaD brain frontal lobe tissue compared with ND. We propose that nitrated hemoglobin may be involved in amyloid-β plaque formation.

PLXDC2 is expressed on the cell surface of macrophage, dendritic and specific mesenchymal and epithelial cells whose activation shifts cellular metabolism and reduces oxidative stress to rebalance the immune response and decrease inflammation. PLXDC2 activation improves disease severity in rheumatoid arthritis models (Tubau-Juni et al. J Immunol 206(Supp)), while loss of PLXDC2 exacerbates the severity of disease in DSS colitis (Tubau-Juni et al. Sci Rep 10(1)). PX-04 is a novel, first-in-class, orally active PLXDC2 agonist. Here, we report on the consequences of activating PLXDC2 using PX-04 in an acute DSS colitis model. Eight-week old mice were given DSS in drinking water for 7 days to induce disruption of the epithelial layer. Mice began once daily dosing with 20mg/kg of PX-04 24 hours after DSS initiation. Mice were weighed and scored daily for symptoms of disease (e.g. weight loss, diarrhea, rectal bleeding, rectal inflammation, pain, & overall behavior). Colons were collected and digested, and the resultant colonic lamina propria immune cell suspensions were isolated by Percoll gradient centrifugation. Cells were labeled with mixtures of extracellular and intracellular antibodies in a sequential live staining. Data were acquired using a FACS Celesta flow cytometer with FACSDiva software. Oral PX-04 treatment decreased the cumulative disease activity of mice challenged with DSS (FIG 1A). Immunologically, PX-04 affected both the adaptive and innate immune responses. First, PX-04 greatly decreased Th1 (FIG. 1B) and Th17 cells in the colon, while providing a slight increase to regulatory CD4+ T cells. A lower proportion of Natural Killer (NK) cells produced IFNγ. Meanwhile, the proportion of TNF producing dendritic cells was decreased by PX-04 treatment (FIG. 1C). In summary, PLXDC2 is expressed in cells relevant to IBD pathogenesis. PX-04, which binds to and activates PLXDC2, effectively decreases levels of effector T cells and myeloid cells, as well as overall disease severity in acute DSS colitis, warranting further investigation.

Oxidised cholesterols, so called oxysterols, have emerged as novel regulators of inflammation in the lung during respiratory infections. We previously discovered that the oxysterols 25-hydroxycholesterol (25-OHC) and 7a,25-Dihydroxycholesterol (7a,25-OHC) are produced in the lung upon SARS-CoV-2 infection in mice and chemotactically attract infiltrating macrophages to the site of infection via the oxysterol-sensing receptor GPR183. However, the therapeutic implications of administering these oxysterols into the lung microenvironment during infection have not been investigated. Given that 25-OHC was reported to possess antiviral properties, we hypothesised that intranasal administration of these oxysterols may promote antiviral and immunomodulatory effects in the lung during SARS-CoV-2 infection. Here we demonstrate that intranasal administration of 25-OHC but not 7a,25-OHC in C57/BL6 mice is antiviral and reduces the severity of SARS-CoV-2 infection-mediated disease. Furthermore, the increased concentrations of these oxysterols in the lung are associated with increased infiltration of macrophages. In vitro, we demonstrated that 25-OHC, but not 7a,25-OHC, exhibits antiviral activity in Calu-3 cells by blocking the early stages of the viral life cycle. Together, our findings highlight the antiviral activity of 25-OHC in the lung during SARS-CoV-2 infection and demonstrate that enhancing local oxysterol concentrations can both promote macrophage infiltration and reduce inflammation into the lungs during SARS-CoV-2 infection.

Type 2 diabetes (T2D) is characterized by low-grade inflammation. Low-density neutrophils (LDNs) represent normally less than 2% of total neutrophils but increase in multiple pathologies, releasing inflammatory cytokines and neutrophil extracellular traps (NETs). We assessed the count and role of high-density neutrophils (HDNs), LDNs, and NET-related activities in patients with T2D. HDNs and LDNs were purified by fluorescence-activated cell sorting (FACS) and counted by flow cytometry. Circulating inflammatory and NETs biomarkers were measured by ELISA (Enzyme Linked Immunosorbent Assay). NET formation was quantified by confocal microscopy. Neutrophil adhesion onto a human extracellular matrix (hECM) was assessed by optical microscopy. We recruited 22 healthy volunteers (HVs) and 18 patients with T2D. LDN counts in patients with diabetes were significantly higher (160%), along with circulating NETs biomarkers (citrullinated H3 histone (H3Cit), myeloperoxidase (MPO), and MPO-DNA (137%, 175%, and 69%, respectively) versus HV. Circulating interleukins (IL-6 and IL-8) and C-Reactive Protein (CRP) were significantly increased by 117%, 171%, and 79%, respectively, in patients compared to HVs. Isolated LDNs from patients expressed more H3Cit, MPO, and NETs, formed more NETs, and adhered more on hECM compared to LDNs from HVs. Patients with T2D pre-sent higher levels of circulating LDN- and NET-related biomarkers and associated pro-inflammatory activities.

Purpose. Activated neutrophils (PMN) expel neutrophil extracellular traps (NET), DNA/proteins fibers. Described as anti-microbial, NET can become immunogenic. Increased NET formation has been reported in rheumatoid arthritis (RA). We have previously shown that NET are pro-inflammatory on resting non-polarized M0 macrophages and this response was enhanced in RA patients. Here, we aimed at comparing the pro-inflammatory activity of NET on cell sub-populations of the myeloid lineage, focusing on the effects of RA NET on target cells from healthy individuals. Results were then confirmed with RA target cells and we analyzed the involved mechanism. Methods. Blood PMN/PBMC were isolated by density centrifugation from healthy donors (HD)/RA patients. Monocytes were purified from PBMC by CD14-positive selection by magnetic sorting. M0 macrophages were differentiated from monocytes with M-CSF, or polarized to M1-like pro-inflammatory macrophages or M2c-like immuno-regulatory macrophages with IFN-γ or IL-10, respectively. Alternatively, pro-inflammatory macrophages and pro-inflammatory dendritic cells (DC) were differentiated from monocytes with cytokines and with an aryl hydrocarbon receptor antagonist or agonist, respectively. NET were induced in vitro by PMA on adherent PMN, then enriched, quantified and characterized. Target cells were cultured with RA NET or LPS/R848, in the presence/absence of ATP to stimulate IL-1β secretion, or 2-deoxy-D-glucose (2-DG)/diphenyleneiodonium (DPI) to inhibit glycolysis/reactive oxygen species (ROS) production. Similar experiments were performed with cells from wild-type and TLR9-deficient mice. Cell purity/phenotype/activation were estimated by flow cytometry. Cytokine secretion was measured by ELISA. Results. RA NET activated monocytes, PMN, macrophages as well as DC, leading to the secretion of pro-inflammatory cytokines. Similar results were obtained both with HD and RA target cells. Particularly, NET triggered the secretion of RA-associated pro-inflammatory cytokines, but not of the immuno-modulatory cytokine IL-10. Both pro-inflammatory macrophage and dendritic cell sub-populations strongly responded to NET. Importantly, even immuno-modulatory macrophages did respond to NET. NET-mediated activation occurred independently of TLR9. In macrophages and PMN, NET induced the secretion of IL-1β, which occurred upon activation of the inflammasome in macrophages and with NET working as the priming stimulus, whereas PMN did not require priming and produced IL-1β directly in response to NET. Finally, NET-mediated activation required ROS in monocytes but not in PMN, whereas it depended on glycolysis in PMN but not in monocytes. Conclusions. Abnormal accumulation of NET in the extracellular space may be a major trigger capable to activate several myeloid cell sub-populations within a pathogenic pro-inflammatory response.

The impact of lipid mediators and dietary polyunsaturated fatty acids (PUFAs) in COPD, is poorly understood. Dietary modifications and interventions may be potential therapeutic interventions in COPD. We hypothesised that lipid mediator production is dysregulated in COPD and a diet high in monounsaturated fatty acid MUFA omega 9 (oleic acid) or omega-3 PUFA (alpha-linoleic acid, ALA) would alleviate inflammation and lung function changes. Female, C57BL/6 mice (n=10) were fed a normal control diet (AIN93G), or high-fat diets (40% fat by energy) composed of predominantly monounsaturated (oleic acid) or omega-3 polyunsaturated (alpha-linolenic acid). Mice were exposed to nose-only cigarette smoke (CS) for (12 weeks) to induce experimental COPD. The hallmark features of COPD, including airway inflammation and fibrosis, alveolar destruction/emphysema, lung function (forced oscillations using Flexivent), and cachexia were assessed. Lipid mediators in bronchoalveolar lavage fluid (BALF) and plasma were profiled using ultra-high-performance lipid chromatography-tandem mass spectrometry (UPLC-MS/MS). MUFA and ALA diets reduced (p<0.05) cigarette smoke-induced lung inflammation and protected against fibrosis but did not alter lung function. Ceramide and sphingomyelin were reduced in MUFA compared to control diets. Both the amount and composition of dietary fats impact disease features in COPD. Dietary increases in oleic acid and ALA and may be beneficial in COPD.

The lipopolysaccharide (LPS)-induced innate immune response in a mouse model follows fundamental principles crucial for understanding the host's defence mechanisms against bacterial infections. Reproducibility of the model is vital to ensure data are consistent and of high value when profiling novel drugs and modalities. Moreover, the ability to measure test drug levels in conjunction with cytokine levels in the same animal's plasma and tissues provides a valuable pharmacokinetic-pharmacodynamic relationship when investigating the in vivo properties of novel therapeutics. Our objective was to develop an LPS-induced innate immune response in a mouse model that serves as a valuable platform for the early development of novel therapeutic strategies in immune-related and chronic inflammatory diseases. Our LPS mouse model involves dose response validation (0.3, 0,5, and 0.8 mg/kg LPS i.p.), drug comparators validation (PK/PD, e.g. TAK-242, Dexamethasone) as well as characterization of multiple organ inflammation such as brain, spinal cord, kidney, liver, and colon. Dexamethasone (5mg/kg s.c. or 10 mg/kg p.o.) was administered prior to LPS. Blood and tissue samples were collected at 4h post-model LPS induction. MSD assessed plasma and tissue cytokines (IL-6, IL-1β, TNF-α, IFN-γ). Test drug levels were quantitated using LC-MS. LPS resulted in an increase of circulating and tissue IL-6, IL-1β, TNF-α, and IFN-γ vs. control. Dexamethasone, as well as TAK-242, significantly reduced their levels, p<0.001. Interestingly, Dexamethasone levels were 10x lower in the brain and 2x higher in the kidney than in the plasma, whilst TAK-242 levels were undetectable at termination time in both plasma and tissues. Understanding LPS-induced responses combined with investigational drug pharmacokinetics enables us to profile a test drug's tissue distribution and efficacy in active inflammation. Importantly, the identification of anti-inflammatory drugs that can cross the blood-brain barrier (BBB) is vital for treating neuroinflammation and improving outcomes of patients with neurodegeneration.

Leptin and adiponectin are adipokines that have been shown to mediate the relationship between systemic inflammation and chronic diseases. However, the inconsistent reporting of leptin and adiponectin data by previous studies hinders the pooling of data across studies for cross-population comparisons or meta-analyses. As such, the aims of this poster are to (1) encourage researchers in the area of systemic inflammation to include standardized measures of leptin and adiponectin and the leptin to adiponectin ratio in future studies, and (2) recommend standardized reporting methods. Following standardized measurement and reporting methods will increase the likelihood that new data generated can be compared across studies and thus, knowledge about the impact of these adipokines on inflammation regulation can be advanced.

Vestibulodynia is a complex pain disorder characterized by chronic discomfort in the vulvar region, often accompanied by tactile allodynia and spontaneous pain. In patients a depressive behaviour is also observed. In this study, we have used a model of vestibulodynia induced by complete Freund's adjuvant (CFA) in female C57BL/6J mice focusing our investigation on the spinal cord neurons and microglia. We investigated tactile allodynia, spontaneous pain, and depressive-like behavior as key behavioral markers of vestibulodynia. In addition, we conducted in vivo electrophysiological recordings to provide, for the first time to our knowledge, the characterization of the spinal sacral neuronal activity in the L6-S1 dorsal horn of the spinal cord. Furthermore, we examined microglia activation in the L6-S1 dorsal horn using immunofluorescence, unveiling hypertrophic phenotypes indicative of neuroinflammation in the spinal cord. This represents a novel insight into the role of microglia in vestibulodynia pathology. To address the therapeutic aspect, we employed pharmacological interventions using GABApentin, amitriptyline, and PeaPol, a combination of palmitoylethanolamide and polydatin. Remarkably, all three drugs, also used in clinic, showed efficacy in alleviating tactile allodynia and depressive-like behavior. Concurrently, we also observed a normalization of the altered neuronal firing and a reduction of microglia hypertrophic phenotypes.

Inflammatory bowel disease (IBD) includes Crohn's disease and ulcerative colitis, both are characterized by chronic inflammation and damage to the GI tract. There is no cure for IBD and the lack of effective long-term treatments means patients may eventually undergo surgery to remove damaged portions of the GI tract. IBD is caused by release of inflammatory proteins from bacteria, blood or gut resident T-cells. Regulatory T-cells (Tregs) are known to be important in preventing IBD. Treg surface activation receptor CD69 and the ecto-ATPase - CD39 (that metabolises inflammatory ATP) in part mediate immunosuppression. Immune cells from the lamina propria isolated from gut biopsy tissue, from treatment naive patients with IBD and non-IBD controls, was incubated with IRL201805 overnight. Mononuclear cells were then stained with a 24-antibody panel and analysed by spectral flow cytometry. IRL201805 increased the generation of double positive CD69+/CD39+ FoxP3 Tregs in inflamed but not non-inflamed tissue. In separate monocyte cultures we observed IRL201805 induced the increase in the cell surface ligands for CD69, galectin-1, and S100A8/9 as detected by quantitative Tandom Mass Tag™ spectrometry. In monocyte-T cell co-cultures IRL201805 led to an increase in STAT5 phosphorylation, which is known to promote Treg differentiation and prevent pro-inflammatory Th1/Th7 cell generation. Therapeutics able to enhance CD69+/CD39+ Tregs in intestinal tissues in vivo, may play an important role in immune system balance and the prevention of inflammation.

Inflammatory bowel disease (IBD) is defined by chronic intestinal inflammation, resulting from perturbation of the intestinal barrier. Disease pathology is driven primarily by a repertoire of T cells including T helper (Th) 1, 17 and T regulatory (Treg) subsets. A considerable number of patients are non-responsive to conventional treatments with biologics and immunomodulators calling for a need for new therapeutic strategies. Growing evidence demonstrates that dietary polyphenols derived from Mangifera indica L. extract (MIE), has the potential to alleviate intestinal inflammation and modulate T cell levels in spleen. This study explores the anti-inflammatory and immuno-modulatory properties of MIE by utilising blood from adult IBD patients followed by a pre-clinical model of T-cell driven colitis. Clinically, MIE demonstrated a significant reduction in TNF levels in an ex-vivo model of intestinal barrier breakdown using LPS-spiked blood from IBD patients. Further investigation in a model of T cell driven colitis revealed a significant improvement in clinical severity with MIE treatment, marked by improved weight loss and levels of faecal calprotectin. This was accompanied by repaired intestinal barrier permeability via restoration of intestinal metabolites and levels of epithelial cell junctional proteins, ZO-1 and occludins. Pathogenic infiltration of T cell subsets in colonic tissue was also reversed by MIE, characterised by the reduced frequency of Th1 and Th17 cells and increase in Treg cells observed via flow cytometry analysis. Additional mechanistic insights uncover direct effects of MIE on vascular endothelium, inhibiting TNF and IFN-induced upregulation of COX and DP2 receptors that contribute to lymphocyte transmigration. Collectively, this study demonstrates the therapeutic benefits of MIE in restoring immunological imbalance during the onset of colitis and its potential use to treat IBD in clinic.

Acute inflammation is a protective mechanism of our body against pathogens or tissue damage, which actively resolves to return the inflamed tissue to homeostasis via the production of endogenous pro-resolving mediators. However, it is now thought that chronic inflammatory diseases such as arthritis may persist due to a failure of resolution responses. Therefore, new therapeutic approaches that promote the resolution of inflammation may offer a useful strategy to tackle the disease burden of Rheumatoid arthritis. The Formyl Peptide Receptor 2 (FPR2, also known as ALX) is a G protein-coupled receptor (GPCR) that promotes the resolution of inflammation through binding of several naturally occurring agonists including small bioactive lipids such as specialized pro-resolving mediators e.g. lipoxin A4 and resolvin D1 and proteins such as annexin A1. In fact, previous studies have demonstrated that mice lacking the Fpr2 gene in all cells and tissues (global Fpr2 knockout mice) present a more exacerbated and prolonged response to K/BxN serum transfer induced arthritis (STIA). Herein, we utilized a humanized FPR2 (hFPR2) mouse colony, bearing an intact or a selective receptor deficiency in myeloid cells (LysM-cre) to dwell on the cellular mechanisms. These mice were characterized by the detection of hFPR2-GFP in whole blood, whereby the GFP (hFPR2) expression in circulating neutrophils was reduced to 30%. hFPR2 flox mice and myeloid cell-specific hFPR2 KO mice were subjected to STIA. Whilst the clinical score was similar between genotypes at day 6 of arthritis, the LysMcre FPR2 KO showed an increased number of neutrophils (Ly6G+) within the arthritic joints at peak disease. Interestingly, whilst hFPR2 expression was absent on joint infiltrating neutrophils, expression on resident macrophages was significantly increased. Further studies investigating whether the resolution phase of arthritis is prolonged in these mice is currently being performed. Taken together, these findings provide further evidence of the protective role of FPR2 in limiting myeloid cell infiltration to the joint during arthritis.

INTRODUCTION. Granulocyte infiltration is a fundamental feature of inflammation. In tissues, they produce/release soluble mediators including eicosanoids derived from 5-lipoxygenase (5-HETE, leukotrienes). They also biosynthesize the endocannabinoid 2-arachidonoyl-glycerol (2-AG) using a pathway involving the acylation of arachidonic acid into glycerophospholipids and its further remodeling into 2-AG (the “Flamand Pathway”). Unlike arachidonic acid, 2-AG is not metabolized by 5-lipoxygenase. Thus, 5-HETE-Glycerol and its oxidized metabolite (5-KETE-Glycerol) should not exist, unless their biosynthesis occurs via the Flamand Pathway. Given that 5-HETE is also acylated into glycerophospholipids, we postulated that 5-HETE-treated granulocytes would generate 5-HETE-Glycerol and possibly 5-KETE-Glycerol. RESULTS. 5-HETE-treated human neutrophils and eosinophils biosynthesized 5-HETE-Glycerol in a concentration- and time-dependent manner. Comparable data were obtained with 5-KETE-treated cells, which led to 5-KETE-Glycerol biosynthesis. Both monoacylglycerols were also detected when neutrophils were treated with arachidonic acid or A23187, indicating that endogenously formed 5-HETE/5-KETE also undergo the Flamand Pathway. We next assessed whether these new monoacylglycerols were found in vivo in joints from arthritic mice, a tissue containing high levels of 5-HETE. We did not detect 5-HETE-Glycerol but 5-KETE-Glycerol was found at comparable levels to 5-KETE. Initial in silico analyses indicated that these new lipids were ligands for cannabinoid receptors but this has not been clearly confirmed by wet lab data yet. CONCLUSIONS. Human granulocytes biosynthesize 5-HETE-Glycerol and 5-KETE-Glycerol. 5-HETE-Glycerol and 5-KETE-Glycerol thus represent two new arachidonic acid-derived metabolites derived from 5-lipoxygenase, with possible biological functions. We are now determining whether these metabolites are present in other inflammatory conditions that are enriched in 5-HETE/5-KETE as well as defining the cellular mechanisms by which they might modulate inflammation. Finally, the validation of the Flamand Pathway indicates that other fatty acid-derived metabolites could be synthesized by this new pathway, including those from the microbiota.

Coronary artery diseases are characterized by chronic inflammatory status and endothelial dysfunction. This involves an increased production of neurotransmitters such as serotonin (5 HT) and acetylcholine. These changes are associated with effects on the vascular function by increasing vasoconstriction. Specialized pro-resolving lipid mediators (SPM), derived from omega-3 polyunsaturated fatty acids: eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) play an active role in the resolution of inflammation. Recent results from our group show that DHA and metabolites (Resolvin D1, D5 and Maresin 1) reduce contractions of human coronary arteries (HCA) induced by PGE2. On the other hand, RvD5 and Mar1 production by human vagus nerve has been measured, their impact on the cardiac neuronal system remains unexplored. The objective of this study is to investigate the impact of the omega-3 on the release and effects of neurotransmitters like acetylcholine and 5-HT in HCA. The HCA were isolated from human hearts (n=6) after transplantation at Bichat Hospital and placed in an organ bath system. They were stimulated with different voltages to release neurotransmitters, before and after 1 or 18 h of incubation with omega-3. In order to evaluate the effect of DHA/EPA on exogenous neurotransmitters, a dose response curves with 5-HT and acetylcholine was realized. Vascular tone variations were analyzed. Our results show that HCA contract after electrical stimulation, with an increased effect at higher voltages. The contractions resulting from this stimulation are attributed to a direct effect on smooth muscle cells and also to the neurotransmitter release, as they are partially blocked by tetrodotoxin (10 µM). DHA (0.1 mM) demonstrates the ability to reduce the contractions induced by stimulations at 10 and 30 volts by 56% and 31%, respectively. Additionally, exogenous neurotransmitters, such as 5-HT and acetylcholine induce contractions in HCA. Acetylcholine induced vasocontractions were reduced by DHA, while the serotonin-induced contractions remain unaffected by DHA/EPA. Our preliminary results indicate that omega-3 may have an effect at the neuronal level in HCA, suggesting potential innovative therapeutic strategies.

The perennial plant Panax Ginseng has been used in traditional medicine in China and Korea for centuries. The preparations of this plant are known to have a wide variety of pharmacological effects, including anti-cancer, anti-diabetes, and anti-inflammatory activities. Although the anti-inflammatory features of the plant have long been described, little is known about the exact components that are responsible for this anti-inflammatory activity. The main active ingredients of Panax Ginseng are seemingly steroid glycosides present in the root of the plant, known as ginsenosides. Here, we present a systematic screening of 23 ginsenosides for their immune-modulating and anti-inflammatory properties by means of lipid-mediator profiling in classically activated macrophages. Among a diversity of cyclooxygenase- and lipoxygenase (LO)-derived lipid mediators, several ginsenosides selectively inhibited the formation of 5-LO products, thus suppressing the formation of the chemotactic and pro-inflammatory leukotriene B4 (LTB4). Three clear structure-activity relationships are evident for ginsenosides to inhibit 5-LO product formation. Firstly, despite their highly similar structure, only PPD-type ginsenosides were able to inhibit 5-LO product formation. Secondly, the potency of PPD-type ginsenosides to inhibit 5-LO product formation was highly correlated with their lipophilicity (R2 = 0.92). Lastly, based on the configuration of the carbon atom at position 20, two epimers of ginsenosides can be created. We observed only (20S)-epimers to potently inhibit 5-LOX product formation, whereas the (20R)-epimers were mostly inactive. The complex and tight regulation of 5-LO activity in the cell allows for multiple mechanisms as point of attack for compounds that suppress 5-LO product formation. Direct inhibition of 5-LO itself by ginsenosides could be excluded, suggesting secondary mechanisms to be at play. Inhibition of cellular 5-LO product formation by ginsenosides was rapid (<1 minute) and irreversible. Interestingly, the aglycon PPD interfered with 5-LO subcellular redistribution necessary for enzyme activation and access towards substrate. Therefore, the aglycon PPD inhibits 5-LO product formation by means of interfering with 5-LO translocation, reflecting an effective strategy to interfere with LT biosynthesis. In conclusion, our study sheds light on (i) the bioactive anti-inflammatory ingredients of Panax Ginseng, and (ii) the underlying anti-inflammatory mechanisms targeting 5-LO product formation in innate immune cells.

INTRODUCTION. Diets enriched in omega-3 polyunsaturated fatty acids fish oils have been linked with beneficial anti-inflammatory effects. However, their daily consumption is not palatable to many, due to their fishy flavour. Additionally, questions regarding the sustainability of fish oils support the exploration of alternative sources. These include plant-derived oils like the stearidonic acid (SDA)-rich oil from Buglossoides arvensis (ahiflower) seeds, which has been shown to be more effective than Flax seed oil at enriching tissues with long-chain omega-3 polyunsaturated fatty acids. We recently documented that unsaturated fatty acids can be acylated into glycerophospholipid and remodeled into monoacylglycerols and N-acyl-ethanolamines in human leukocytes. In order to better understand the putative beneficial effects of a SDA-enriched diet and the cellular/molecular mechanisms involved, we postulated that, and investigated if, SDA also undergoes such metabolism, possibly yielding undocumented bioactive effectors. METHOD. We chemoenzymatically synthesized 1-stearidonoyl-glycerol (SDG) and N stearidonoyl-ethanolamine (SDEA). We optimized their detection/quantification by tandem mass spectrometry and assessed their functional impact in different models. RESULTS. In line with out previous data with several PUFAs, SDA-treated neutrophils biosynthesized a significant amount of 1/2-SDG as well as a limited amount of SDEA. This was concentration-dependent and the levels of 1/2-SDG obtained were in between those obtained for 2 arachidonoyl-glycerol and 1/2-linoleoyl-glycerol when cells were treated with their respective precursors, arachidonic acid and linoleic acid. In contrast to 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine, neither 1-SDG nor SDEA were ligands for the cannabinoid receptors 1 and 2. However, 1-SDG activated both PPARα and PPARγ while SDEA selectively activated PPARα. Of note, neither the monoacylgycerol nor the N-acyl-ethanolamine of linoleic acid had an impact on these transcription factors. Finally, an ahiflower oil-enriched diet led to an increase in 1/2-SDG levels in the joints of arthritic mice (K/BxN model), which coincided with a decrease in inflammatory score. CONCLUSIONS. 1) Human neutrophils biosynthesize the SDA metabolites 1/2-SDG and SDEA. 2) 1/2-SDG activates both PPARα and PPARγ while SDEA only activates PPARα. 3) Neither 1-SDG nor SDEA bound to the cannabinoid 1 or 2 receptors. 4) An SDA-enriched diet diminishes the severity of experimental arthritis. While the cellular/molecular mechanisms involved in the beneficial effect of SDA and its metabolites remain to be solved in arthritic mice, their impact on PPAR(s) might explain, at least in part, the beneficial effect of this PUFA in experimental arthritis and other inflammatory conditions. We are currently exploring this possibility.

Background: Numerous studies indicate an association between vitamin D status and inflammatory biomarkers in patients with asthma, but findings are inconsistent. This review aims to summarize the relationship between serum vitamin D status, assessed by 25-hydroxyvitamin D (25(OH)D) level, and inflammatory biomarkers. Methods: A literature search of interventional and observational studies on 25(OH)D up to November 2022 was conducted across six electronic databases. The outcomes of interest included a range of inflammatory biomarkers classified in four distinct categories (T helper 2 (Th2) pro-inflammatory biomarkers, non-Th2 pro-inflammatory biomarkers, anti-inflammatory biomarkers, and non-specific biomarkers). The characteristics and the risk of bias of the studies were extracted and evaluated by independent reviewers. Meta-analysis was conducted on studies with a low risk of bias, while narrative reporting was used to present the direction of associations (positive, no association, or negative) for each biomarker, within the subset of low-risk studies and across all included studies. Results: We included 71 studies (3 interventional, 68 observational) investigating the association between vitamin D status and Th2 pro-inflammatory biomarkers (N=58), non-Th2 pro-inflammatory biomarkers (N=18), anti-inflammatory biomarkers (N=16), and non-specific biomarkers (N=10). Thirteen (18.3%) studies, 50 (70.4%) and only 8 (11.3%) were at high, neutral, and low risk of bias, respectively. Only one meta-analysis could be performed. The pooled estimate for 25(OH)D and serum IgE showed a negative association (β (95% CI) = –0.33 (–0.65 to –0.01); I2 = 88%; P < 0.01; N=4 studies). In studies at low risk of bias reporting statistically significant results, there was a negative correlation between vitamin D status and both serum IgE and blood eosinophil, whereas a positive correlation with LL-37 was observed. When considering all studies regardless of bias risk, those with statistically significant results predominantly indicated a negative association of 25(OH)D with pro-inflammatory biomarkers and a positive association with anti-inflammatory biomarkers. Nevertheless, the majority of studies reported non-statistically significant relationships with 25(OH)D. Conclusion: Serum 25(OH)D is negatively associated with serum IgE. Whereas incomplete reporting and non-adjustment for confounders prevented a meta-analysis of other biomarkers, most studies with statistically significant results support a potential anti-inflammatory effect of 25(OH)D.

Inflammatory bowel disease (IBD) is a multifaceted disease involving the integrity of the epithelial barrier, the gut microbiome, and its mucosal immune cells. Therefore, investigating the regulatory mechanisms that command recruitment and infiltration of circulating leukocytes, without contributing to persistent inflammation, are gaining traction as potential therapeutic targets. Facilitating crucial interactions between the microbiota and the intestinal epithelium, glycans remain a facet of significant interest in the alteration of mucosal immunity. Specifically implicated in disease regulation, we investigated the impact of Galectin-9 (Gal-9) supplementation and deficiency, and the synergetic influence of Gal-3, on disease progression of murine Gal-3 knockout (KO), Gal-9 KO, and Gal- 3/Gal-9 double KO models of colitis. The KO models with DSS-induced injury reduced the associated weight loss compared to the wildtype (WT) DSS injury model, suggesting a potentially pro-inflammatory role of both galectins. However, there was no notable negative impact of intraperitoneal introduction of Gal-9 and Gal-3 respectively into WT DSS injury models. Considering the extensive involvement of disrupted T-cell homeostasis in IBD, the impact of Gal-9 was examined on T-cell driven model of colitis. Curiously, the receipient Rag (-/-) mice which were supplemented with recombinant Gal-9 were found to have reduced intestinal inflammation and better a clinical outcome in comparison to the diseased control. This investigation unveils a double-edged functionality for Gal-9, perhaps hingeing on the initial injury trigger. Finally, our studies indicate no detectable differences in the cytokine profile of bone marrow-derived macrophages (BMDMs) from WT and Gal- 9 KO mice, in response to pathogen-associated molecular patterns (PAMPs). However, human monocyte-derived macrophages supplemented with exogenous Gal-9 can markedly stimulate TNF-α release. When further stimulated with increasing concentrations of LPS, a dose-dependent increase in TNF-α was observed. While no such pattern of secretion of TNF-α was found with Gal-3 supplementation, modest reduction of IL-6 levels was noted. Therefore, our results indicate that while Gal-9 and Gal-3 may not be central drivers of colonic inflammation, they present as viable therapeutic targets for intestinal inflammation.

Nurr1 is a member of the orphan nuclear receptor family NR4A which regulates different cellular processes such as differentiation, proliferation, survival, and inflammation. Nurr1 restricts the inflammatory response mainly through transrepression mechanisms, by interacting and inhibiting the function of transcription factors such as NF-κB p65. The peroxisome proliferator-activated receptor (PPAR)γ is another member of the nuclear receptor superfamily which is involved in the regulation of glucose and lipid metabolism, and shows anti-inflammatory properties. Nurr1 was found to be able to bind PPARγ, as well as to be recruited to the PPARG promoter in LPS-activated mouse microglial cells, although the functional relationship between Nurr1 and PPARγ remains unclear. In this study we aimed to investigate the association between Nurr1 and PPARγ1 in human macrophages. Pro- (GM-MDMs) and anti-inflammatory (M-MDMs) macrophages were generated by culture of human monocytes with GM-CSF or M-CSF, respectively. The protein levels of PPARγ1 and Nurr1 were elevated in GM-MDMs in comparison to M-MDMs, and their expression was positively correlated. This positive correlation was also detected in macrophages from adipose tissue samples. PPARγ1 activation in GM-MDMs with the agonist rosiglitazone did not modify Nurr1 expression. However, Nurr1 activation with the agonist C-DIM12 increased PPARγ1 protein levels through stabilization of PPARγ1 protein. Further, C-DIM12 decreased the number of PPARγ1 molecules phosphorylated at Ser84, which is a repressive mark for PPARγ transcriptional activity, similar to that observed with the rosiglitazone treatment. Co-exposure of GM-MDMs to C-DIM12 and rosiglitazone synergistically enhanced the transcriptional activity of PPARγ and the expression of two PPARγ target genes, CD36 and PLIN2. Both PPARγ and Nurr1 agonists exhibited anti-inflammatory effects on GM-MDMs when administered alone, and they demonstrated some synergistic activity in combination. Collectively, these findings indicate that Nurr1 enhances PPARγ1 transcriptional activity, potentially through stabilizing PPARγ1 protein levels and by decreasing its repressive phosphorylation at Ser84. This novel mechanism highlights the role of Nurr1 targeting not only inflammation but also metabolic pathways in macrophages.

Rationale: Cigarette smoke (CS) is a potent pro-inflammatory stimulus that impairs normal mechanisms that resolve inflammation, leading to chronic inflammation that contributes to many diseases including lung cancer and Chronic Obstructive Pulmonary Disease (COPD). CS also directly damages the lung epithelium. Maresin conjugate of tissue regeneration 3 (MCTR3) is a novel specialized pro-resolving mediator (SPM) derived from dietary fatty acids that has the property of promoting tissue repair and regeneration in addition to promoting resolution of inflammation. Here, we investigated the ability of MCTR3 to promote repair of airway epithelium injured by CS. Methods: Primary human small airway epithelial cells were differentiated at the air-liquid interface over 4 weeks and exposed CS for 30 min/day for 4 days. MCTR3 (1 to 100 nM) was added immediately after CS exposure on days 1-4. On day 5, culture media was collected, a FITC-dextran leak assay was performed to measure permeability of the differentiated cell layer, and the cells were fixed for immunofluorescence. Results: CS induced production of the pro-inflammatory cytokine IL-8 and damaged the epithelial barrier, indicated by increased FITC-dextran in the bottom compartment. MCTR3 significantly reduced both IL-8 production and barrier leak (Fig. A and data not shown). CS induced apoptosis and decreased cell proliferation, as indicated by immunostaining for caspase-3 and Ki67, respectively. MCTR3 treatment after CS exposure restored Ki67 expression and blocked apoptosis (Fig. B). Conclusions: MCTR3 protects the airway epithelium from CS-induced pro-inflammatory responses, cell death and epithelial barrier damage. MCTR3 demonstrates exciting, strong potential to be a novel therapeutic for CS-related diseases, including COPD.

While treatments targeting only tumor cells are not effective for many cancer types, tissue stroma as a critical driver of cancer offers an opportunity to target the tumor microenvironment. Cytotoxic chemotherapy and checkpoint inhibitors create tumor cell debris that may stimulate or induce tumor growth and metastasis. A paradigm shift is emerging in cancer therapy with the discovery of novel specialized pro-resolving mediators (SPMs), such as resolvin E1 (RvE1). However, no “pro-resolving” based therapy has been approved for cancer to date. Resolvin receptors are expressed by the major leukocyte types in the tumor stroma, i.e, macrophages, T lymphocytes, natural killer (NK), and dendritic cells. We hypothesize that RvE1, delivered as an active ingredient in a novel small molecule by subcutaneous injection, would reprogram leukocytes and enhance the clearance of cell debris. We demonstrate here that nanogram dosing of RvE1 (i.e. 30 ug/kg to 300 ug/kg) was efficacious when given QD, Q3D or Q6D and induced sustained tumor inhibition in pancreatic, lung, melanoma, and colon cancer in murine models. Remarkably, RvE1 enhanced immunotherapy (anti-PD1 or anti-CTLA-4) to transform a “cold” tumor into a “hot tumor” in pancreatic adenocarcinoma, Lewis lung carcinoma, and B16F10 melanoma. This suggests that RvE1 synergizes with chemotherapy and/or immune checkpoint inhibitors to potently inhibit tumor growth. We also evaluated RvE1 anti-tumor activity in the orthotopic KPC (KrasG12D/+/P53-/-/Pdx1-Cre) cell line, a robust model of human pancreatic ductal adenocarcinoma. In this model, RvE1 in combination with chemotherapy prolonged survival over 380 days, reduced tumor weight and prevented chemotherapy-induced metastasis. RNA-seq profiling revealed enrichment of CD8 T, dendritic, and NK cells, suggesting that RvE1 triggers an adaptive immune response. Depletion of CD8 T lymphocytes or NK cells abrogated the anti-tumor activity of RvE1. LC-MS-MS analysis revealed increased RvE1 concentrations in orthotopic pancreas tumors in treated mice compared to control. RvE1 also stimulated immune cell-mediated clearance of tumor debris. Taken together, this study introduces a novel molecular strategy for reprogramming tumor stroma via CD8 T lymphocyte and NK cell response. Thus, resolution-based SPM-directed stromal reprogramming with tumor-directed cytotoxic and immunologic drugs is a promising novel therapeutic approach for cancer.

Resolution of inflammation is a crucial process for restoring tissue homeostasis following an injury. Formyl peptide receptor 2 (FPR2) is a G-protein coupled receptor (GPCR) that plays a fundamental role in the resolution of inflammation by binding to various pro-resolving molecules. Misplaced inflammation is a major contributor to tissue damage and mortality associated to various infectious agents, such as SARS-CoV-2. Here, we assessed the role of the FPR2 receptor during Betacoronavirus infection. Wild-type (WT) mice and FPR2/3 knock-out mice (FPR2/3KO) were intranasally infected with a strain of murine betacoronavirus (MHV-3), which mimics severe COVID-19, by causing pneumonia and death in mice. FPR2/3KO mice showed approximately 50% protection from lethality when exposed to a lethal MHV-3 inoculum, compared to WT mice, in which 100% succumbed to infection by days 5-7. Interestingly, a logarithmic reduction in MHV-3 inoculum resulted in 100% protection against lethality in FPR2/3KO mice, while all WT mice were deceased by day 6-10 post-infection. FPR2/3KO mice also exhibited reduced viral titers in the lungs, liver, plasma and spleen, decreased lung damage and diminished production of inflammatory mediators. FPR2/3KO mice also displayed higher numbers of eosinophils in the lungs and increased expression of eosinophil peroxidase (EPO) when compared to WT mice. Mechanistically, eosinophils, alveolar macrophages and activated dendritic cells of FPR2/3KO mice exhibit increased expression of iNOS as evaluated by flow cytometry. Pharmacological blockade of FPR2 using a selective inhibitor WRW4 (8mg/kg, intraperitoneal route, daily) resulted in reduced systemic viral titers in the spleen without exerting any additional protective effects on the lungs. Surprisingly, these findings suggest that inhibition of FPR2/3 receptors could represent a promising therapeutic target against Betacoronavirus infection.

Microglia are the resident immune cells of the central nervous system (CNS). Their involvement in phagocytosis of myelin debris and remyelination of the CNS is particularly relevant in demyelinating neurological pathologies such as multiple sclerosis (MS). In this context, microglia adopt phenotypes associated with damage, which are frequently linked to the expression of the Igf1 gene. However, the mechanisms by which microglia aid in remyelination are still unclear. Given that Insulin-like Growth Factor 1 (IGF-1) is involved in cellular signaling, regulating proliferation, differentiation, migration, and survival, it is logical to question whether IGF-1 could play a pivotal role in CNS repair efforts by microglia. Our hypothesis is that IGF-1 derived from microglia would be a mediator of microglial effectiveness in repairing the CNS. To test this hypothesis, we generated transgenic mice Cx3cr1CreER::Igf1fl/fl in which microglia are conditionally invalidated in IGF-1 after tamoxifen treatment. We studied the effects of this mutation in combination with cuprizone treatment, during which the death of mature oligodendrocytes and subsequent demyelination are followed by robust microgliosis. After 5 weeks of cuprizone treatment followed by 1 week of recovery, Igf1 gene deletion is associated with an increase in the amount of intact/non-phagocytosed myelin and a decrease in the number of oligodendrocytes in the corpus callosum. In parallel, the cKO mice show an increase in the number of proliferating microglia and those expressing the surface markers Cd11c and Ly9. Our results suggest that IGF-1 derived from microglia plays a role in the microglial polarization towards a reparative phenotype.

Pulmonary hypertension (PH) secondary to lung diseases (PH Group 3) is associated with the highest incidence and mortality. This pathology is frequently associated with an inflammatory response where nucleoside phosphates could be involved [1]. Adenosine triphosphate (ATP) and diphosphate (ADP) or uridine triphosphate (UTP) and diphosphate (UDP) are known to regulate vascular tone. However, the cells, the receptor subtypes, and the mechanisms underlying the responses to these nucleoside phosphates on human pulmonary arteries (HPAs) have not been fully characterized, either in preparations derived from non-PH or PH patients. In this study, we evaluated the vasorelaxant or contractile responses of different nucleoside phosphates (ATP, ADP, UTP, UDP, ATPγS, Adenosine) in HPAs derived from PH Group 3 and Non-PH patients in the presence or absence of endothelium. A pharmacological study (organ bath system) was performed using selective purinergic receptor (P2Y/X) antagonists or agonists [2]. Moreover, we examined the contribution of nitric oxide (NO) and prostacyclin (PGI2) pathways to ATP-induced relaxation of HPAs using inhibitors of these pathways (L-NOARG and indomethacin, respectively). This study showed that ATP, UTP and ATPγS produced greater relaxations of HPAs than ADP , UDP or Adenosine . The relaxant responses were mostly blocked by either MRS2279 (P2Y1), ARC118925 (P2Y2), ATP (10 µM, P2Y4) or AR-C118925 (P2Y2) while MRS4062 (P2Y4 agonist) was inactive. In addition, on HPAs derived from Group 3 PH patients, ATP and ADP produced a less potent relaxation (~ 50 % less compared to control). UTP and UDP did not produce any relaxation, suggesting a uridine purinergic dysfunction on HPAs derived from Group 3 PH patients. Together, our results indicate that the purinergic agonists inducing-relaxation are endothelium, PGI2, and NO dependent. Moreover, we are currently able to identify two (2) purinergic receptors (P2Y1, P2Y2) involved in the relaxation, and we are currently investigating three (3) others (P2Y11 P2Y6, P2Y4). Finally, our results show that these relaxations are significantly reduced in HPAs from Group 3 PH patients, particularly the UTP relaxation, probably due to P2Y2 and P2Y4 dysfunction and might be a possible pathological mechanism underlying Group 3 PH. [1] Cai Z et al 2020, PMID : 32867554 [2]. Jacobson KA et al 2020, PMID : 32037507

Annexin A1 (AnxA1) is a glucocorticoid-induced protein with potent anti-inflammatory by regulation of inflammatory mediators and activation of NLRP3 inflammasome. Considering that the role of AnxA1 in normal and inflamed skin is not completely defined, this study evaluated AnxA1 and NLRP3 levels in skin biopsies from control and atopic dermatitis (AD) patients, and examined their effects on human keratinocytes stimulated with IL-4. Two studies containing publicly available transcriptome data (GSE16161 and GSE1120721) were individually analyzed using the GEO2R tool to detect AnxA1 and NLRP3 mRNA levels. For protein detection, paraffin-embedded human skin biopsies from AD patients and controls (n=10/group) were processed for immunohistochemical analyses. In vitro, IL-4-stimulated keratinocytes, HaCaT lineage, were treated with or without AnxA1-derived peptide Ac2-26 (5 or 25 ng/mL); some cells received 15 min before Ac2-26, the pan-formyl peptide receptor (FPR) antagonist Boc2 (10 µM). Transcriptome analyses of GSE16161 showed a significant increase in ANXA1 and NLRP3 transcripts in AD skins compared to the control skins, while in study GSE120721 only NLRP3 transcripts showed a significant increase. In study GSE120721, separate evaluation of the epidermal transcriptome showed elevated levels of ANXA1 transcripts in AD lesional skin relative to non-lesional AD skin, while transcriptional levels of NLRP3 were reduced in the lesional AD epidermis compared to controls. Immunohistochemical analysis of AD skin biopsies showed coexpression of AnxA1 and NLRP3 in the cytoplasm of keratinocytes. Under IL4 stimulation, Ac2-26 at 25ng produced a marked decrease of keratinocyte proliferation rate, effect abrogated by Boc2. This highest concentration of Ac2-26 also decreased NLRP3 levels, IL-1β release and ROS production by IL-4-stimulated keratinocytes in comparison to non-treated cells. Altogether, these findings suggest that AnxA1 exerts an immunomodulatory effect on keratinocytes and contributes to epidermal homeostasis through regulation of cell proliferation provoked by AD-induced inflammatory microenvironment. Funding: FAPESP, CAPES, CNPq.

NETosis is a specialized mechanism of cell death, achieved through the formation of neutrophil extracellular traps (NETs). NETs are lattices of extracellular fibers formed from released nuclear chromatin and cellular granular peptides, which can immobilize and destroy invading micro-organisms. However, aberrant formation or accumulation of NETs causes inappropriate activation of the host immune response and can contribute to the pathogenesis of several diseases, including diabetes, rheumatoid arthritis, and COVID-19. The development of inhibitors that directly target NETs (e.g. DNase 1) or inhibit upstream activation and signaling events provide an attractive therapeutic approach to alleviate immune-inflammatory disorders. Ongoing commercial activity in this field includes the Phase 1 trial of a first-in-class anti-histone therapeutic CIT-013 (Citryll), and Brensocatib (Insmed Inc.), a DDP-1 inhibitor, undergoing Phase 3 trials for non-cystic fibrosis bronchiectasis. A high-throughput compound screening platform was developed at Sygnature to quantify NETosis and differentiate NET formation from other cell death pathways such as apoptosis or necrosis. This was achieved using human primary neutrophils sourced from the in-house blood donor panel, in conjunction with IncuCyte® ZOOM live cell imaging and ImageXpress confocal microscopy platforms. Multiplex cell imaging assays were established to analyze the nuclear and plasma membrane morphology combined with fluorescent tagging of nuclear and extracellular DNA fibers. This approach enabled the quantitative detection of NET formation and the determination of literature inhibitor activity, giving scope for new targeted drug discovery programs. Furthermore, neutrophil reactive oxygen species (ROS) production, an important mediator in the NETosis pathway, was detected using a cytochrome C reduction assay, and inhibited by the MAPK-p38 inhibitor SB203580. To enable higher throughput screening, the NETosis assay was successfully transferred to a differentiated HL-60 human leukocyte cell line, enabling more efficient screening to help identify novel NETosis inhibitors.