Evaluation of Anti-Inflammatory Activity of the New Cardiotonic Steroid γ-Benzylidene Digoxin 8 (BD-8) in Mice.

Cardiotonic steroids are known to bind to Na+/K+-ATPase and regulate several biological processes, including the immune response. The synthetic cardiotonic steroid γ-Benzylidene Digoxin 8 (BD-8) is emerging as a promising immunomodulatory molecule, although it has remained largely unexplored. Therefore, we tested the immunomodulatory potential of BD-8 both in vitro and in vivo. Hence, primary mouse macrophages were incubated with combinations of BD-8 and the pro-inflammatory fungal protein zymosan (ZYM). Nitric oxide (NO) production was determined by Griess reagent and cytokines production was assessed by enzyme-linked immunosorbent assay. Inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), p-nuclear factor kappa B p65 (NF-κB p65), p-extracellular signal-regulated kinase (p-ERK), and p-p38 were evaluated by flow cytometry. Macrophages exposed to BD-8 displayed reduced phagocytic activity, NO levels, and production of the proinflammatory cytokine IL-1ß induced by ZYM. Furthermore, BD-8 diminished the expression of iNOS and phosphorylation of NF-κB p65, ERK, and p38. Additionally, BD-8 exhibited anti-inflammatory capacity in vivo in a carrageenan-induced mouse paw edema model. Taken together, these findings demonstrate the anti-inflammatory activity of BD-8 and further reinforce the potential of cardiotonic steroids and their derivatives as immunomodulatory molecules.

Interleukins IL33/ST2 and IL1-ß in Intrauterine Growth Restriction and Seropositivity of Anti-Toxoplasma gondii Antibodies.

Toxoplasma gondii (T. gondii) is the causal agent of toxoplasmosis. It may produce severe damage in immunocompromised individuals, as well as congenital infection and intrauterine growth restriction (IUGR). Previous reports have associated interleukin IL-33 with miscarriage, fetal damage, and premature delivery due to infections with various microorganisms. However, IL-33 has not been associated with congenital toxoplasmosis. The sST2 receptor has been reported in patients who have had recurrent miscarriages. On the other hand, IL-1ß was not found in acute Toxoplasma infection. Our aim was to analyze the associations between the serum levels of IL-33 and IL-1ß in IUGR and toxoplasmosis during pregnancy. Eighty-four serum samples from pregnant women who had undergone 26 weeks of gestation were grouped as follows: with anti-Toxoplasma antibodies, without anti-Toxoplasma antibodies, IUGR, and the control group. IgG and IgM anti-T. gondii antibodies, as well as IL-33, ST2, and IL-1ß, were determined using an ELISA assay. Statistical analyses were performed using the Pearson and Chi-square correlation coefficients, as well as the risk factors and Odds Ratios (ORs), with a confidence interval of 95% (CI 95). The results showed that 15/84 (17.8%) of cases were positive for IgG anti-Toxoplasma antibodies and 2/84 (2.38%) of cases were positive for IgM. A statistically significant difference was found between IUGR and IL-33 (p < 0.001), as well as between ST2 and IUGR (p < 0.001). In conclusion, IUGR was significantly associated with IL-33 and ST2 positivity based on the overall IUGR grade. No significant association was found between IUGR and the presence of anti-Toxoplasma antibodies. There was no association between IL-1ß and IUGR. More research is needed to strengthen the utility of IL-33 and ST2 as biomarkers of IUGR.

Cutaneous inflammasome driving ASC / gasdermin-D activation and IL-1ß-secreting macrophages in severe atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus, and chronic skin colonization by Staphylococcus aureus. To understand the inflammatory status in AD, we investigated the inflammasome complex, that activates ASC (Apoptosis-associated speck-like protein containing a CARD), caspase-1 and GSDMD (gasdermin-D), and production of IL-1ß and IL-18. We aimed to evaluate the expression of the inflammasome pathway in the skin of adults with AD. Thirty patients with moderate to severe AD and 20 healthy controls were enrolled in the study. We performed the analysis of the inflammasome components NLRP1, NLRP3, AIM-2, IL-1ß, IL-18, Caspase-1, ASC, GSDMD, and CD68 expression (macrophage marker) by immunohistochemistry and immunofluorescence. The main findings included increased expression of NLRP3, NLRP1 and AIM-2 at dermal level of severe AD; augmented IL-18 and IL-1ß expression at epidermis of moderate and severe patients, and in the dermis of severe AD; augmented expression of ASC, caspase-1 and GSDMD in both epidermis and dermis of moderate and severe AD. We detected positive correlation between caspase-1, GSDMD and IL-1ß (epidermis) and caspase-1 (dermis) and AD severity; NLRP3, AIM-2 and IL-1ß, and NLRP3 with IL-18 in the epidermis; ASC, GSDMD and IL-1ß, and NLRP3, AIM-2, caspase-1, and IL-18 in the dermis. We also evidenced the presence of CD68+ macrophages secreting GSDMD, ASC and IL-1ß in moderate and severe AD. Cutaneous macrophages, early detected in moderate AD, have its role in the disease inflammatory mechanisms. Our study indicates a canonical activation pathway of inflammasomes, reinforced by the chronic status of inflammation in AD. The analysis of the inflammasome complex evidenced an imbalance in its regulation, with increased expression of the evaluated components, which is remarkably in severe AD, emphasizing its relevance as potential disease biomarkers and targets for immunomodulatory interventions.

Tanshinone IIA alleviates IL-1ß-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression.

OBJECTIVE: This study explored the pharmacological mechanism of Tanshinone IIA (TAN IIA) in the treatment of Osteoarthritis (OA), which provided a certain reference for further research and clinical application of Tan IIA in OA. METHODS: CHON-001 cells were stimulated with 10 µg/mL IL-1ß for 48 h and treated with 10 µM TAN IIA for 48 h. Cellular viability and apoptosis were evaluated by CCK-8 assay and flow cytometry, and Cleaved caspase-3 was measured by Immunoblot assay and RT-qPCR. TNF-α, IL-6, and iNOS in CHON-001 cells were determined by RT-qPCR and ELISA. To further verify the effect of TAN IIA on OA, a rat model of OA in vivo was established by right anterior cruciate ligament transection. TAN IIA was administered at 50 mg/kg or 150 mg/kg for 7 weeks. The degree of cartilage destruction in OA rats was observed by TUNEL and HE staining. Cleaved caspase-3 and FBXO11 were measured by immunohistochemical staining, RT-qPCR, and Immunoblot. TNF-α, IL-6, and iNOS in chondrocytes of OA rats were detected by ELISA. RESULTS: IL-1ß stimulated CHON-001 cell apoptosis and inflammation, and TAN IIA had anti-apoptosis and anti-inflammatory effects on IL-1ß-regulated CHON-001 cells. TAN IIA down-regulated FBXO11 and inhibited PI3K/AKT and NF-κB pathways, thereby alleviating apoptotic and inflammatory reactions in CHON-001 cells under IL-1ß treatment. Moreover, TAN IIA treatment improved chondrocyte apoptosis and inflammations in OA rats. CONCLUSION: TAN IIA inhibits PI3K/Akt and NF-κB pathways by down-regulating FBXO11 expression, alleviates chondrocyte apoptosis and inflammation, and delays the progression of OA.

Cardiac hypertrophy that affects hyperthyroidism occurs independently of the NLRP3 inflammasome.

Cardiac hypertrophy (CH) is an adaptive response to maintain cardiac function; however, persistent stress responses lead to contractile dysfunction and heart failure. Although inflammation is involved in these processes, the mechanisms that control cardiac inflammation and hypertrophy still need to be clarified. The NLRP3 inflammasome is a cytosolic multiprotein complex that mediates IL-1ß production. The priming step of NLRP3 is essential for increasing the expression of its components and occurs following NF-κB activation. Hyperthyroidism triggers CH, which can progress to maladaptive CH and even heart failure. We have shown in a previous study that thyroid hormone (TH)-induced CH is linked to the upregulation of S100A8, leading to NF-κB activation. Therefore, we aimed to investigate whether the NLRP3 inflammasome is involved in TH-induced CH and its potential role in CH pathophysiology. Hyperthyroidism was induced in NLRP3 knockout (NLRP3-KO), Caspase-1-KO and Wild Type (WT) male mice of the C57Bl/6J strain, aged 8-12 weeks, by triiodothyronine (7 µg/100 g BW, i.p.) administered daily for 14 days. Morphological and cardiac functional analysis besides molecular assays showed, for the first time, that TH-induced CH is accompanied by reduced NLRP3 expression in the heart and that it occurs independently of the NLRP3 inflammasome and caspase 1-related pathways. However, NLRP3 is important for the maintenance of basal cardiac function since NLRP3-KO mice had impaired diastolic function and reduced heart rate, ejection fraction, and fractional shortening compared with WT mice.

In Situ versus Systemic Immune Response in the Pathogenesis of Cutaneous Leishmaniasis.

The role of the immune response in the pathogenesis of cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis is predominantly carried out via blood cells. Here, we evaluate whether cytokine production by peripheral blood mononuclear cells (PBMCs) reflects what has been documented at the lesion site. The participants included 22 CL patients diagnosed with a positive PCR. PBMCs were stimulated for 72 h with a soluble leishmania antigen (SLA). Biopsies obtained from the edge of the ulcers were incubated for the same period. Cytokines in supernatants were assessed via ELISA. TNF, IL-1ß, IL-6, IL-17, and granzyme B (GzmB) were higher in the supernatants of biopsies than in PBMCs, but IFN-γ was higher in the supernatants of PBMCs than in biopsies. There was a positive correlation between IFN-γ and TNF in PBMCs, and an inverse correlation between TNF and IL-10 in the cells from the lesion site. A strong correlation between IL-1ß, IL-17, and GzmB was observed in the biopsies, and a positive correlation was detected between these cytokines and the lesion size. Our results indicate that the immune response in L. braziliensis lesions is different from that observed in peripheral blood, and our data suggest that in addition to IL-1ß and GzmB, IL-17 participates in the pathology of CL.

Lack of Annexin A1 Exacerbates Inflammatory Response in Acute Endometritis Model.

Annexin A1 (AnxA1) is a glucocorticoid-inducible protein and an important endogenous modulator of inflammation. However, its effect in the endometrial microenvironment is poorly explained. This study aimed to evaluate the role of endogenous AnxA1 in an endometritis mouse model induced by lipopolysaccharide (LPS). Female C57BL/6 wild-type (WT) and AnxA1-/- mice were divided into two groups: SHAM and LPS. To induce endometritis, mice received a vaginal infusion of 50 µL of LPS (1 mg/mL) dissolved in phosphate-buffered saline. After 24 h, the mice were euthanized, and blood and uteri samples were collected. The endometrium inflammatory scores were significantly increased in the LPS-treated group. AnxA1-/- mice from the LPS group demonstrated a significant increase in the number of degranulated mast cell levels compared to AnxA1-/- SHAM mice. The Western blotting analysis revealed that a lack of AnxA1 promoted the upregulation of NLRP3 and pro-IL-1ß in the acute endometritis animal model compared to WT LPS animals. LPS-induced endometritis increased the number of blood peripheral leukocytes in both WT and AnxA1-/- mice compared with SHAM group mice (p < 0.001). AnxA1-/- mice also showed increased plasma levels of IL-1ß (p < 0.01), IL-6, IL-10, IL-17, and TNF-α (p < 0.05) following LPS-induced endometritis. In conclusion, a lack of endogenous AnxA1 exacerbated the inflammatory response in an endometritis model via NLRP3 dysregulation, increased uterine mast cell activation, and plasma pro-inflammatory cytokine release.

Hepatic SPARC Expression Is Associated with Inflammasome Activation during the Progression of Non-Alcoholic Fatty Liver Disease in Both Mice and Morbidly Obese Patients.

The severity of non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to steatohepatitis, and it is not yet clearly understood which patients will progress to liver fibrosis or cirrhosis. SPARC (Secreted Protein Acidic and Rich in Cysteine) has been involved in NAFLD pathogenesis in mice and humans. The aim of this study was to investigate the role of SPARC in inflammasome activation, and to evaluate the relationship between the hepatic expression of inflammasome genes and the biochemical and histological characteristics of NAFLD in obese patients. In vitro studies were conducted in a macrophage cell line and primary hepatocyte cultures to assess the effect of SPARC on inflammasome. A NAFLD model was established in SPARC knockout (SPARC-/-) and SPARC+/+ mice to explore inflammasome activation. A hepatic RNAseq database from NAFLD patients was analyzed to identify genes associated with SPARC expression. The results were validated in a prospective cohort of 59 morbidly obese patients with NAFLD undergoing bariatric surgery. Our results reveal that SPARC alone or in combination with saturated fatty acids promoted IL-1ß expression in cell cultures. SPARC-/- mice had reduced hepatic inflammasome activation during the progression of NAFLD. NAFLD patients showed increased expression of SPARC, NLRP3, CASP1, and IL-1ß. Gene ontology analysis revealed that genes positively correlated with SPARC are linked to inflammasome-related pathways during the progression of the disease, enabling the differentiation of patients between steatosis and steatohepatitis. In conclusion, SPARC may play a role in hepatic inflammasome activation in NAFLD.

Noncanonical NLRP3 Inflammasome Activation: Standard Protocols.

The canonical activation of multimeric inflammasomes usually occurs through caspase-1 activation, and it is characterized by the presence of extracellular IL-1ß and IL-18 or measuring danger signal proteins, such as HMGB1 using enzyme-linked immunosorbent assay (ELISA) or Western blots; these assays differentiate non-cleaved and cleaved forms of these two cytokines (the cleaved form is the mature and active form). Similar techniques can be used to assess noncanonical inflammasome activation. Real-time PCR can measure the relative mRNA expression for a specific gene, whereas Western blots or immunocytochemistry can detect the presence of proteins by binding of specific antibodies to their antigens in biological samples. Moreover, noncanonical inflammasome activation can be evaluated through the cleavage of the amino and the carboxy terminals of one important component, gasdermin D (GSDMD), whose cleavage induces its pyroptotic activity. Thus, the analysis of cleaved GSDMD is an ideal pathway to study the noncanonical inflammasome. ELISA and immunoblot can be performed on cell culture supernatants or cell extracts.

Cervical carcinoma induces NLRP3 inflammasome activation and IL-1ß release in human peripheral blood monocytes affecting patients' overall survival.

PURPOSE: Our group previously demonstrated that genetic variants in inflammasome genes contribute to protection against the establishment of human papilloma virus (HPV)-associated cervical carcinoma (CC). The objective of this study was to better understand the contribution of inflammasome and its cytokines in the CC microenvironment. METHODS: The inflammasome activation was analyzed in CC tumoral cell lines and healthy donors (HD)' monocytes in co-culture. In vitro results were then compared to CC patients' public databases. RESULTS: CC cells did not produce IL-1ß or IL-18 themselves, however, when in co-culture with HD monocytes, induced IL-1ß release in those leucocytes. Inflammasome activation appears to be partially dependent on the NLRP3 receptor. Public data analysis revealed that IL1B expression is increased in the CC compared to normal uterine cervix, and that patients with high IL1B expression had a shorter overall survival. CONCLUSION: CC microenvironment can activate the inflammasome and IL-1ß release in surrounding monocytes, which could be detrimental for CC prognosis.

The p,p'-DDE disturbs the M1 function without affecting the M2 phenotype nor unstimulated bone marrow-derived macrophages from BALB/c mice.

DDT, a persistent organic pollutant, remains affecting human health worldwide. DDT and its most persistent metabolite (p,p'-DDE) negatively affect the immune response regulation and mechanisms involved in protecting against pathogens Such metabolite decreases the capability to limit intracellular growth of Mycobacterium microti and yeast. However, the effect on unstimulated (M0) and anti-inflammatory macrophages (M2) has been evaluated scanty. Herein, we evaluated the impact of p,p'-DDE at environmentally relevant concentrations (0.125, 1.25, 2.5, and 5 µg/mL) on bone marrow-derived macrophages stimulated with IFNγ+LPS to M1 or with IL-4 +IL-13 to M2. Thus we study whether the p,p'-DDE induces M0 to a specific phenotype or modulates activation of the macrophage phenotypes and explains, at least partly, the reported effects of p,p'-DDE on the M1 function. The p,p'-DDE did not affect the cell viability of M0 or the macrophage phenotypes. In M1, the p,p'-DDE decreased NO•- production and IL-1ß secretion, but increasing cellular ROS and mitochondrial O2•-, but did not alter iNOS, TNF-α, MHCII, and CD86 protein expression nor affect M2 markers arginase activity, TGF-ß1, and CD206; p,p'-DDE, did not affect marker expression in M0 or M2, supporting that its effects on M1 parameters are not dependent on M0 nor M2 modulation. The decreasing of NO•- production by the p,p'-DDE without altering iNOS levels, Arginase activity, or TNF-α, but increasing cellular ROS and mitochondrial O2 suggests that p,p'-DDE interferes with the iNOS function but not with its transcription. The p,p'-DDE decreasing of IL-1ß secretion, without any effect on TNF-α, suggest that an alteration of specific targets involved in IL-1ß secretion may be affected and related to ROS induction. The p,p'-DDE effect on iNOS function and the IL-1ß secretion process, as the NLRP3 activation, deserves further study.

Transcriptional Activation of a Pro-Inflammatory Response (NF-κB, AP-1, IL-1ß) by the Vibrio cholerae Cytotoxin (VCC) Monomer through the MAPK Signaling Pathway in the THP-1 Human Macrophage Cell Line.

This study describes, to some extent, the VCC contribution as an early stimulation of the macrophage lineage. Regarding the onset of the innate immune response caused by infection, the ß form of IL-1 is the most important interleukin involved in the onset of the inflammatory innate response. Activated macrophages treated in vitro with VCC induced the activation of the MAPK signaling pathway in a one-hour period, with the activation of transcriptional regulators for a surviving and pro-inflammatory response, suggesting an explanation inspired and supported by the inflammasome physiology. The mechanism of IL-1ß production induced by VCC has been gracefully outlined in murine models, using bacterial knockdown mutants and purified molecules; nevertheless, the knowledge of this mechanism in the human immune system is still under study. This work shows the soluble form of 65 kDa of the Vibrio cholerae cytotoxin (also known as hemolysin), as it is secreted by the bacteria, inducing the production of IL-1ß in the human macrophage cell line THP-1. The mechanism involves triggering the early activation of the signaling pathway MAPKs pERK and p38, with the subsequent activation of (p50) NF-κB and AP-1 (cJun and cFos), determined by real-time quantitation. The evidence shown here supports that the monomeric soluble form of the VCC in the macrophage acts as a modulator of the innate immune response, which is consistent with the assembly of the NLRP3 inflammasome actively releasing IL-1ß.

Synergism between chikungunya virus infection and rheumatoid arthritis on cytokine levels: Clinical implications?

Post-chikungunya virus (CHIKV) chronic arthritis shares several immunopathogenic mechanisms with rheumatoid arthritis (RA), which has led to discussions about the probable relationship between the two diseases. Indeed, some studies have suggested a role for CHIKV infection in RA development. However, to the best of our knowledge, the influence of CHIKV on previous RA has not yet been demonstrated. Herein, we analyzed the potential synergism between CHIKV infection and RA on cytokine and chemokine levels. For this, we compared the IL-1ß, IL-6, IL-10, IL-17A, CCL2, CXCL8, CXCL9 and CXCL10 levels, in addition to rheumatoid factor (RF) and C-reactive protein (CRP), in patients with post-CHIKV chronic arthritis (named CHIKV group), patients with RA (RA group), and patients with previous RA who were later infected by CHIKV (RA-CHIKV). History of CHIKV infection was confirmed by serology (IgG, ELISA). Cytokines/chemokines were quantified by flow cytometry. RF, CRP, age and sex data were obtained from medical records. IL-1ß, IL-6, IL-10 and IL-17A levels were significantly higher in RA-CHIKV compared to the other groups. CXCL8 levels were higher in the CHIKV group than in RA. CXCL9 was higher in CHIKV than in the RA-CHIKV group. CXCL10 was higher in CHIKV than in the other groups. FR levels were higher in RA than in the CHIKV group, and in RA-CHIKV than in CHIKV. No significant difference was observed in CCL2 and CRP, as well as in age and sex. Finally, our findings suggest an interplay between CHIKV infection and RA, which must be analyzed for its possible clinical impact.

Genetic and Epigenetic Regulation of the Innate Immune Response to Gout.

Gout is a disease caused by uric acid (UA) accumulation in the joints, causing inflammation. Two UA forms - monosodium urate (MSU) and soluble uric acid (sUA) have been shown to interact physically with inflammasomes, especially with the nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3), albeit the role of the immune response to UA is poorly understood, given that asymptomatic hyperuricemia does also exist. Macrophage phagocytosis of UA activate NLRP3, lead to cytokines release, and ultimately, lead to chemoattract neutrophils and lymphocytes to the gout flare joint spot. Genetic variants of inflammasome genes and of genes encoding their molecular partners may influence hyperuricemia and gout susceptibility, while also influencing other comorbidities such as metabolic syndrome and cardiovascular diseases. In this review, we summarize the inflammatory responses in acute and chronic gout, specifically focusing on innate immune cell mechanisms and genetic and epigenetic characteristics of participating molecules. Unprecedently, a novel UA binding protein - the neuronal apoptosis inhibitor protein (NAIP) - is suggested as responsible for the asymptomatic hyperuricemia paradox.Abbreviation: ß2-integrins: leukocyte-specific adhesion molecules; ABCG2: ATP-binding cassete family/breast cancer-resistant protein; ACR: American college of rheumatology; AIM2: absent in melanoma 2, type of pattern recognition receptor; ALPK1: alpha-protein kinase 1; ANGPTL2: angiopoietin-like protein 2; ASC: apoptosis-associated speck-like protein; BIR: baculovirus inhibitor of apoptosis protein repeat; BIRC1: baculovirus IAP repeat-containing protein 1; BIRC2: baculoviral IAP repeat-containing protein 2; C5a: complement anaphylatoxin; cAMP: cyclic adenosine monophosphate; CARD: caspase activation and recruitment domains; CARD8: caspase recruitment domain-containing protein 8; CASP1: caspase 1; CCL3: chemokine (C-C motif) ligand 3; CD14: cluster of differentiation 14; CD44: cluster of differentiation 44; Cg05102552: DNA-methylation site, usually cytosine followed by guanine nucleotides; contains arbitrary identification code; CIDEC: cell death-inducing DNA fragmentation factor-like effector family; CKD: chronic kidney disease; CNV: copy number variation; CPT1A: carnitine palmitoyl transferase - type 1a; CXCL1: chemokine (CXC motif) ligand 1; DAMPs: damage associated molecular patterns; DC: dendritic cells; DNMT(1): maintenance DNA methyltransferase; eQTL: expression quantitative trait loci; ERK1: extracellular signal-regulated kinase 1; ERK2: extracellular signal-regulated kinase 2; EULAR: European league against rheumatism; GMCSF: granulocyte-macrophage colony-stimulating factor; GWAS: global wide association studies; H3K27me3: tri-methylation at the 27th lysine residue of the histone h3 protein; H3K4me1: mono-methylation at the 4th lysine residue of the histone h3 protein; H3K4me3: tri-methylation at the 4th lysine residue of the histone h3 protein; HOTAIR: human gene located between hoxc11 and hoxc12 on chromosome 12; IκBα: cytoplasmatic protein/Nf-κb transcription inhibitor; IAP: inhibitory apoptosis protein; IFNγ: interferon gamma; IL-1ß: interleukin 1 beta; IL-12: interleukin 12; IL-17: interleukin 17; IL18: interleukin 18; IL1R1: interleukin-1 receptor; IL-1Ra: interleukin-1 receptor antagonist; IL-22: interleukin 22; IL-23: interleukin 23; IL23R: interleukin 23 receptor; IL-33: interleukin 33; IL-6: interleukin 6; IMP: inosine monophosphate; INSIG1: insulin-induced gene 1; JNK1: c-jun n-terminal kinase 1; lncRNA: long non-coding ribonucleic acid; LRR: leucine-rich repeats; miR: mature non-coding microRNAs measuring from 20 to 24 nucleotides, animal origin; miR-1: miR followed by arbitrary identification code; miR-145: miR followed by arbitrary identification code; miR-146a: miR followed by arbitrary identification code, "a" stands for mir family; "a" family presents similar mir sequence to "b" family, but different precursors; miR-20b: miR followed by arbitrary identification code; "b" stands for mir family; "b" family presents similar mir sequence to "a" family, but different precursors; miR-221: miR - followed by arbitrary identification code; miR-221-5p: miR followed by arbitrary identification code; "5p" indicates different mature miRNAs generated from the 5' arm of the pre-miRNA hairpin; miR-223: miR followed by arbitrary identification code; miR-223-3p: mir followed by arbitrary identification code; "3p" indicates different mature miRNAs generated from the 3' arm of the pre-miRNA hairpin; miR-22-3p: miR followed by arbitrary identification code, "3p" indicates different mature miRNAs generated from the 3' arm of the pre-miRNA hairpin; MLKL: mixed lineage kinase domain-like pseudo kinase; MM2P: inductor of m2-macrophage polarization; MSU: monosodium urate; mTOR: mammalian target of rapamycin; MyD88: myeloid differentiation primary response 88; n-3-PUFAs: n-3-polyunsaturated fatty-acids; NACHT: acronym for NAIP (neuronal apoptosis inhibitor protein), C2TA (MHC class 2 transcription activator), HET-E (incompatibility locus protein from podospora anserina) and TP1 (telomerase-associated protein); NAIP: neuronal apoptosis inhibitory protein (human); Naip1: neuronal apoptosis inhibitory protein type 1 (murine); Naip5: neuronal apoptosis inhibitory protein type 5 (murine); Naip6: neuronal apoptosis inhibitory protein type 6 (murine); NBD: nucleotide-binding domain; Nek7: smallest NIMA-related kinase; NET: neutrophil extracellular traps; Nf-κB: nuclear factor kappa-light-chain-enhancer of activated b cells; NFIL3: nuclear-factor, interleukin 3 regulated protein; NIIMA: network of immunity in infection, malignancy, and autoimmunity; NLR: nod-like receptor; NLRA: nod-like receptor NLRA containing acidic domain; NLRB: nod-like receptor NLRA containing BIR domain; NLRC: nod-like receptor NLRA containing CARD domain; NLRC4: nod-like receptor family CARD domain containing 4; NLRP: nod-like receptor NLRA containing PYD domain; NLRP1: nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 1; NLRP12: nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 12; NLRP3: nod-like receptor family pyrin domain containing 3; NOD2: nucleotide-binding oligomerization domain; NRBP1: nuclear receptor-binding protein; Nrf2: nuclear factor erythroid 2-related factor 2; OR: odds ratio; P2X: group of membrane ion channels activated by the binding of extracellular; P2X7: p2x purinoceptor 7 gene; p38: member of the mitogen-activated protein kinase family; PAMPs: pathogen associated molecular patters; PBMC: peripheral blood mononuclear cells; PGGT1B: geranylgeranyl transferase type-1 subunit beta; PHGDH: phosphoglycerate dehydrogenase; PI3-K: phospho-inositol; PPARγ: peroxisome proliferator-activated receptor gamma; PPARGC1B: peroxisome proliferative activated receptor, gamma, coactivator 1 beta; PR3: proteinase 3 antigen; Pro-CASP1: inactive precursor of caspase 1; Pro-IL1ß: inactive precursor of interleukin 1 beta; PRR: pattern recognition receptors; PYD: pyrin domain; RAPTOR: regulatory associated protein of mTOR complex 1; RAS: renin-angiotensin system; REDD1: regulated in DNA damage and development 1; ROS: reactive oxygen species; rs000*G: single nuclear polymorphism, "*G" is related to snp where replaced nucleotide is guanine, usually preceded by an id number; SLC2A9: solute carrier family 2, member 9; SLC7A11: solute carrier family 7, member 11; SMA: smooth muscular atrophy; Smac: second mitochondrial-derived activator of caspases; SNP: single nuclear polymorphism; Sp3: specificity protein 3; ST2: serum stimulation-2; STK11: serine/threonine kinase 11; sUA: soluble uric acid; Syk: spleen tyrosine kinase; TAK1: transforming growth factor beta activated kinase; Th1: type 1 helper T cells; Th17: type 17 helper T cells; Th2: type 2 helper T cells; Th22: type 22 helper T cells; TLR: tool-like receptor; TLR2: toll-like receptor 2; TLR4: toll-like receptor 4; TNFα: tumor necrosis factor alpha; TNFR1: tumor necrosis factor receptor 1; TNFR2: tumor necrosis factor receptor 2; UA: uric acid; UBAP1: ubiquitin associated protein; ULT: urate-lowering therapy; URAT1: urate transporter 1; VDAC1: voltage-dependent anion-selective channel 1.

Doxycycline attenuates l-DOPA-induced dyskinesia through an anti-inflammatory effect in a hemiparkinsonian mouse model.

The pharmacological manipulation of neuroinflammation appears to be a promising strategy to alleviate l-DOPA-induced dyskinesia (LID) in Parkinson's disease (PD). Doxycycline (Doxy), a semisynthetic brain-penetrant tetracycline antibiotic having interesting anti-inflammatory properties, we addressed the possibility that this compound could resolve LID in l-DOPA-treated C57BL/6 mice presenting either moderate or intermediate lesions of the mesostriatal dopaminergic pathway generated by intrastriatal injections of 6-OHDA. Doxy, when given subcutaneously before l-DOPA at doses of 20 mg kg-1 and 40 mg kg-1, led to significant LID reduction in mice with moderate and intermediate dopaminergic lesions, respectively. Importantly, Doxy did not reduce locomotor activity improved by l-DOPA. To address the molecular mechanism of Doxy, we sacrificed mice with mild lesions 1) to perform the immunodetection of tyrosine hydroxylase (TH) and Fos-B and 2) to evaluate a panel of inflammation markers in the striatum, such as cyclooxygenase-2 and its downstream product Prostaglandin E2 along with the cytokines TNF-α, IL-1ß and IL-6. TH-immunodetection revealed that vehicle and Doxy-treated mice had similar striatal lesions, excluding that LID improvement by Doxy could result from neurorestorative effects. Importantly, LID inhibition by Doxy was associated with decreased Fos-B and COX-2 expression and reduced levels of PGE2, TNF-α, and IL-1ß in the dorsolateral striatum of dyskinetic mice. We conclude 1) that Doxy has the potential to prevent LID regardless of the intensity of dopaminergic lesioning and 2) that the anti-inflammatory effects of Doxy probably account for LID attenuation. Overall, the present results further indicate that Doxy might represent an attractive and alternative treatment for LID in PD.

IL-1ß expression driven by androgen receptor absence or inactivation promotes prostate cancer bone metastasis.

We report the inverse association between the expression of androgen receptor (AR) and interleukin-1beta (IL-1ß) in a cohort of patients with metastatic castration resistant prostate cancer (mCRPC). We also discovered that AR represses the IL-1ß gene by binding an androgen response element (ARE) half-site located within the promoter, which explains the IL-1ß expression in AR-negative (ARNEG) cancer cells. Consistently, androgen-depletion or AR-pathway inhibitors (ARIs) de-repressed IL-1ß in ARPOS cancer cells, both in vitro and in vivo. The AR transcriptional repression is sustained by histone de-acetylation at the H3K27 mark in the IL-1ß promoter. Notably, patients' data suggest that DNA methylation prevents IL-1ß expression, even if the AR-signaling axis is inactive. Our previous studies show that secreted IL-1ß supports metastatic progression in mice by altering the transcriptome of tumor-associated bone stroma. Thus, in prostate cancer patients harboring ARNEG tumor cells or treated with ADT/ARIs, and with the IL-1ß gene unmethylated, IL-1ß could condition the metastatic microenvironment to sustain disease progression.

Early effects of LPS-induced neuroinflammation on the rat hippocampal glycolytic pathway.

Neuroinflammation is a common feature during the development of neurological disorders and neurodegenerative diseases, where glial cells, such as microglia and astrocytes, play key roles in the activation and maintenance of inflammatory responses in the central nervous system. Neuroinflammation is now known to involve a neurometabolic shift, in addition to an increase in energy consumption. We used two approaches (in vivo and ex vivo) to evaluate the effects of lipopolysaccharide (LPS)-induced neuroinflammation on neurometabolic reprogramming, and on the modulation of the glycolytic pathway during the neuroinflammatory response. For this, we investigated inflammatory cytokines and receptors in the rat hippocampus, as well as markers of glial reactivity. Mitochondrial respirometry and the glycolytic pathway were evaluated by multiple parameters, including enzymatic activity, gene expression and regulation by protein kinases. Metabolic (e.g., metformin, 3PO, oxamic acid, fluorocitrate) and inflammatory (e.g., minocycline, MCC950, arundic acid) inhibitors were used in ex vivo hippocampal slices. The induction of early inflammatory changes by LPS (both in vivo and ex vivo) enhanced glycolytic parameters, such as glucose uptake, PFK1 activity and lactate release. This increased glucose consumption was independent of the energy expenditure for glutamate uptake, which was in fact diverted for the maintenance of the immune response. Accordingly, inhibitors of the glycolytic pathway and Krebs cycle reverted neuroinflammation (reducing IL-1ß and S100B) and the changes in glycolytic parameters induced by LPS in acute hippocampal slices. Moreover, the inhibition of S100B, a protein predominantly synthesized and secreted by astrocytes, inhibition of microglia activation and abrogation of NLRP3 inflammasome assembly confirmed the role of neuroinflammation in the upregulation of glycolysis in the hippocampus. Our data indicate a neurometabolic glycolytic shift, induced by inflammatory activation, as well as a central and integrative role of astrocytes, and suggest that interference in the control of neurometabolism may be a promising strategy for downregulating neuroinflammation and consequently for diminishing negative neurological outcomes.

IL-1ß blockade prevents cell death and mucosal damage of the small intestine in a model of sterile inflammation.

The intestinal mucosa is covered by a layer of epithelial cells that is constantly challenged by commensal, opportunistic, and pathogenic microorganisms, their components, and harmful compounds. Any inflammatory response to these materials must be tightly controlled to limit tissue damage and restore the integrity of the mucosal barrier. We have shown previously that production of IL-1ß via activation of the inflammasome can lead to mucosal damage in the small intestinal pathology that occurs after intragastric administration of a gluten derived peptide, p31-43. Here we show that specific inhibition of caspase-1 or NLRP3 abolishes the damage induced by p31-43, and that antibody-mediated blocking of IL-1ß inhibits the both the histological changes and the induction of apoptosis and caspase-3 activation driven by p31-43. Understanding the role of IL-1ß in sterile inflammation may help to understand chronic inflammatory pathological processes, and design new intervention strategies.

Inflammasome activation and assembly in Huntington's disease.

Huntington's disease (HD) is a rare neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms. Inflammasomes are multiprotein complexes capable of sensing pathogen-associated and damage-associated molecular patterns, triggering innate immune pathways. Activation of inflammasomes results in a pro-inflammatory cascade involving, among other molecules, caspases and interleukins. NLRP3 (nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3) is the most studied inflammasome complex, and its activation results in caspase-1 mediated cleavage of the pro-interleukins IL-1ß and IL-18 into their mature forms, also inducing a gasdermin D mediated form of pro-inflammatory cell death, i.e. pyroptosis. Accumulating evidence has implicated NLRP3 inflammasome complex in neurodegenerative diseases. The evidence in HD is still scant and mostly derived from pre-clinical studies. This review aims to present the available evidence on NLRP3 inflammasome activation in HD and to discuss whether targeting this innate immune system complex might be a promising therapeutic strategy to alleviate its symptoms.

Angiotensin type 2 receptor antagonism as a new target to manage gout.

BACKGROUND: There is a growing search for therapeutic targets in the treatment of gout. The present study aimed to evaluate the analgesic and anti-inflammatory potential of angiotensin type 2 receptor (AT2R) antagonism in an acute gout attack mouse model. METHODS: Male wild-type (WT) C57BL/6 mice either with the AT2R antagonist, PD123319 (10 pmol/joint), or with vehicle injections, or AT2R KO mice, received intra-articular (IA) injection of monosodium urate (MSU) crystals (100 µg/joint), that induce the acute gout attack, and were tested for mechanical allodynia, thermal hyperalgesia, spontaneous nociception and ankle edema development at several times after the injections. To test an involvement of AT2R in joint pain, mice received an IA administration of angiotensin II (0.05-5 nmol/joint) with or without PD123319, and were also evaluated for pain and edema development. Ankle joint tissue samples from mice undergoing the above treatments were assessed for myeloperoxidase activity, IL-1ß release, mRNA expression analyses and nitrite/nitrate levels, 4 h after injections. RESULTS: AT2R antagonism has robust antinociceptive effects on mechanical allodynia (44% reduction) and spontaneous nociception (56%), as well as anti-inflammatory effects preventing edema formation (45%), reducing myeloperoxidase activity (54%) and IL-1ß levels (32%). Additionally, Agtr2tm1a mutant mice have largely reduced painful signs of gout. Angiotensin II administration causes pain and inflammation, which was prevented by AT2R antagonism, as observed in mechanical allodynia 4 h (100%), spontaneous nociception (46%), cold nociceptive response (54%), edema formation (83%), myeloperoxidase activity (48%), and IL-1ß levels (89%). PD123319 treatment also reduces NO concentrations (74%) and AT2R mRNA levels in comparison with MSU untreated mice. CONCLUSION: Our findings show that AT2R activation contributes to acute pain in experimental mouse models of gout. Therefore, the antagonism of AT2R may be a potential therapeutic option to manage gout arthritis.