IL-12p40 Expression Research Articles

Effects of Morphine on Gp120-induced Neuroinflammation Under Immunocompetent Vs. Immunodeficient Conditions.

HIV-associated neurocognitive disorder (HAND) is a common complication of HIV infection, whose development is known to be facilitated by inflammation and exacerbated by morphine. Previously, using the gp120 transgenic (tg) mouse model in combination with LP-BM5 (a murine retrovirus that can cause systemic immunodeficiency in susceptible mouse strains) we demonstrated differential gp120-associated central nervous system (CNS) neuroinflammatory responses under immunocompetent (-LP-BM5) vs. immunocompromised (+LP-BM5) conditions. Here, we further investigated the effects of morphine on gp120-associated neuroinflammatory response within the hippocampus under differential immune status. First, we confirmed that morphine treatment (2 × 25mg pellets) did not significantly affect the development of immunodeficiency induced by LP-BM5 and all brain regions examined (hippocampus, striatum, and frontal lobe) had detectable LP-BM5 viral gag genes. Morphine notably reduced the performance of gp120tg+ mice in the alteration T-maze assay when 2-minute retention was used, regardless of LP-BM5 treatment. Morphine further enhanced GFAP expression in gp120tg+ mice regardless of host immune status, while promoted CD11b expression only in immunocompetent mice, regardless of gp120tg expression. In immunocompetent gp120tg+ mice, morphine increased the RNA expression of CCL2, CCL5, CXCL10, IL-12p40, and IFNβ; while under the immunodeficient condition, morphine downregulated the expression of CCL2, CCL5, CXCL10, IL-12p40, and IL-1β. Further, expression of TNFα and IFNγ were enhanced by morphine regardless of host immune status. Altogether, our results suggest that the effects of morphine are complex and dependent on the immune status of the host, and host immune status-specific, targeted anti-neuroinflammatory strategies are required for effective treatment of HAND.

Cadmium and wild boar: Environmental exposure and immunological impact on macrophages

Cadmium (Cd2+) is regarded as one of the most toxic heavy metals, which can enter the food chain through environmental contamination and be bioaccumulated. Its exposure in Ligurian wild boars was monitored between 2016–2020 and revealed high level of this heavy metal in different provinces. In one of these polluted area, 21 wild boars were additionally sampled and the relationship between hepatic and renal Cd2+ concentration suggested that majority of these animals presented chronic intoxication. Cd2+ exposure of wild boar might lead to an immunosuppression status, thus in vitro experiments on wild boar monocyte-derived macrophages (moMФ) were carried out. Effects of Cd2+ scalar doses were evaluated through viability and adsorption assays, ELISA, qPCR. Moderate doses of this environmental pollutant (20 μM) were absorbed by moMФ, with subsequent reduction of their viability. This heavy metal did not trigger release of either IFN- β, anti-inflammatory or pro-inflammatory cytokines by moMФ, instead 24 h treatment with 20 μM of Cd2+ resulted in down-regulated expression of TNF-α, IL-12p40, several TLRs, CD14, MD2, BD2, MyD88, p65, and NOS2. The results of our monitoring activity suggested that wild boar can be useful to monitor environmental exposure of this heavy metal and can help in understanding the type of contamination. In addition, in vitro experiments on wild boar moMФ revealed that Cd2+ exposure negatively affected the immune function of these cells, likely leading to increased susceptibility to infection.

Expression and variation of serum cytokines in mouse model with different types of bacteremia

Abstract To investigate the expression and variation of MIP-1β, MIP-2, IL-12p40 and IL-12p70 in mice with bloodstream infection caused by four types of bacteria. ICR mouse models of bloodstream infection with Staphylococcus aureus (Sa), Enterococcus faecalis (Ef), Escherichia coli (Ec) and Klebsiella pneumonia (Kp) were established. Mice were infected by bacteria for 0.5h, 1h, 3h, 6h, 12h, 24h and 48h, while mice in control group was injected with PBS. MIP-1β, MIP-2, IL-12p40 and IL-12p70 in serum were detected by Luminex xMAP assay and were compared among different groups at different time points. MIP-1β increased significantly after bacteria were injected for each time point (p < 0.05). MIP-1β and MIP-2 in Ec and Kp groups were significantly higher than those in Sa and Ef groups (p < 0.01). IL-12p40 peaked at 3h after injection and remained at high level for12h in four groups. IL-12p70 in Sa group increased significantly and reached the maximum value 6h after infection, while in the other three groups, it increased significantly 3h after infection (P < 0.05).

Abstract P006: Intratumoral electroporation of IL-12 and SARS-Cov-2 spike plasmids drives a coordinated vaccine response and elicits robust anti-tumor immunity

Abstract Despite extensive clinical evidence on the efficacy and safety of SARS-CoV-2 vaccines, there remains a paucity of data on their effectiveness in cancer patients who are actively receiving antineoplastic therapeutics. A recent study demonstrated only ~30% of cancer patients had positive serologic test following 2 doses of FDA-authorized SARS-CoV-2 vaccines, in contrast to ~80% positivity rate in healthy individuals, regardless of the age. Therefore, further investigation into novel approaches to boost immune response to SARS-CoV-2 vaccines in cancer patients is required. Our previous preclinical and clinical studies have established intratumoral IL-12 plasmid (TAVO) electroporation (EP) induces localized expression of IL-12p70, converting immune-excluded tumors into inflamed immunogenic lesions, thereby generating objective responses in both treated and untreated, distant tumors. Based on the enhancement of immunotherapy efficacy by IL-12, we leveraged the flexibility of our DNA plasmid-EP platform to express SARS-CoV-2 spike protein in addition to IL-12 (CORVax12) as an intratumoral vaccine candidate which we hypothesized could not only drive anti-SARS-CoV-2 immune responses but also generate a productive anti-tumor response. Naïve mice were vaccinated via intradermal injection of SARS-CoV-2 spike plasmid followed immediately by EP with or without plasmid-encoded mIL-12 on days 1 and 21. Longitudinal serum samples were collected to interrogate virus-specific cellular responses as well anti-spike IgG antibody. A surrogate viral neutralization test (sVNT) assessed serum blockade of soluble human ACE2 binding to immobilized SARS-CoV-2 spike. Our data demonstrated that intradermally electroporated CORVax12 elicits significantly higher anti-SARS-CoV-2 spike IgG antibodies and neutralization when compared with EP of SARS-CoV-2 spike alone. Next, we asked if improved SARS-CoV-2 immune response may be observed when CORVax12 is incorporated into intratumoral EP in single-tumor bearing mice. CORVax12 robustly inhibited tumor growth, induced high percentages of germinal-center B cells and class switched B cells in tumor draining lymph nodes, and generated high of anti-spike IgG and neutralization antibodies. To further investigate systemic effects of this combination, we continued with contralateral tumor mice models. In both CT26 and B16-F10 tumor models, CORVax12 intratumoral EP induced strong systemic anti-tumor responses similar to IL-12 EP alone while also producing high serum levels of anti-SARS-CoV-2 spike IgG and neutralization antibodies. Critically, this anti-viral immunity did not limit this IL-12-based intratumoral anti-tumor therapy. In summary, our preclinical data indicates that intratumoral EP of CORVax12 can induce IgG responses to SARS-CoV-2 spike as well as apparent viral neutralizing activity all while maintaining local and systemic anti-tumor effects expected from TAVO Treatment. This combined intratumoral therapy represents a novel strategy to address both tumor burden and anti-SARS-CoV-2 immunity in patients with cancer. Citation Format: Mia Han, Jack Y. Lee, Vincent Wu, Kurt Sakurada, Bianca Nguyen, David A. Canton, Christopher G. Twitty. Intratumoral electroporation of IL-12 and SARS-Cov-2 spike plasmids drives a coordinated vaccine response and elicits robust anti-tumor immunity [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P006.

Impact of intrarectal chromofungin treatment on dendritic cells-related markers in different immune compartments in colonic inflammatory conditions.

BACKGROUNDChromofungin (CHR: chromogranin-A 47-66) is a chromogranin-A derived peptide with anti-inflammatory and anti-microbial properties. Ulcerative colitis (UC) is characterized by a colonic decrease of CHR and a dysregulation of dendritic CD11c+ cells.AIMTo investigate the association between CHR treatment and dendritic cells (DCs)-related markers in different immune compartments in colitis.METHODSA model of acute UC-like colitis using dextran sulphate sodium (DSS) was used in addition to biopsies collected from UC patients.RESULTSIntrarectal CHR treatment reduced the severity of DSS-induced colitis and was associated with a significant decrease in the expression of CD11c, CD40, CD80, CD86 and interleukin (IL)-12p40 in the inflamed colonic mucosa and CD11c, CD80, CD86 IL-6 and IL-12p40 within the mesenteric lymph nodes and the spleen. Furthermore, CHR treatment decreased CD80 and CD86 expression markers of splenic CD11c+ cells and decreased NF-κB expression in the colon and of splenic CD11c+ cells. In vitro, CHR decreased CD40, CD80, CD86 IL-6 and IL-12p40 expression in naïve bone marrow-derived CD11c+ DCs stimulated with lipopolysaccharide. Pharmacological studies demonstrated an impact of CHR on the NF-κB pathway. In patients with active UC, CHR level was reduced and showed a negative linear relationship with CD11c and CD86.CONCLUSIONCHR has protective properties against intestinal inflammation via the regulation of DC-related markers and CD11c+ cells. CHR could be a potential therapy of UC.

KDP, a Lactobacilli Product from Kimchi, Enhances Mucosal Immunity by Increasing Secretory IgA in Mice and Exhibits Antimicrobial Activity.

The potential of KDP, a lactic acid bacterial strain of Lactobacillus sakei, to enhance the production of mucosal specific immunoglobulin A (IgA) in mice and thereby enhance gut mucosal immunity was examined. KDP is composed of dead cells isolated from the Korean traditional food kimchi. Female BALB/c mice orally received 0.25 mg KDP once daily for 5 weeks and were co-administrated ovalbumin (OVA) for negative control and cholera toxin for positive control. Mice administered KDP exhibited increased secretory IgA (sIgA) contents in the small intestine, Peyer’s patches, serum, colon, and lungs as examined by ELISA. KDP also significantly increased the gene expression of Bcl-6, IL-10, IL-12p40, IL-21, and STAT4. In addition, KDP acted as a potent antioxidant, as indicated by its significant inhibitory effects in the range of 16.5–59.4% for DPPH, nitric oxide, maximum total antioxidant capacity, and maximum reducing power. Finally, KDP exhibited potent antimicrobial activity as evidenced by a significant decrease in the growth of 7 samples of gram-negative and gram-positive bacteria and Candida albicans. KDP’s adjuvant effect is shown to be comparable to that of cholera toxin. We conclude that KDP can significantly enhance the intestine’s secretory immunity to OVA, as well as act as a potent antioxidant and antimicrobial agent. These results suggest that orally administered KDP should be studied in clinical trials for antigen-specific IgA production.

Immunomodulatory Responses of Two Synthetic Peptides against Salmonella Typhimurium Infection.

In vitro assays of phagocytic activity showed that the peptide Pin2[G] stimulates phagocytosis in BMDM cells from 0.15 to 1.25 μg/mL, and in RAW 264.7 cells at 0.31 μg/mL. In the same way, the peptide FA1 induced phagocytosis in BMDM cells from 1.17 to 4.69 μg/mL and in RAW 264.7 cells at 150 μg/mL. Cytokine profiles of uninfected RAW 264.7 showed that Pin2[G] increased liberation TNF (from 1.25 to 10 μg/mL) and MCP-1 (10 μg/mL), and FA1 also increased the release of TNF (from 18.75 to 75 μg/mL) but did not increase the liberation of MCP-1. In RAW 264.7 macrophages infected with Salmonella enterica serovar Typhimurium, the expression of TNF increases with Pin2[G] (1.25–10 μg/mL) or FA1 (18.75–75 μg/mL). In these cells, FA1 also increases the expression of IL-12p70, IL-10 and IFN-γ when applied at concentrations of 37.5, 75 and 150 μg/mL, respectively. On the other hand, stimulation with 1.25 and 10 μg/mL of Pin2[G] promotes the expression of MCP-1 and IL-12p70, respectively. Finally, peptides treatment did not resolve murine gastric infection, but improves their physical condition. Cytokine profiles showed that FA1 reduces IFN-γ and MCP-1 but increases IL-10, while Pin2[G] reduces IFN-γ but increases the liberation of IL-6 and IL-12p70. This data suggests a promising activity of FA1 and Pin2[G] as immunomodulators of gastric infections in S. Typhimurium.

​Expression Profiling of Cytokine-related Genes in the Small Intestine of Healthy Pre and Post-weaned Piglets Administered Orally with Probiotic and Zinc

Background: The present study was designed to evaluate alterations in the immunological status of the small intestine after dietary inclusion of probiotic and zinc in pre and post-weaned piglets. Methods: For the present study, 18 healthy Large White Yorkshire (LWY) piglets, irrespective of sex, were divided into three different age groups [pre-weaning (20 days old, n = 3), weaning (30 days old, n = 3) and post-weaning (60 days old, n = 3)]. The piglets were weaned at 28 days of age. They were divided into control group (C) fed with basal diet and treatment group (T) fed with combined probiotic and zinc oral supplement along with the basal diet. A probiotic mixture consisted of Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium longum (1.25x109cfu/d for 10 days) along with zinc supplement (ZnO @ 2000 ppm/d for 10 days) administered orally to the treatment group of piglets. Result: The relative expression of TNF-α, IL-1β and IL-6 genes was significantly higher (P less than 0.05) in the treatment group of piglets as compared to the control group. However, the expression of IL-8, IL-12p40 and IL-18 genes were significantly lower (P less than 0.05) after oral administration of probiotic and zinc to the piglets.

A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

SummaryTurning non-inflamed (cold) tumors into inflamed (hot) tumors is important for maximizing the effect of immune checkpoint inhibitors (ICIs) against malignancies. We showed that lactate, a product of the Warburg effect, inhibited the efficacy of ICIs and suppressed IL-12 p40 expression in dendritic cells (DCs) through reducing NF-κB p65, p50, and c-Rel DNA-binding activity to the IL-12 p40 promoter. Additionally, lactate promoted the expression of early growth response protein 1 (EGR1), whose expression was increased in human invasive melanoma compared with non-invasive melanoma. We also found that EGR1 interacts with serum response factor (SRF) and represses the expression of CD80 in DCs. These findings suggest that lactate and its induced EGR1 are key factors that turn hot tumors into cold tumors and may represent targets in cancer treatment with ICIs.

Early Immune Initiation by Porcine Cells following Toxoplasma gondii Infection versus TLR Ligation.

Containment of acute Toxoplasma gondii infection is dependent on an efficient interferon gamma response. However, the earliest steps of immune response initiation immediately following exposure to the parasite have not been previously characterized in pigs. Murine and human myeloid cells produce large quantities of interleukin (IL)-12 during early T. gondii infection. We therefore examined IL-12 expression by porcine peripheral blood monocytes and dendritic cell (DC) subsets following toll-like receptor (TLR) ligation and controlled T. gondii tachyzoite infection. We detected IL-12p40 expression by porcine plasmacytoid DC, but not conventional or monocyte-derived DC following TLR ligation. Unexpectedly, we also observed considerable IL-12p40 production by porcine CD3– NKp46+ cells—a classical natural killer cell phenotype—following TLR ligation. However, in response to T. gondii exposure, no IL-12 production was observed by either DC or CD3– NKp46+ cells. Despite this, IL-18 production by DC-enriched peripheral blood mononuclear cells was detected following live T. gondii tachyzoite exposure. Only combined stimulation of porcine peripheral blood mononuclear cells with recombinant IL-12p70 and IL-18 induced innate interferon gamma production by natural killer cells, while T cells and myeloid cells did not respond. Therefore, porcine CD3– NKp46+ cells serve as important IL-12 producers following TLR ligation, while IL-18 likely plays a prominent role in early immune response initiation in the pig following T. gondii infection.

Interleukin 23 Produced by Hepatic Monocyte-Derived Macrophages Is Essential for the Development of Murine Primary Biliary Cholangitis.

Background and AimsPrimary Biliary Cholangitis (PBC) is an organ-specific autoimmune liver disease. Mononuclear phagocytes (MNPs), comprise of monocyte, dendritic cells and monocyte-derived macrophages, constitute major arm of the innate immune system known to be involved in the pathogenesis of autoimmune disorders. MNPs were shown to accumulate around intra-hepatic bile ducts in livers of PBC patients. Interleukin 23 (IL-23) is a pro-inflammatory cytokine. IL-23-positive cells were detected in livers of patients with advanced stage PBC and IL-23 serum levels found to be in correlation with PBC disease severity. Our overall goal was to assess the importance of IL-23 derived from MNPs in PBC pathogenesis.MethodsWe utilized an inducible murine model of PBC and took advantage of transgenic mice targeting expression of IL-23 by specific MNP populations. Analysis included liver histology assessment, flow cytometry of hepatic immune cells and hepatic cytokine profile evaluation. Specific MNPs sub-populations were sorted and assessed for IL-23 expression levels.ResultsFlow cytometry analysis of non-parenchymal liver cells in autoimmune cholangitis revealed massive infiltration of the liver by MNPs and neutrophils and a decrease in Kupffer cells numbers. In addition, a 4-fold increase in the incidence of hepatic IL-17A producing CD4+ T cells was found to be associated with an increase in hepatic IL23-p19 and IL17A expression levels. Disease severity was significantly ameliorated in both CD11ccreP19flox/flox and CX3CR1creP19 flox/flox mice as assessed by reduced portal inflammation and decreased hepatic expression of various inflammatory cytokines. Amelioration of disease severity was associated with reduction in IL-17A producing CD4+ T cells percentages and decreased hepatic IL23-p19 and IL17A expression levels. qRT-PCR analysis of sorted hepatic MNPs demonstrated high expression levels of IL-23 mRNA specifically by CX3CR1hiCD11c+ monocyte-derived macrophages.ConclusionOur results indicate a major role for IL-23 produced by hepatic monocyte-derived macrophages in the pathogenesis of PBC. These results may pave the road for the development of new immune-based and cell specific therapeutic modalities for PBC patients not responding to current therapies.

The effects of exercise training on the lungs and cardiovascular function of animals exposed to diesel exhaust particles and gases

Air pollution has been identified as one of the main environmental risks to health. Since exercise training seems to act as an anti-inflammatory modulator, our hypothesis is that exercise training prevents damage to respiratory and cardiovascular function caused by diesel exhaust particle (DEP) exposure. This study aimed to evaluate whether aerobic exercise training prior to DEP exposure prevents inflammatory processes in the pulmonary and cardiovascular systems. Therefore, BALB/C male mice were or were not submitted to a 10-week exercise training protocol (5×/week, 1 h/d), and after four weeks, they were exposed to DEP in a chamber with 24 μg/m3 PM2.5 or filtered air. Heart rate variability, lung mechanics and bronchoalveolar lavage fluid, cytokines and polymorphonuclear cells in the lung parenchyma were evaluated. Exposure to DEPs reduced heart rate variability and the elastance of the respiratory system and increased the number of cells in bronchoalveolar lavage fluid, as well as macrophages, neutrophils and lymphocytes, the density of polymorphonuclear cells and the proportion of collagen fibres in the lung parenchyma. Additionally, DEP-exposed animals showed increased expression of IL-23 and IL-12p40 (proinflammatory cytokines) and inducible nitric oxide synthase. Exercise training avoided the increases in all these inflammatory parameters, except the elastance of the respiratory system, the amount of collagen fibres and the expression of inducible nitric oxide synthase. Additionally, trained animals showed increased expression of the anti-inflammatory cytokine IL-1ra. Although our data showed a reduction in proinflammatory markers and an increase in markers of the anti-inflammatory pathway, these changes were not sufficient to prevent damage to the lung and cardiovascular function induced by DEPs. Based on these data, we propose that aerobic exercise training prevents the lung inflammatory process induced by DEPs, although it was not sufficient to avoid chronic damage, such as a loss of lung function or cardiovascular events.

Investigation of Newly Diagnosed Drug-Naive Patients with Systemic Autoimmune Diseases Revealed the Cleaved Peptide Tyrosine Tyrosine (PYY 3-36) as a Specific Plasma Biomarker of Rheumatoid Arthritis.

There is a current imperative to reveal more precisely the molecular pathways of early onset of systemic autoimmune diseases (SADs). The investigation of newly diagnosed drug-naive SAD patients might contribute to identify novel disease-specific and prognostic markers. The multiplex analysis of 30 plasma proteins in 60 newly diagnosed drug-naive SADs, such as RA (rheumatoid arthritis, n = 31), SLE (systemic lupus erythematosus, n = 19), and SSc (systemic scleroderma, n = 10) patients, versus healthy controls (HCs, n = 40) was addressed. Thirty plasma cytokines were quantified using the Procarta Plex™ panel. The higher expression of IL-12p40, IL-10, IL-13, IFN-γ, M-CSF, IL-4, NTproBNP, IL-17A, BMP-9, PYY (3-36), GITRL, MMP-12, and TNFRSF6 was associated with RA; IL-12p40, M-CSF, IL-4, GITRL, and NTproBNP were higher in SLE; or NTproBNP, PYY (3-36), and MMP-12 were increased in SSc over HCs, respectively. The cleaved peptide tyrosine tyrosine (PYY 3-36) was elevated in RA (361.6 ± 47.7 pg/ml) vs. HCs (163.96 ± 14.5 pg/ml, mean ± SEM, ∗∗∗p = 4 × 10−5). The CI (95%) was 268.05-455.16 pg/ml for RA vs. 135.55-192.37 pg/ml for HCs. The elevated PYY (3-36) level correlated significantly with the increased IL-4 or GITRL concentration but not with the clinical scores (DAS28, CRP, ESR, RF, aMCV). We are the first to report cleaved PYY (3-36) as a specific plasma marker of therapy-naive RA. Additionally, the multiplex plasma protein analysis supported a disease-specific cytokine pattern in RA, SLE, and SSc, respectively.

Effect of chemically modified tetracycline-8 (CMT-8) on hematology, blood chemistry, cytokines and peripheral blood lymphocyte subsets of healthy dogs

Tetracyclines are antibiotics widely used in human and veterinary medicine. Effects on the immune system and inflammatory response, including effects on blood leukocytes proliferation and function and in cytokines synthesis, have been described. Chemically modified tetracyclines (CMT) have lost their antimicrobial activity, but maintain these other properties. This study analyzes the effect of chemically modified tetracycline-8 (CMT-8) on the evolution of complete blood count, blood chemistry, the mRNA expression of selected cytokines and peripheral blood lymphocyte subpopulations distribution in healthy dogs. CMT-8 at a dose of 10 mg/kg once daily was administered per os to six healthy dogs. A control group of five healthy dogs, living in the same conditions than dogs treated with CMT-8, received placebo with an identical therapeutic regimen. When given at the doses used in this study, no side effects of CMT-8 were detected, suggesting a good tolerance and a limited toxicity of the drug. Dogs treated with CMT-8 showed a gradual increase in mean corpuscular hemoglobin. The administration of CMT-8 in healthy dogs did not affect blood mRNA expression of IFN-γ, TNFα, IL-4, IL-6, IL-10, IL-12 p40 and IL-13. However, the lymphocytes expressing class II MHC on their surface decreased during the first two weeks of CMT-8 treatment and subsequently increased for the next three months. Considering the absence of antimicrobial properties of the drug, the effects of CMT-8 detected in this study seem to be unrelated to the classical antimicrobial activity attributed to tetracyclines.

Rv3722c Promotes Mycobacterium tuberculosis Survival in Macrophages by Interacting With TRAF3.

Mycobacterium tuberculosis (M.tb) secretes numerous proteins to interfere with host immune response for its long-term survival. As one of the top abundant M.tb secreted proteins, Rv3722c was found to be essential for bacilli growth. However, it remains elusive how this protein interferes with the host immune response and regulates M.tb survival. Here, we confirmed that Rv3722c interacted with host TRAF3 to promote M.tb replication in macrophages. Knock-down of TRAF3 attenuated the effect of Rv3722c on the intracellular M.tb survival. The interaction between Rv3722c and TRAF3 hampered MAPK and NF-κB pathways, resulting in a significant increase of IFN-β expression and decrease of IL-1β, IL-6, IL-12p40, and TNF-α expression. Our study revealed that Rv3722c interacted with TRAF3 and interrupted its downstream pathways to promote M.tb survival in macrophages. These findings facilitate further understanding of the mechanism of M.tb secreted proteins in regulating the host cell immune response and promoting its intracellular survival.

Down-Regulation of LOC645166 in T Cells of Ankylosing Spondylitis Patients Promotes the NF-κB Signaling via Decreasingly Blocking Recruitment of the IKK Complex to K63-Linked Polyubiquitin Chains.

Ankylosing spondylitis (AS) is a chronic inflammatory disease that mainly affects the spine. AS is highly associated with the expression of HLA-B27. Up to 95% AS patients are HLA-B27-positive. However, only 1%–2% of the HLA-B27-positive carriers suffer from AS, implying that other factors may also govern the development of AS. Long non-coding RNAs (lncRNAs) can regulate the immune response via their binding proteins. In the present study, we have identified that the levels of lncRNA, LOC645166, in T cells of AS patients were reduced. Overexpression of LOC645166 in Jurkat cells down-regulated the IL-23p19 expression and suppressed the JAK2/STAT3 signaling in response to stimulation by phorbol 12-myristate 13-acetate. Suppression of STAT3 activation by LOC645166 was also observed when Jurkat cells or T cells of AS patient were treated with anti-CD3/CD28 antibodies. In order to explore the role of LOC645166 in the pathogenesis of AS, RNA pull-down assay plus proteomic approach and western blotting were performed and identified that LOC645166 prefers binding the K63-linked polyubiquitin chains. LOC645166 can suppress recruitment of the IKK complex to K63-linked polyubiquitin chains and diminish IKK2 activation, leading to down-regulation of NF-κB activation. Down-regulation of LOC645166 expression in T cells of AS patients up-regulates NF-kB activation via decreasingly impeding recruitment of the IKK complex to K63-linked polyubiquitin chains, allowing AS patients to exhibit more sensitivity to stimulation by the proinflammatory cytokines or by TLR ligands.

Extensive Histopathological Characterization of Inflamed Bowel in the Dextran Sulfate Sodium Mouse Model with Emphasis on Clinically Relevant Biomarkers and Targets for Drug Development.

This study aims to develop a reliable and reproducible inflammatory bowel disease (IBD) murine model based on a careful spatial–temporal histological characterization. Secondary aims included extensive preclinical studies focused on the in situ expression of clinically relevant biomarkers and targets involved in IBD. C57BL/6 female mice were used to establish the IBD model. Colitis was induced by the oral administration of 2% Dextran Sulfate Sodium (DSS) for 5 days, followed by 2, 4 or 9 days of water. Histological analysis was performed by sectioning the whole colon into rings of 5 mm each. Immunohistochemical analyses were performed for molecular targets of interest for monitoring disease activity, treatment response and predicting outcome. Data reported here allowed us to develop an original scoring method useful as a tool for the histological assessment of preclinical models of DSS-induced IBD. Immunohistochemical data showed a significant increase in TNF-α, α4β7, VEGFRII, GR-1, CD25, CD3 and IL-12p40 expression in DSS mice if compared to controls. No difference was observed for IL-17, IL-23R, IL-36R or F480. Knowledge of the spatial–temporal pattern distribution of the pathological lesions of a well-characterized disease model lays the foundation for the study of the tissue expression of meaningful predictive biomarkers, thereby improving translational success rates of preclinical studies for a personalized management of IBD patients.

Colonic mucosal and cytobrush sample cytokine mRNA expression in canine inflammatory bowel disease and their correlation with disease activity, endoscopic and histopathologic score.

Canine inflammatory bowel disease (IBD) is a group of chronic gastrointestinal disorders, the pathogenesis of which remains elusive, but it possibly involves the interaction of the intestinal immune system with luminal microbiota and food-derived antigens. Mucosal cytokines profiles in canine IBD have been investigated mainly in small intestinal disease, while data on cytokine profiles in large intestinal IBD are limited. The objective of this study was to measure colonic mucosal and cytobrush sample messenger (m)RNA expression of interleukin (IL)-1β, IL-2, IL-12p40, IL-23p19, tumor necrosis factor-alpha (TNF-α) and chemokine C‐C motif ligand (CCL28) in dogs with IBD and healthy controls using quantitative real-time polymerase chain reaction (PCR), and assess their correlation with clinical disease activity, endoscopic and histopathologic score. Dogs with IBD had a significantly increased mRNA expression of IL-1β, IL-23p19 and CCL28 in the colonic mucosa, compared to healthy controls. None of the selected cytokines had significantly different mRNA expression in the colonic cytobrush samples between the two groups or between the colonic mucosa and cytobrush samples of dogs with IBD. Finally, there was a statistically significant correlation of clinical disease activity with endoscopic activity score and fibrosis and atrophy of the colonic mucosa in dogs with large intestinal IBD. IL-1β, IL-23p19 and CCL28 could play a role in the pathogenesis of canine large intestinal IBD. Colonic cytokine expression does not correlate with clinical disease activity and/or endoscopic score. However, clinical signs reflect the severity of endoscopic lesions.

Epicutaneous challenge with protease allergen requires its protease activity to recall TH2 and TH17/TH22 responses in mice pre-sensitized via distant skin

Epicutaneous exposure to allergenic proteins is an important sensitization route for skin diseases like protein contact dermatitis, immunologic contact urticaria, and atopic dermatitis. Environmental allergen sources such as house dust mites contain proteases, which are frequent allergens themselves. Here, the dependency of T-helper (TH) cell recall responses on allergen protease activity in the elicitation phase in mice pre-sensitized via distant skin was investigated. Repeated epicutaneous administration of a model protease allergen, i.e. papain, to the back skin of hairless mice induced skin inflammation, serum papain-specific IgE and TH2 and TH17 cytokine responses in the sensitization sites, and antigen-restimulated draining lymph node cells. In the papain-sensitized but not vehicle-treated mice, subsequent single challenge on the ear skin with papain, but not with protease inhibitor-treated papain, up-regulated the gene expression of TH2 and TH17/TH22 cytokines along with cytokines promoting these TH cytokine responses (TSLP, IL-33, IL-17C, and IL-23p19). Up-regulation of IL-17A gene expression and cells expressing RORγt occurred in the ear skin of the presensitized mice even before the challenge. In a reconstructed epidermal model with a three-dimensional culture of human keratinocytes, papain but not protease inhibitor-treated papain exhibited increasing transdermal permeability and stimulating the gene expression of TSLP, IL-17C, and IL-23p19. This study demonstrated that allergen protease activity contributed to the onset of cutaneous TH2 and TH17/TH22 recall responses on allergen re-encounter at sites distant from the original epicutaneous sensitization exposures. This finding suggested the contribution of protease-dependent barrier disruption and induction of keratinocyte-derived cytokines to the recall responses.

Fusobacterium nucleatum drives a pro-inflammatory intestinal microenvironment through metabolite receptor-dependent modulation of IL-17 expression

ABSTRACT The colorectal cancer (CRC)-associated microbiota creates a pro-tumorigenic intestinal milieu and shapes immune responses within the tumor microenvironment. However, how oncomicrobes – like Fusobacterium nucleatum, found in the oral cavity and associated with CRC tissues– affect these distinct aspects of tumorigenesis is difficult to parse. Herein, we found that neonatal inoculation of ApcMin/+ mice with F. nucleatum strain Fn7-1 circumvents technical barriers preventing its intestinal colonization, drives colonic Il17a expression prior to tumor formation, and potentiates intestinal tumorigenesis. Using gnotobiotic mice colonized with a minimal complexity microbiota (the altered Schaedler’s flora), we observed that intestinal Fn7-1 colonization increases colonic Th17 cell frequency and their IL-17A and IL-17F expression, along with a concurrent increase in colonic lamina propria Il23p19 expression. As Fn7-1 stably colonizes the intestinal tract in our models, we posited that microbial metabolites, specifically short-chain fatty acids (SCFA) that F. nucleatum abundantly produces in culture and, as we demonstrate, in the intestinal tract, might mediate part of its immunomodulatory effects in vivo. Supporting this hypothesis, we found that Fn7-1 did not alter RORγt+ CD4+T cell frequency in the absence of the SCFA receptor FFAR2. Taken together, our work suggests that F. nucleatum influences intestinal immunity by shaping Th17 responses in an FFAR2-dependent manner, although further studies are necessary to clarify the precise and multifaceted roles of FFAR2. The potential to increase intestinal Th17 responses is shared by another oncomicrobe, enterotoxigenic Bacteroides fragilis, highlighting a conserved pathway that could potentially be targeted to slow oncomicrobe-mediated CRC.