Induction Of Interleukin Research Articles

Escherichia coli isolated from food sources and irrigation water: A potential risk for causing intestinal dysfunction?

We have previously shown that diarrheagenic Escherichia coli (DEC) and non-DEC are prevalent in food sources and irrigation water in South Africa. Recent data suggest that an increased relative abundance of faecal Enterobacteriaceae is associated with poorer health outcomes among children in developing countries. Thus, exposure to non-DEC from environmental sources may incur adverse effects, although the mechanisms underlying these effects remain obscure. To further elucidate this phenomenon, we assayed non-DEC strains from environmental sources in South Africa for phenotypes that may be associated with intestinal dysfunction (ID). DEC strains were also used. The strains had previously been isolated from Producer Distributor Bulk Milk (PDBM), irrigated lettuce, street vendor coleslaw and irrigation water.In-vitro assays identified; biofilm formation (n = 38), extracellular polymeric substance (EPS) formation (n = 38), cytotoxic activity (n = 10), disruption of tight junctions and induction of Interleukin 8 (IL-8) on polarized T-84 cells (n = 20). The number of strains tested for each assay differed, depending on prior molecular and phenotypic characterization that signalled potential pathogenicity in-vitro. Subsequently, all strains having data points for all analyses were used to compute Principal Component Analysis (PCA) plot curves to infer potential associations amongst test strains and a standard DEC pathogenic strain (042).Biofilm formation on glass coverslips after strains were grown in nutrient-rich media (LB and DMEM-F12 + 0.5% D-Mannose) at 37 °C varied based on pathotype (DEC and non-DEC) and source of isolation (food, irrigation water, clinical) suggesting that pathotype and source isolation influence persistence within a defined environmental niche. Additionally, DEC isolated from irrigated lettuce had a significantly higher (p ≤ 0.05) propensity for biofilm formation in both media compared to all strains including DEC standard controls. This suggested the propensity for irrigated lettuce as a potential source of persistent pathogenic strains. Furthermore, all strains were able to form EPS suggesting the ability to form mature biofilms under conditions relevant for food processing (20–25 °C). Of the (60%, 6 out of 10) strains that showed cytotoxic activity, most (83%, 5 out of 6 strains) were non-DEC isolated from food sources many of which are consumed with minimal processing.Mean percentage reduction in initial TEER (a measure of intestinal disruption), did not significantly differ (p = 0.05) in all test strains from that observed in the standard DEC. Additionally, IL-8 induction from strains isolated from PDBM (139 pg/mL), irrigation water (231.93 pg/mL) and irrigated lettuce (152.98 pg/mL) was significantly higher (p ≤ 0.05) than in the commensal strain aafa. PCA categorized strains based on sources of isolation showed potential for use in source tracking especially when comparing many strains from various environmental sources. We show that non-DEC strains along the food chain possess characteristics that may lead to ID. Further investigations using a larger collection of strains may provide a clearer link to these reported observations that could be associated with the high diarrheal disease burden within the country, especially among infants.

Rg6, a rare ginsenoside, inhibits systemic inflammation through the induction of interleukin-10 and microRNA-146a

The immunobiological functions of Rg6, a rare ginsenoside from ginseng, have been largely unreported. In this paper, we demonstrate that Rg6 has a significant immunosuppressive function on Toll-like receptor (TLR) 4-induced systemic inflammatory responses. Rg6 was found to negatively regulate pro-inflammatory responses and severity in vivo, and thus induced recovery in mice with lipopolysaccharide (LPS)-induced septic shock and cecal ligation and puncture (CLP)-induced sepsis. Rg6 treatment also facilitated recovery in mice with LPS-induced lung damage via reduced neutrophil infiltration and tumor necrosis factor-α expression in lung tissues. Rg6 injection also downregulated pro-inflammatory cytokines and increased the levels of interleukin (IL)-10 in the serum of septic mice. Mechanistically, Rg6 did not induce TLR negative regulators, such as A20 and IRAK-M, in bone marrow-derived macrophages (BMDMs). Instead, addition of Rg6 to LPS-activated BMDMs augmented IL-10 expression, whereas it inhibited inflammatory signaling, such as by nuclear factor κB activation and mitogen-activated protein kinases. Furthermore, Rg6 significantly induced miR-146a, an operator miRNA for anti-inflammation, in BMDMs. Collectively, these data indicate that Rg6 inhibits inflammatory responses through the induction of IL-10 and miR-146a.

Cell-Type-Specific Interleukin 1 Receptor 1 Signaling in the Brain Regulates Distinct Neuroimmune Activities

Interleukin-1 (IL-1) signaling is important for multiple potentially pathogenic processes in the central nervous system (CNS), but the cell-type-specific roles of IL-1 signaling are unclear. We used a genetic knockin reporter system in mice to track and reciprocally delete or express IL-1 receptor 1 (IL-1R1) in specific cell types, including endothelial cells, ventricular cells, peripheral myeloid cells, microglia, astrocytes, and neurons. We found that endothelial IL-1R1 was necessary and sufficient for mediating sickness behavior and drove leukocyte recruitment to the CNS and impaired neurogenesis, whereas ventricular IL-1R1 was critical for monocyte recruitment to the CNS. Although microglia did not express IL-1R1, IL-1 stimulation of endothelial cells led to the induction of IL-1 in microglia. Together, these findings describe the structure and functions of the brain's IL-1R1-expressing system and lay a foundation for the dissection and identification of IL-1R1 signaling pathways in the pathogenesis of CNS diseases.

Secretory immunoglobulin A induces human lung fibroblasts to produce inflammatory cytokines and undergo activation.

Immunoglobulin (Ig)A is the most abundant immunoglobulin in humans, and in the airway mucosa secretory IgA (sIgA) plays a pivotal role in first-line defense against invading pathogens and antigens. IgA has been reported to also have pathogenic effects, including possible worsening of the prognosis of idiopathic pulmonary fibrosis (IPF). However, the precise effects of IgA on lung fibroblasts remain unclear, and we aimed to elucidate how IgA activates human lung fibroblasts. We found that sIgA, but not monomeric IgA (mIgA), induced interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production by normal human lung fibroblasts (NHLFs) at both the protein and mRNA levels. sIgA also promoted proliferation of NHLFs and collagen gel contraction comparable to with transforming growth factor (TGF)-β, which is involved in fibrogenesis in IPF. Also, Western blot analysis and real-time quantitative polymerase chain reaction (PCR) revealed that sIgA enhanced production of α-smooth muscle actin (α-SMA) and collagen type I (Col I) by NHLFs. Flow cytometry showed that NHLFs bound sIgA, and among the known IgA receptors, NHLFs significantly expressed CD71 (transferrin receptor). Transfection of siRNA targeting CD71 partially but significantly suppressed cytokine production by NHLFs co-cultured with sIgA. Our findings suggest that sIgA may promote human lung inflammation and fibrosis by enhancing production of inflammatory or fibrogenic cytokines as well as extracellular matrix, inducing fibroblast differentiation into myofibroblasts and promoting human lung fibroblast proliferation. sIgA's enhancement of cytokine production may be due partially to its binding to CD71 or the secretory component.

Induction of interleukin 6 impairs the anti-HBV efficiency of IFN-α in human hepatocytes through upregulation of SOCS3.

Interleukin 6 (IL-6) is a pleiotropic cytokine with pivotal functions in the regulation of the biological responses of several target cells, including hepatocytes. Previous studies have shown that serum IL-6 levels are increased in hepatitis B patients. However, the role of IL-6 in modulating the anti-hepatitis B virus (HBV) activity of interferon-α (IFN-α) remains unclear. In this study, we found that both HBV and viral proteins could induce the expression of IL-6 in hepatocytes (LO2 and HepG2). Exogenous IL-6 had no effect on HBV replication, whereas knockdown of IL-6 expression by RNAi inhibited that. Interestingly, IFN-α markedly induced IL-6 expression in hepatocytes, especially in HBV replicating hepatocytes. In turn, IL-6 impaired the anti-HBV efficiency of IFN-α by decreases the expression of IFN-α downstream effectors by upregulation of suppressor of cytokine signaling-3 (SOCS3). Furthermore, we demonstrated that downregulation of SOCS3 improved IFN antiviral activity to some extent in HBV replicating hepatocytes. These data provided new insights for a better understanding of the mechanism of IFN-α resistance and may represent a novel therapeutic strategy to efficiently target HBV infection.

EBV LMP1 in Gingival Epithelium Potentially Contributes to Human Chronic Periodontitis via Inducible IL8 Production.

Human chronic periodontitis is a major health problem. Although some oral bacteria have been reported to be putative pathogens, Epstein-Barr virus (EBV) is reported to be associated with the progression of periodontitis. However, the role of EBV in the aetiology of periodontitis is unknown. Therefore, we investigated periodontal pathogenesis of EBV to confirm whether EBV-encoded latent membrane protein 1 (LMP1) induces Interleukin-8 (IL8) production in human gingival cells. Real-time polymerase chain reaction, luciferase assay, enzyme-linked immunosorbent assay (ELISA), and western blotting were performed for determining IL8 mRNA expression, nuclear factor kappa B (NF-ĸB) transcription, IL8 production, and the phosphorylation of NF-ĸB p65 and Inhibitor of kappa B alpha (IĸBα), respectively, in Ca9-22 human gingival epithelial cells. Two LMP1 mutants lacking C-terminal activating region (CATR) domains responsible for activating NF-ĸB were used. Extremely high IL8 production was induced by LMP1 in time- and dose-dependent manner, where simultaneous phosphorylation of NF-κB p65 and IĸBα and transcription of NF-ĸB were observed. On the contrary, IL8 production and NF-ĸB transcription were drastically inhibited by dominant negative mutant of IĸBα. Moreover, the LMP1 mutants failed to induce IL8 production. Our findings suggest that due to CATR domains, LMP1 contributes to the progression of periodontitis via IL8 production attributable to NF-ĸB activation.

Mesenchymal stromal cells from infants with simple polydactyly modulate immune responses more efficiently than adult mesenchymal stromal cells

Bone marrow–derived stromal cells or mesenchymal stromal cells (BMSCs or MSCs, as we will call them in this work) are multipotent progenitor cells that can differentiate into osteoblasts, adipocytes and chondrocytes. In addition, MSCs have been shown to modulate the function of a variety of immune cells. Donor age has been shown to affect the regenerative potential, differentiation, proliferation and anti-inflammatory potency of MSCs; however, the impact of donor age on their immunosuppressive activity is unknown. In this study, we evaluated the ability of MSCs derived from very young children and adults on T-cell suppression and cytokine secretion by monocytes/macrophages. MSCs were obtained from extra digits of children between 10 and 21 months and adults between 28 and 64 years of age. We studied cell surface marker expression, doubling time, lineage differentiation potential and immunosuppressive function of the MSCs. Young MSCs double more quickly and differentiate into bone and fat cells more efficiently than those from older donors. They also form more and dense colonies of fibroblasts (colony forming unit–fibroblast [CFU-F]). MSCs from both young and adult subjects suppressed T-cell proliferation in a mitogen-induced assay at 1:3 and 1:30 ratios. At a 1:30 ratio, however, MSCs from adults did not, but MSCs from infants did suppress T-cell proliferation. In the mixed lymphocyte reaction assay, MSCs from infants produced similar levels of suppression at all three MSC/T-cell ratios, but adult MSCs only inhibited T-cell proliferation at a 1:3 ratio. Cytokine analyses of co-cultures of MSCs and macrophages showed that both adult and young MSCs suppress tumor necrosis factor alpha (TNF-α) and induce interleukin-10 (IL-10) production in macrophage co-culture assay in a similar manner. Overall, this work shows that developing MSCs display a higher level of immunosuppression than mature MSCs.

Elevated Notch1 enhances interleukin-22 production by CD4+ T cells via aryl hydrocarbon receptor in patients with lung adenocarcinoma.

Notch signaling induced interleukin (IL)-22 secretion by CD4+ T cells via retinoid-related orphan nuclear receptor γt (RORγt) or aryl hydrocarbon receptor (AhR). Previous studies have demonstrated that Notch-AhR-IL-22 axis took part in the pathogenesis of chronic viral infection, however, its role in cancer has not been fully elucidated. Thus, the aim of current study was to investigate the involvement of Notch-AhR-IL-22 axis in the pathogenesis of lung adenocarcinoma. A total of 37 late-stage lung adenocarcinoma patients and 17 healthy individuals were enrolled. CD4+ T cells were purified from peripheral bloods and bronchoalveolar lavage fluids (BALF), and were stimulated with γ-secretase inhibitor (GSI). mRNA corresponding to Notch receptors and transcriptional factors were measured by real-time PCR. IL-22 concentration was investigated by ELISA. The bioactivity (including cellular proliferation, cell cycle, apoptosis, and invasion) of lung adenocarcinoma cell line A549 was also assessed in response to recombinant IL-22 stimulation in vitro. Notch1 mRNA expression was significantly elevated in CD4+ T cells purified from peripheral bloods and tumor site BALF in lung adenocarcinoma patients. IL-22 expression and RORγt/AhR mRNA in BALF was also remarkably increased in tumor site. Inhibition of Notch signaling by GSI did not affect cellular proliferation, but reduced IL-22 production in CD4+ T cells from BALF, along with down-regulation of AhR, but not RORγt. Moreover, IL-22 stimulation promoted A549 cells invasion. The current data indicated that elevated Notch1 induced higher IL-22 secretion by CD4+ T cells in lung adenocarcinoma patients, and Notch-AhR-IL-22 axis took part in the pathogenesis of lung adenocarcinoma.

Angiotensin II Causes Biphasic STAT3 Activation Through TLR4 to Initiate Cardiac Remodeling

Evidence indicates that Ang II (angiotensin II) activates STAT3 (signal transducer and activator of transcription 3) in cardiomyocytes. However, the mechanisms underlying STAT3 activation and downstream responses are not fully known. In this study, we show that Ang II caused biphasic STAT3 activation in cardiomyocytes. A rapid and early activation was mediated by direct association between TLR4 (toll-like receptor-4) and STAT3. This early activation increased IL-6 (interleukin-6) production, which in turn, induced the second STAT3 activation through the IL-6/gp130 (glycoprotein 130)/JAK2 (Janus-family tyrosine kinases 2) pathway, resulting in unregulated expression of genes for cardiac remodeling. Moreover, STAT3 inhibition or TLR4 knockout in mice protected against Ang II-induced hypertrophy, fibrosis, and cardiac functional deficits. Thus, Ang II-induced STAT3 activation in cardiomyocytes was biphasic, providing a sequential induction of IL-6 and myocardial remodeling genes, respectively. This work supports a novel mechanism on STAT3 activation in Ang II-induced cardiac dysfunction and remodeling.

OK-432 Administration Inhibits Murine Allergic Rhinitis at the Induction Phase, through the Macrophage Activation with TLR2 Signaling Pathway.

OK-432, a preparation of a low-virulence strain (Su) of Streptococcus pyogenes (Group A) killed by a penicillin and lyophilized, is a stiff inducer of Th1 cytokines, and exerts anti-cancer effects in tumor-bearing mice. OK-432 has been reported to consist of many bacterial components, such as peptidoglycan, M-protein, etc. However, it is yet to be ascertained which bacterial component induces T helper 1 (Th1) responses. For the last decade, Toll-like receptor (TLR) family proteins are well elucidated to play a role in recognizing bacterial components and inducing interleukin (IL)-12 from macrophages. Above all, peptidoglycan seems to be the agonist of TLR2 rather than the obverse. In our present study, the role of TLR2 for the recognition of OK-432 by macrophages and the effects of OK-432 are examined on murine allergic rhinitis model. Interestingly, results show IL-12 production by macrophages derived from TLR2 knock-out (ko) mice was significantly decreased, in comparison with that of macrophages derived from wild-type mice. Moreover, in TLR2 ko mice, no regulatory effect of OK-432 was observed on an allergic rhinitis model. These data indicate that TLR2 signaling is involved in regulating OK-432-induced anti-T helper 2 (Th2) immunity, and may offer a new prophylactic and therapeutic approach using OK-432 to downregulate allergic disorders, such as allergic rhinitis.

IVIg and LPS Co-stimulation Induces IL-10 Production by Human Monocytes, Which Is Compromised by an FcγRIIA Disease-Associated Gene Variant.

Intravenous Immunoglobulin (IVIg) is used to treat autoimmune or inflammatory diseases, but its mechanism of action is not completely understood. We asked whether IVIg can induce interleukin-10 (IL-10) and reduce pro-inflammatory cytokine production in human monocytes, and whether this response is reduced in monocytes from people with an Fcγ receptor IIA (FcγRIIA) gene variant, which is associated with increased risk of inflammatory diseases and poor response to antibody-based biological therapy. IVIg increased IL-10 production and reduced pro-inflammatory cytokine production in response to bacterial lipopolysaccharide (LPS), which required FcγRI and FcγRIIB and activation of MAPKs, extracellular signal-regulated kinase 1/2 (ERK1/2), and p38. IL-10 production was lower and pro-inflammatory cytokine production was higher in monocytes from people with the FcγRIIA risk variant and the risk variant prevented IL-10 production in response to (IVIg+LPS). Finally, we show that IVIg did not induce MAPK activation in monocytes from people with the risk variant. Our results demonstrate that IVIg can skew human monocytes to an anti-inflammatory, IL-10-producing activation state, which is compromised in monocytes from people with the FcγRIIA risk variant. This research has profound implications for the use of IVIg because 25% of the population is homozygous for the FcγRIIA risk variant and its efficacy may be reduced in those individuals. In addition, this research may be useful to develop new therapeutic strategies to replace IVIg by cross-linking FcγRIs and FcγRIIBs to promote anti-inflammatory macrophage activation, independent of the FcγRIIA genotype.

Involvement of ERK1/2 activation in the gene expression of senescence-associated secretory factors in human hepatic stellate cells.

Senescent hepatic stellate cells (senescent HSCs) are found in patients with liver cirrhosis and have been thought to be involved in the development of hepatocellular carcinoma (HCC) in mice via the senescence-associated secretory proteins. However, in humans, which secretory proteins are involved and what regulate their expression remain unclear. In the current study, we characterized senescence-associated β-galactosidase-positive senescent human HSCs (hHSCs) induced by repetitive passaging. They exhibited enhanced expression of 14 genes for secretory protein and persistent phosphorylation of ERK1/2 protein but not JNK or p38 MAPK proteins. Enhanced nuclear ERK1/2 phosphorylation was observed in senescent hHSCs. Treatment of the senescent hHSCs with ERK1/2 inhibitor, SCH772984, significantly decreased the levels of angiopoietin like 4 (ANGPTL4), C-C motif chemokine ligand 7 (CCL7), Interleukin-8 (IL-8), platelet factor 4 variant 1 (PF4V1), and TNF superfamily member 15 (TNFSF15) mRNA levels in a dose-dependent manner. The enhanced phosphorylation of ERK1/2 and expression of ANGPTL4, IL-8 and PF4V1 genes were observed in both of senescent human dermal fibroblasts and X-ray-induced senescent hHSCs. However, transient ERK1/2 activation induced by epidermal growth factor could not mimic the gene profile of the senescent hHSCs. These results revealed involvement of ERK1/2 signaling in the regulation of senescence-associated secretory factors, suggesting that simultaneous induction of ANGPTL4, IL-8, and PF4V1 genes is a marker of hHSC senescence. This study will contribute to understanding roles of senescent hHSCs in liver diseases.

NF‑κB inhibitor DHMEQ inhibits titanium dioxide nanoparticle‑induced interleukin‑1β production: Inhibition of the PM2.5‑induced inflammation model.

PM2.5 is a particle with a diameter <2.5µm that is often involved in air pollution. Nanoparticles <100nm are thought to invade the trachea and lungs to cause inflammation, possibly through the activation of macrophages. On the other hand, titanium dioxide (TiO2) particles can be used in models of nano‑micro‑sized particles, as one can prepare the particles with such sizes. TiO2 particles are classified into Rutile, Anatase, and Brookite types by their crystal structure. Among them, Anatase‑type TiO2 particles with a primary diameter of 50nm (A50) were reported to induce interleukin (IL)‑1β production and secretion effectively in phorbol 12‑myristate 13‑acetate‑treated human monocytic leukemia THP‑1 cells (THP‑1 macrophages). We previously designed and synthesized dehydroxymethyl‑epoxyqinomicin (DHMEQ) as an inhibitor of NF‑κB. The present study investigated whether the NF‑κB inhibitor DHMEQ inhibits TiO2 nanoparticle‑induced IL‑1β production in THP‑1 macrophages, and determined the mechanism. As a result, DHMEQ inhibited A50‑induced IL‑1β secretion in ELISA assays at nontoxic concentrations. It decreased the expression of IL‑1β mRNA, which was dependent on NF‑κB. Although NLR family pyrin domain containing 3 (NLRP3)‑inflammasome‑caspase‑1 activation is required for the maturation of IL‑1β, and DHMEQ reduced the NLRP3 mRNA expression and caspase‑1 activity; a caspase‑1 inhibitor did not influence the A50‑induced IL‑1β production. Therefore, it is likely that inhibition of pro‑IL‑1β expression by DHMEQ may be sufficient to inhibit mature IL‑1β production. Thus, DHMEQ may be useful for the amelioration of inflammation in the trachea and lungs caused by inhalation of PM2.5.

Endoplasmic reticulum-mediated induction of interleukin-8 occurs by hepatitis B virus infection and contributes to suppression of interferon responsiveness in human hepatocytes

The events in the immune response to hepatitis B virus (HBV) remain unclear. We analyzed the direct influence of HBV on gene expression in human hepatocytes under immunodeficient conditions using a human hepatocyte chimeric mouse model. HBV-infected or non-infected chimeric mouse livers were collected, and gene expression profiles were compared. Since IL-8 was the most significantly up-regulated gene at 8 weeks after HBV infection, we focused on IL-8 and found that HBx and the large HBs (L-HBs) protein induce transcription of IL-8 via endoplasmic reticulum stress. This stress induces IL-8 transcription via NFAT activation and contributes to suppression of interferon responsiveness in HBV-infected human hepatocytes. In the present study, we identified a novel regulatory mechanism in which the L-HBs protein activates IL-8 via endoplasmic reticulum stress, suggesting a key role for IL-8 in the immune response to HBV and a potential new target for antiviral treatments of HBV infection.

Gamma-irradiation-killed Streptococcus pneumoniae potently induces the expression of IL-6 and IL-8 in human bronchial epithelial cells

Streptococcus pneumoniae is a major respiratory pathogen that can cause pneumonia, meningitis, and otitis media. Although capsular polysaccharide-based vaccines are commercially available, there is a need for broad-spectrum, serotype-independent, and cost-effective vaccines. Recently, an intranasal vaccine formulated with gamma-irradiated nonencapsulated S. pneumoniae whole cells has been developed and its immunogenicity is under investigation. Since innate immunity influences the subsequent adaptive immunity, in the present study, we investigated the immunostimulatory activity of gamma-irradiated S. pneumoniae (r-SP) in the human bronchial epithelial cell-line, BEAS-2B, by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP). r-SP potently induced interleukin (IL)-6 and IL-8 at both mRNA and protein levels in a dose- and time-dependent manner, whereas h-SP and f-SP poorly induced them. Of note, the mRNA levels of IL-6 and IL-8 were approximately two-fold higher when cells were stimulated with 3 × 107 CFU/ml of r-SP for 3 h, while the protein levels of IL-6 and IL-8 were approximately five-fold higher after stimulation with 3 × 107 CFU/ml of r-SP for 24 h. Furthermore, r-SP exhibited potent activation of Toll-like receptor 2 compared with h-SP or f-SP. The expression of IL-6 and IL-8 induced by r-SP was mediated through the activation of mitogen-activated protein kinases. Remarkably, when r-SP was further treated with heat or formalin, there was a decrease in the aforementioned activities. Taken together, we suggest that r-SP stimulates the human respiratory epithelial cells to produce the cytokines IL-6 and IL-8, which might influence the induction of adaptive immune responses.

Immune system-mediated atherosclerosis caused by deficiency of long non-coding RNAMALAT1in ApoE−/−mice

The immune system is considered a key driver of atherosclerosis, and beyond proteins and microRNAs (miRs), long non-coding RNAs (lncRNAs) are implicated in immune control. We previously described that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is involved in cardiac innate immunity in a myocarditis model. Here, we investigated the impact of MALAT1 deficiency upon atherosclerosis development. Heterozygous MALAT1-deficient ApoE-/- mice displayed massive immune system dysregulation and atherosclerosis within 2 months even when kept on normal diet. Aortic plaque area (P < 0.05) and aortic root plaque size (P < 0.001) were increased in MALAT1-deficient vs. MALAT1-wildtype ApoE-/- mice. Serum levels of interferon-γ (IFN-γ), tumour necrosis factor (TNF), and interleukin 6 (IL6) were elevated (P < 0.001) in MALAT1-deficient animals. MALAT1-deficient bone marrow-derived macrophages showed enhanced expression of TNF (P = 0.001) and inducible NO synthase (NOS2) (P = 0.002), suppressed MMP9 (P < 0.001), and impaired phagocytic activity (P < 0.001) upon lipopolysaccharide stimulation. RNA-sequencing revealed grossly altered transcriptomes of MALAT1-deficient splenocytes already at baseline, with massive induction of IFN- γ, TNF, NOS2, and granzyme B; CC and CXC chemokines and CCR8; and innate immunity genes interferon-induced protein with tetratricopeptide repeats (IFIT)1/3, interferon-induced transmembrane protein (IFITM)1/3, ISG15. Multiple miRs were up to 45-fold upregulated. Further, selective ablation of the cytosolic part of the MALAT1 system only, the enzymatically MALAT1-derived mascRNA, resulted in massive induction of TNF (P = 0.004) and IL6 (P = 0.028) in macrophages. Northern analysis of post-myocardial infarction patient vs. control peripheral blood mononuclear cells showed reduced (P = 0.005) mascRNA in the patients. CHART-enriched RNA-sequencing reads at the genomic loci of MALAT1 and neighbouring nuclear enriched abundant transcript (NEAT1) documented direct interaction between these lncRNA transcripts. The data suggest a molecular circuit involving the MALAT1-mascRNA system, interactions between MALAT1 and NEAT1, and key immune effector molecules, cumulatively impacting upon the development of atherosclerosis. It appears reasonable to look for therapeutic targets in this circuit and to screen for anomalies in the NEAT1-MALAT1 region in humans, too, as possible novel disease risk factors.

Calcimycin induced IL-12 production inhibits intracellular mycobacterial growth by enhancing autophagy

Previously, we reported pivotal role of P2RX7 in augmenting autophagy in THP-1 cells upon Calcimycin treatment by modulating intracellular Calcium regulated ATP production but the role of immune modulators in Calcimycin induced autophagy is not known. In this study, we demonstrate that treatment with Calcimycin in PMA (Phorbol 12-myristate 13-acetate) differentiated THP-1 (dTHP-1) cells significantly induced interleukin (IL)-12 mRNA expression and its release. IL-12 receptor (IL-12Rβ1 and IL-12Rβ2) was also significantly expressed on the cell surface in dTHP-1 cells upon Calcimycin treatment. We report that small molecule or siRNA based P2RX7 inhibition abrogated IL-12 release upon Calcimycin treatment. P2RX7 inhibition also resulted in reduced Jun N-terminal kinase (JNK) activation, IκBα phosphorylation, p65 translocation and NF-κB expression. Further, inhibition of NF-κB activation or IL-12-IL-12R interaction led to down-regulation of the expression of autophagy related markers such as Beclin-1, autophagy-related gene (Atg) 3, Atg 7 and impairment of microtubule-associated protein 1A/1B-light chain 3-I (LC3-I) to LC3-II conversion. Finally, blocking of autophagy led to significant growth of intracellular mycobacteria in Calcimycin treated macrophages. Overall, these results reveal that interaction of Calcimycin with P2RX7 modulates intracellular JNK-NF-κB signaling pathway. This modulation results in IL-12 release that restricts the mycobacterial growth in THP-1 macrophages.

Absence of IL-10 production by human PBMCs co-cultivated with human cells expressing or secreting retroviral immunosuppressive domains.

Immunosuppression by retroviruses including the human immunodeficiency virus—1 (HIV-1) is well known, however the mechanisms how retroviruses induce this immunosuppression is not fully investigated. It was shown that non-infectious retroviral particles as well as retroviral or recombinant retroviral transmembrane envelope (TM) proteins demonstrated immunosuppressive properties. The same was shown for peptides corresponding to a highly conserved domain in the TM protein. This domain is called immunosuppressive (ISU) domain and it induces modulation of the cytokine release of peripheral blood mononuclear cells (PBMCs) from healthy donors. In addition, it changes the gene expression of these cells. Common indications for the immunosuppressive activity were tumour growth in vivo and interleukin—10 (IL-10) release from human PBMCs in vitro. Single mutations in the ISU domain abrogated the immunosuppressive activity. In order to develop a new model system for the expression of the ISU domain and presentation to PBMCs which is not prone to possible endotoxin contaminations, two expression systems were developed. In the first system, designated pOUT, retroviral proteins containing the ISU domain were expressed and released into the cell culture medium, and in the second system, tANCHOR, the ISU domain was presented by a tetraspanin-anchored sequence on the cell surface of human cells. Both systems were exploited to express the wild-type (wt) ISU domains of HIV-1, of the porcine endogenous retrovirus (PERV) and of the murine leukaemia virus (MuLV) as well as to express mutants (mut) of these ISU domains. PERV is of special interest in the context of virus safety of xenotransplantation using pig organs. Expression of the TM proteins was demonstrated by confocal laser scanning microscopy, ELISA and Western blot analyses using specific antibodies. However, when cells expressing and releasing the ISU were co-incubated with human PBMCs, no increased production of IL-10 was observed when compared with the mutants. Similar results were obtained when the released TM proteins were concentrated by immunoprecipitation and added to PBMCs. We suggest that the absence of IL-10 induction can be explained by a low amount of protein, by the lack of a biologically active conformation or the absence of additional factors.

IL22 Promotes Kras-Mutant Lung Cancer by Induction of a Protumor Immune Response and Protection of Stemness Properties.

Somatic KRAS mutations are the most common oncogenic variants in lung cancer and are associated with poor prognosis. Using a Kras-induced lung cancer mouse model, CC-LR, we previously showed a role for inflammation in lung tumorigenesis through activation of the NF-κB pathway, along with induction of interleukin 6 (IL6) and an IL17-producing CD4+ T-helper cell response. IL22 is an effector molecule secreted by CD4+ and γδ T cells that we previously found to be expressed in CC-LR mice. IL22 mostly signals through the STAT3 pathway and is thought to act exclusively on nonhematopoietic cells with basal IL22 receptor (IL22R) expression on epithelial cells. Here, we found that higher expression of IL22R1 in patients with KRAS-mutant lung adenocarcinoma was an independent indicator of poor recurrence-free survival. We then showed that genetic ablation of Il22 in CC-LR mice (CC-LR/IL22KO mice) caused a significant reduction in tumor number and size. This was accompanied by significantly lower tumor cell proliferation, angiogenesis, and STAT3 activation. Il22 ablation was also associated with significant reduction in lung-infiltrating inflammatory cells and expression of protumor inflammatory cytokines. Conversely, this was accompanied with increased antitumor Th1 and cytotoxic CD8+ T-cell responses, while suppressing the protumor immunosuppressive T regulatory cell response. In CC-LR/IL22KO mice, we found significantly reduced expression of core stemness genes and the number of prototypical SPC+CCSP+ stem cells. Thus, we conclude that IL22 promotes Kras-mutant lung tumorigenesis by driving a protumor inflammatory microenvironment with proliferative, angiogenic, and stemness contextual cues in epithelial/tumor cells. Cancer Immunol Res; 6(7); 788-97. ©2018 AACR.

Adeno-associated Virus Vector-mediated Interleukin-10 Induction Prevents Vascular Inflammation in a Murine Model of Kawasaki Disease

Kawasaki disease (KD), which is the leading cause of pediatric heart disease, is characterized by coronary vasculitis and subsequent aneurysm formation. Although intravenous immunoglobulin therapy is effective for reducing aneurysm formation, a certain number of patients are resistant to this therapy. Because interleukin-10 (IL-10) was identified as a negative regulator of cardiac inflammation in a murine model of KD induced by Candida albicans water-soluble fraction (CAWS), we investigated the effect of IL-10 supplementation in CAWS-induced vasculitis. Mice were injected intramuscularly with adeno-associated virus (AAV) vector encoding IL-10, then treated with CAWS. The induction of AAV-mediated IL-10 (AAV-IL-10) significantly attenuated the vascular inflammation and fibrosis in the aortic root and coronary artery, resulting in the improvement of cardiac dysfunction and lethality. The predominant infiltrating inflammatory cells in the vascular walls were Dectin-2+CD11b+ macrophages. In vitro experiments revealed that granulocyte/macrophage colony-stimulating factor (GM-CSF) induced Dectin-2 expression in bone marrow-derived macrophages and enhanced the CAWS-induced production of tumor necrosis factor-α (TNF-α) and IL-6. IL-10 had no effect on the Dectin-2 expression but significantly inhibited the production of cytokines. IL-10 also inhibited CAWS-induced phosphorylation of ERK1/2, but not Syk. Furthermore, the induction of AAV-IL-10 prevented the expression of TNF-α and IL-6, but not GM-CSF and Dectin-2 at the early phase of CAWS-induced vasculitis. These findings demonstrate that AAV-IL-10 may have therapeutic application in the prevention of coronary vasculitis and aneurysm formation, and provide new insights into the mechanism underlying the pathogenesis of KD.