Upregulation Of Cytokine Production Research Articles

Selenopolysaccharide Isolated from Lentinula edodes Mycelium Affects Human T-Cell Function.

Lentinula edodes polysaccharides are natural immunomodulators. SeLe30, analyzed in this study, is a new mixture of selenium-enriched linear 1,4-α-glucans and 1,3-β- and 1,6-β-glucans isolated from L. edodes mycelium. In the present study, we evaluated its immunomodulatory properties in human T cells. Peripheral blood mononuclear cells (PBMCs) and T cells were isolated from healthy donors' buffy coats. The effects of SeLe30 on CD25, CD366, and CD279 expression, the subsets of CD8+ T cells, and IFN-γ, IL-6, and TNF-α production were analyzed. SeLe30 downregulated CD25, CD279, and CD366 expression on T cells stimulated by the anti-CD3 antibody (Ab) and upregulated in unstimulated and anti-CD3/CD28-Abs-stimulated T cells. It increased the percentage of central memory CD8+ T cells in unstimulated PBMCs and naïve and central memory T cells in anti-CD3-Ab-stimulated PBMCs. SeLe30 decreased the number of central memory and naïve CD8+ T cells in anti-CD3/CD28-stimulated T cells, whereas, in PBMCs, it reduced the percentage of effector memory CD8+ T cells. Moreover, SeLe30 upregulated cytokine production. SeLe30 exhibits context-dependent effects on T cells. It acts on unstimulated T cells, affecting their activation while increasing the expression of immune checkpoints, which sensitizes them to inhibitory signals that can silence this activation. In the case of a lack of costimulation, SeLe30 exhibits an inhibitory effect, reducing T-cell activation. In cells stimulated by dual signals, its effect is further enhanced, again increasing the "safety brake" of CD366 and CD279. However, the final SeLe30 effect is mediated by its indirect impacts by altering interactions with other immune cells.

Curcumin attenuates neuroinflammatory damage induced by LPS: Implications for the role of S100B

Inflammation is a common feature of neurological disorders that alters cell function in microglia and astrocytes as well as other neuronal cell types. Astrocytes modulate blood flow, regulate glutamate metabolism, and exert antioxidant protection. When responding to inflammatory damage, astrocytes enhance immune cell infiltration and amplify inflammatory responses via the upregulation of cytokine production. Several molecules have been proposed to attenuate neuroinflammation and control neurological diseases. Curcumin gained attention due to its capacity to cross the blood-brain barrier and its well-described anti-inflammatory and antioxidant activities. Our study aimed to understand if oral curcumin administration could protect against central nervous system inflammatory damage induced by intracerebroventricular injection of LPS while focusing on astrocyte function. Despite its poor bioavailability, we found that curcumin reaches the central nervous system, prevents the locomotory damage caused by LPS, and reduces inflammatory signaling via IL-1β and COX-2. Furthermore, we observed that curcumin was protective against LPS-induced S100B secretion in the cerebrospinal fluid and GSH reduction in the hippocampal tissue. However, curcumin could not protect the animals from anhedonia, assessed by the sucrose preference test, and weight loss induced by LPS. Our results indicate that oral curcumin administration exerts a protective anti-inflammatory action in the central nervous system, attenuating the sickness behavior induced by ICV LPS. This work demonstrates that curcumin has an important modulative effect on astrocytes, thus suggesting that astrocytes are critical to the anti-inflammatory effects of curcumin.

Gintonin upregulates cytokine production and expression of NKp30, NKp44 and NKp44 related to natural killer cell activity on immunosuppressive rat

The objective of the study is to estimate the potential of gintonin, as an immune enhancing agent through natural killer cell (NK cell) activity in cyclophosphamide (CY)-induced immunosuppressive animals. Accumulated results reveals that, gintonin attenuated CY-induced immunosuppression and it might modulate NK cell activity to boost the immunity.

Efficient non-viral CAR-T cell generation via silicon-nanotube-mediated transfection

Cell-based immunotherapy such as chimeric antigen receptor (CAR)-T therapy holds great promise in treating cancer and other diseases; but the current viral-based method represents a significant cost and safety hurdle. Here, we show for the first time successful CAR transfection into primary T cells via vertically aligned silicon nanotube (SiNT) arrays. SiNT-mediated transfection achieves comparable or even higher delivery efficiency (20–37%) and expression efficiency (18–24%) to that achieved by electroporation. Scanning electron microscopy imaging after focused ion beam milling demonstrated the tight T cell–SiNT interface. The induced membrane invaginations and the proximity between individual SiNTs and the nucleus might enhance endocytic pathways, and enable direct delivery of CAR construct into the nucleus, thus resulting in higher CAR expression efficiency. SiNT-interfacing also results in faster proliferation of T cells compared to cells transfected by electroporation; non-activated T (N_SiNT) cells undergo higher numbers of cell division than pre-activated ones (A_SiNT). By co-culturing with target lymphoma Raji cells, we prove that SiNT-transfected CAR-T cells can suppress Raji cell growth, indicated by significant increase in effector:target (E:T) ratio (by up to 30.7-fold). While SiNTs induce an overall upregulation of cytokine production in T cells, N_SiNT T cells exhibited high increase in secretion of IFNγ and IL-6, and relatively high in TNFα, which could contribute to their enhanced killing ability (∼96% cytotoxicity), demonstrated by their stronger inhibition on target Raji cells through luciferase assay. The results demonstrate the capacity of SiNT-mediated transfection of generating effective anti-lymphoma CAR-T cells. Considering the growing potential of cell-based therapies, we expect that a non-viral nanoinjection platform such as ours will facilitate the full realization of their therapeutic promise.

Effects of nasal inflammation on the olfactory bulb.

Sinonasal diseases, such as rhinosinusitis, affect up to 12% of individuals each year which constitutes these diseases as some of the most common medical conditions in the world. Exposure to environmental pathogens and toxicants via the nasal cavity can result in a severe inflammatory state commonly observed in these conditions. It is well understood that the epithelial and neuronal cells lining the olfactory mucosa, including olfactory sensory neurons (OSNs), are significantly damaged in these diseases. Prolonged inflammation of the nasal cavity may also lead to hyposmia or anosmia. Although various environmental agents induce inflammation in different ways via distinct cellular and molecular interactions, nasal inflammation has similar consequences on the structure and homeostatic function of the olfactory bulb (OB) which is the first relay center for olfactory information in the brain. Atrophy of the OB occurs via thinning of the superficial OB layers including the olfactory nerve layer, glomerular layer, and superficial external plexiform layer. Intrabulbar circuits of the OB which include connectivity between OB projection neurons, OSNs, and interneurons become significantly dysregulated in which synaptic pruning and dendritic retraction take place. Furthermore, glial cells and other immune cells become hyperactivated and induce a state of inflammation in the OB which results in upregulated cytokine production. Moreover, many of these features of nasal inflammation are present in the case of SARS-CoV-2 infection. This review summarizes the impact of nasal inflammation on the morphological and physiological features of the rodent OB.

Lactococcus lactis subsp. cremoris C60 induces macrophages activation that enhances CD4+ T cell-based adaptive immunity.

Lactococcus lactis subsp. cremoris C60 is a probiotic strain that induces diverse functional modifications in immune cells. In this report, as a novel effect of C60 on myeloid lineage cells, we show that C60 enhances the immunological function of macrophages that consequently promotes CD4+ T cell activity in an antigen-dependent manner. Heat-killed (HK) C60 induced the production of pro-inflammatory cytokines in thioglycolate-elicited peritoneal macrophages (TPMs) much stronger than Toll-like receptor (TLR) ligand stimulation. The HK-C60 treatment also augmented the expression of antigen-presenting and co-stimulatory molecules, such as major histocompatibility complex (MHC) class II, CD80, and CD86, as well as antigen uptake in TPMs. These HK-C60-mediated functional upregulations in TPMs resulted in the promotion of CD4+ T cell activation in an antigen-dependent manner. Interestingly, the TPMs that originated from the mice fed the HK-C60 diet showed pre-activated characteristics, which was confirmed by the upregulation of cytokine production and antigen presentation-related molecule expression under lipopolysaccharide (LPS) stimulation. Furthermore, the antigen-dependent CD4+ T cell activation was also enhanced by the TPMs. This implied that antigen presentation activity was enhanced in the TPMs that originated from the HK-C60 diet mice. Thus, C60 effectively upregulates the immunological function of macrophages that directly connects to CD4+ T cell-based adaptive immunity.

Peripheral Cytokine Levels as a Prognostic Indicator in Gastric Cancer: A Review of Existing Literature.

Although strong connections exist between the carcinogenesis of gastric cancer and chronic inflammation, gastric cancer is unique in that the chronic gastritis which frequently precedes carcinogenesis is strongly associated with H. pylori infection. The interplay between H. pylori virulence factors and host immune cells is complex but culminates in the activation of inflammatory pathways and transcription factors such as NF-κB, STAT3, and AP-1, all of which upregulate cytokine production. Due to the key role of cytokines in modulating the immune response against tumour cells as well as possibly stimulating tumour growth and proliferation, different patterns of cytokine secretion may be associated with varying patient outcomes. In relation to gastric cancer, interleukin-6, 8, 10, 17A, TNF, and IFN-γ may have pro-tumour properties, although interleukin-10, TNF, and IFN-γ may have anti-tumour effects. However, due to the lack of studies investigating patient outcomes, only a link between higher interleukin-6 levels and poorer prognosis has been demonstrated. Further investigations which link peripheral cytokine levels to patient prognosis may elucidate important pathological mechanisms in gastric cancer which adversely impact patient survival and allow treatments targeting these processes to be developed.

Zika Virus Infection Activates Proinflammatory Cytokines and Triggers Monocyte Differentiation

In 2015, Zika virus (ZIKV) outbreak in several countries of South America was linked to microcephaly in newborns. Microcephaly, a severe clinical presentation, was first diagnosed in association with ZIKV, which primarily considered self-limiting infection. From infected keratinocytes, virus disseminates crossing blood-tissue barriers and is detected in various tissues including brain, muscles and placenta. One of the mechanisms utilized by viruses to cross tissue barriers involves leukocytes, where monocytes are often identified as the carriers. Recently, circulating monocytes were shown to harbor ZIKV, suggesting that these leukocyte populations could serve as vehicle to transport ZIKV to the fetus. We speculate that ZIKV infection could promote monocyte activation and release of cytokines causing tissue damage. However, little is known about monocyte susceptibility to ZIKV infection. Also, effect of ZIKV infection on transcriptional activation and production remains unclear.Materials and methods. Monocytes were isolated from cord blood buffy coats (University of Colorado Cord Blood Bank, Aurora) using Miltenyi magnetic bead separation kit (CD14 Microbeads; Miltenyi, Auburn, CA). Monocytes were infected with two ZIKV strains, PRVABC59 (South America) and IBH30656 (Nigeria), ATCC (Manassas, VA). Total RNA was extracted and used for Next generation Sequencing (Illustra RNAspin Mini kit (GE Healthcare, Marlborough, MA)). FastQ data generated by the MiSeq (Illumina, San Diego CA) was annotated and the sequence reads were analyzed by CLC workbench 9.0.1 (Qiagen, Germantown, MD) for the detection of viral and cellular genes. Differential expressions of cellular genes based on the Reads Per Kilobase of transcript per Million mapped reads were determined by comparing with mock-infected cells using RNA-seq analysis tool of CLC Workbench. Differentially regulated genes due to ZIKV infection, identified by CLC Workbench, were subjected for determining the deregulated pathways using IPA Ingenuity Pathways analysis tool (Qiagen Germantown, MD). Viral replication was determined by qPCR, Western blot and immunofluorescence. Cytokine activation was analyzed using Bio-Plex Pro Human Cytokine 27-plex Panel (Bio-Rad, Hercules, CA, USA) following manufacturer's instructions.Results. We have shown that South American and Nigerian strains of ZIKV can infect monocytes and actively replicate. Presence of capsid protein and accumulation of viral transcripts was demonstrated in vitro. Infection with PRVABC59 strain changes transcriptional activity of 332 cellular genes, while the Nigerian strain affected the transcription of 521 cellular genes compared to the mock-infected controls. Interestingly, 195 genes were commonly deregulated in cells infected with both strains. Analysis of these commonly affected genes identified multiple pathways including apoptosis, chemotaxis, cell proliferation, inflammation, acute phase response, mitochondria activation. The most amazing observation was upregulation of mitochondrial and inflammasome genes. Mitochondrial genes (MT-ND2, MT-ND5 and MT-ATP8) activated in ZIKV infected monocytes were shown to play a key role in pathogenesis of other neurodegenerative disease.Analysis of cytokine production revealed early activation of proinflammatory cytokines (IL-1β, IL-6 and IL-8) (Table 1). Upregulation of IL-1β by ZIKV infected monocytes is evident for inflammasome activation. Upregulation of IL-6, IL-10 and IL-17α suggested an activation of Th17 pathway in ZIKV infected monocytes. Besides only Nigerian ZIKV strain increased the production of IL-4 in monocytes. IL-4 together with IL-10 could promote Th2 type immune response to ZIKV. Finally, both strains increased production of FGF, while levels of VEGF were increased only in supernatants of PRVABC59 infected monocytes.Conclusions. Human monocytes are susceptible to infection with ZIKV strains, PRVABC59 and IBH30656 and support viral replication. ZIKV infection affects cellular pathways, including mitochondrial and inflammasome ones that could play significant role in pathogenesis. ZIKV infection of monocytes upregulates cytokine production, which can trigger inflammation and leukocyte migration. Analysis of cytokine production by ZIKV infected monocytes suggests activation of Th2 and Th17 leukocytes, supporting the role of this signaling in ZIKV pathogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phase I study of SEA-CD40, gemcitabine, nab-paclitaxel, and pembrolizumab in patients (pts) with metastatic pancreatic ductal adenocarcinoma (PDAC) (trial in progress).

TPS451 Background: SEA-CD40 is an investigational non-fucosylated, humanized IgG1 monoclonal antibody directed against CD40, a co-stimulatory receptor expressed on antigen-presenting cells (APCs). Activation of CD40 on APCs upregulates cytokine production and co-stimulatory receptors, enhancing tumor antigen presentation to T cells. Preclinical data indicate that treatment of PDAC with chemotherapy in conjunction with a CD40 agonist could enhance antigen presentation and initiate an antitumor immune response (Byrne KT and Vonderheide RH, Cell Rep 2016;15, 2719–2732). A Phase 1 study (SGNS40-001) is evaluating SEA-CD40 monotherapy and in combination with other agents in pts with advanced solid or hematologic malignancies. A cohort is enrolling to evaluate the combination of SEA-CD40, gemcitabine, nab-paclitaxel, and pembrolizumab in PDAC. Methods: The cohort consists of pts with metastatic PDAC who have had no prior therapy for metastatic disease. Pts must be ≥18 years old, with (neo)adjuvant therapy completed > 4 months prior to enrollment; ECOG status ≤1; adequate renal, hepatic, and hematologic function; and measurable disease per RECIST v 1.1 criteria. A standard regimen of gemcitabine and nab-paclitaxel on Days 1, 8, and 15 of each 28-day cycle is administered with SEA-CD40 IV on Day 3. Pembrolizumab is administered every 42 days starting on Day 8. The primary objective is to evaluate antitumor activity; secondary objectives are to evaluate safety and tolerability and SEA-CD40 and pembrolizumab pharmacokinetics. Efficacy endpoints are confirmed RECIST objective response rate per investigator (primary), disease control rate (response or stable disease ≥16 weeks), duration of response, progression-free survival, and overall survival. Disease is assessed every 8 weeks using RECIST and immune-based RECIST (iRECIST). Treatment continues until unacceptable toxicity, progressive disease per iRECIST, consent withdrawal, or study closure, whichever occurs first. Assessment of dose-limiting toxicity will occur initially in groups of 6 pts to identify the recommended phase 2 dose of SEA-CD40 for the cohort. Enrollment to this cohort began in November 2019. Clinical trial information: NCT02376699.

Phase I study of SEA-CD40, gemcitabine, nab-paclitaxel, and pembrolizumab in patients with metastatic pancreatic ductal adenocarcinoma (PDAC).

TPS4671 Background: SEA-CD40 is an investigational non-fucosylated, humanized IgG1 monoclonal antibody directed against CD40, a co-stimulatory receptor expressed on antigen-presenting cells (APCs). Activation of CD40 on APCs upregulates cytokine production and co-stimulatory receptors, enhancing tumor antigen presentation to T cells. Preclinical data indicate that treatment of PDAC with chemotherapy in conjunction with a CD40 agonist could enhance antigen presentation and initiate an antitumor immune response (Byrne KT and Vonderheide RH, Cell Rep 2016;15, 2719–32). An ongoing Phase 1 study (SGNS40-001) is evaluating SEA-CD40 as monotherapy and in combination with pembrolizumab in patients with advanced solid or hematologic malignancies. A new cohort is enrolling to evaluate the combination of SEA-CD40, gemcitabine, nab-paclitaxel, and pembrolizumab in PDAC. Methods: The cohort consists of patients with metastatic PDAC who have had no prior therapy for metastatic disease. Patients must be ≥18 years old, with (neo)adjuvant therapy completed >4 months prior to enrollment, ECOG status ≤1, adequate renal, hepatic, and hematologic function, and measurable disease per RECIST v 1.1 criteria. A standard regimen of gemcitabine and nab-paclitaxel on Days 1, 8, and 15 of each 28-day cycle is administered with SEA-CD40 IV on Day 3. Pembrolizumab is administered every 42 days starting on Day 8. The primary objective is antitumor activity; secondary objectives are safety and tolerability and SEA-CD40 and pembrolizumab pharmacokinetics. Efficacy endpoints are confirmed RECIST ORR per investigator (primary), disease control rate (response or stable disease ≥16 weeks), duration of response, PFS, and OS. Disease is assessed every 8 weeks using RECIST and immune-based RECIST (iRECIST). Treatment continues until occurrence of unacceptable toxicity, progressive disease per iRECIST, consent withdrawal, or study closure. Assessment of dose-limiting toxicity will occur initially in groups of 6 patients to identify the recommended phase 2 dose of SEA-CD40 for the cohort. Enrollment to this cohort began in November 2019. Clinical trial information: NCT02376699 .

Non-toxic cross-reactivity with healthy tissues supports in vivo expansion and therapeutic efficacy of T cells engineered with synthetic antigen receptors

Background & Aim Clinical success of engineered T cell therapeutics against blood cancers has fueled rapid evolution of the field of T cell-based therapies and created demand for new antigen-binding domains. Among different tumor-targeting domains, single-chain antibodies (scFvs) remain the most prevalent. Extensive screening of scFvs for unwanted cross-reactivities with healthy tissues can reduce the likelihood of selecting ligands reactive to host tissues but not completely eliminate the possibility. Here we describe our experience with two BCMA-specific scFvs and the effect of non-toxic tissue cross-reactivity on the performance of engineered T cells. Methods, Results & Conclusion BCMA-specific scFvs derived from the monoclonal antibodies C11D5.3 and J22.9-xi were used as antigen-binding domains on two synthetic antigen receptor frameworks, T cell antigen coupler (TAC) receptor and chimeric antigen receptor (CAR), and tested against multiple myeloma xenografts. When T cells were engineered with the TAC framework, both scFvs yielded TAC T cells with equivalent in vitro performance; however, the J22.9-xi TAC T cells were more therapeutically robust in vivo. Further analysis revealed superior in vivo expansion of J22.9-xi TAC T cells, compared to C11D5.3 TAC T cells. Surprisingly, J22.9-xi TAC T cells expanded equally well in tumor-free and tumor-bearing mice in the absence of toxicity, suggesting that cross-reactivity to the host supported in vivo expansion and efficacy of J22.9-xi TAC T cells. In the context of a CD28-based CAR, T cells engineered with the J22.9-xi CAR displayed markedly greater levels of exhaustion, compared with T cells engineered with the C11D5.3 CAR, as evidenced by upregulation of checkpoint receptors, higher basal cytokine production, and reduced ability to upregulate cytokine production upon stimulation. Despite functional in vitro impairments, the J22.9-xi-28ζ CAR T cells proved to be the most efficacious and persistent in vivo when compared to the other engineered T cell populations; again, therapeutic efficacy was not associated with overt toxicities. Taken together, these observations suggest that non-lethal cross-reactivity of engineered T cells can augment in vivo potency and enable therapeutic efficacy of T cell products with evidence of functional exhaustion.

MiRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy.

Dendritic cell (DC) based immunotherapy is a promising approach to clinical cancer treatment. miRNAs are a class of small non-coding RNA molecules that bind to RNAs to mediate multiple events which are important in diverse biological processes. miRNA mimics and antagomirs may be potent agents to enhance DC-based immunotherapy against cancers. miRNA array analysis was used to identify a representative miR-5119 potentially regulating PD-L1 in DCs. We evaluated levels of ligands of immune cell inhibitory receptors (IRs) and miR-5119 in DCs from immunocompetent mouse breast tumor-bearing mice, and examined the molecular targets of miR-5119. We report that miRNA-5119 was downregulated in spleen DCs from mouse breast cancer-bearing mice. In silico analysis and qPCR data showed that miRNA-5119 targeted mRNAs encoding multiple negative immune regulatory molecules, including ligands of IRs such as PD-L1 and IDO2. DCs engineered to express a miR-5119 mimic downregulated PD-L1 and prevented T cell exhaustion in mice with breast cancer homografts. Moreover, miR-5119 mimic-engineered DCs effectively restored function to exhausted CD8+ T cells in vitro and in vivo, resulting in robust anti-tumor cell immune response, upregulated cytokine production, reduced T cell apoptosis, and exhaustion. Treatment of 4T1 breast tumor-bearing mice with miR-5119 mimic-engineered DC vaccine reduced T cell exhaustion and suppressed mouse breast tumor homograft growth. This study provides evidence supporting a novel therapeutic approach using miRNA-5119 mimic-engineered DC vaccines to regulate inhibitory receptors and enhance anti-tumor immune response in a mouse model of breast cancer. miRNA/DC-based immunotherapy has potential for advancement to the clinic as a new strategy for DC-based anti-breast cancer immunotherapy.

Complement Receptor-Mediated Phagocytosis Induces Proinflammatory Cytokine Production in Murine Macrophages.

Macrophages are professional phagocytes that are uniquely situated between the innate and adaptive arms of immunity with a high capacity for phagocytosis and proinflammatory cytokine production as well as antigen presentation. Phagocytosis is a critical process to eliminate microbes, apoptotic cells and other foreign particles and is accelerated by host-generated opsonins, such as antibodies and complement. Early phagocytosis studies established the paradigm that FcγR-mediated phagocytosis was more proinflammatory than Complement Receptor (CR)-mediated uptake in macrophages. Using qPCR, cytokine antibody arrays and ELISA, we revisited this research question in primary macrophages. Using qPCR we determined that CR-mediated phagocytosis increases levels of TNF-α, IL-1β, IL-6, and MMP-9, compared to FcγR-mediated phagocytosis and control unstimulated cells. We confirmed these findings at the protein level using cytokine antibody arrays and ELISAs. We next investigated the mechanism behind upregulated cytokine production during CR-mediated phagocytosis. IκBα protein levels were reduced after phagocytosis of both IgG- and C3bi-sRBCs indicating proteolytic degradation and implicating NF-κB activation. Inhibition of NF-κB activation impacted IL-6 production during phagocytosis in macrophages. Due to the roles of calpain in IκBα and integrin degradation, we hypothesized that CR-mediated phagocytosis may utilize calpain for proinflammatory mediator enhancement. Using qPCR and cytokine antibody array analysis, we saw significant reduction of cytokine expression during CR-mediated phagocytosis following the addition of the calpain inhibitor, PD150606, compared to untreated cells. These results suggest that the upregulation of proinflammatory mediators during CR-mediated phagocytosis is potentially dependent upon calpain-mediated activation of NF-κB.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response. This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 µL basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFNγ and TNFα) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFNγ+, TNFα+ and CD107a+ CD4+ and CD8+ T cells was quantified. This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Human papillomavirus type 16 E6 oncoprotein promotes proliferation and invasion of non-small cell lung cancer cells through Toll-like receptor 3 signaling pathway.

Human papillomavirus (HPV) oncoproteins play vital roles in non-small cell lung cancer (NSCLC) pathogenesis, and Toll-like receptors (TLRs) contribute to tumor progression. However, interaction between HPV oncoproteins and TLR signaling in NSCLC progression remains unclear. Thus, the aim of the study was to explore effects of HPV16 E6 oncoprotein-induced TLRs pathway on growth and invasion of NSCLC cells and to examine potential mechanisms being involved. Recombinant plasmid (pcDNA-HPV16 E6) expressing HPV16 E6 protein was constructed. The expression prolife of TLRs was measured in NSCLC cell line A549 with or without pcDNA-HPV16 E6 transfection by real-time reverse polymerase chain reaction and Western blot. Cellular proliferation, invasion, cytokine productions, and downstream signaling pathways were also examined in TLR3-silencing/pcDNA-HPV16 E6 transfect A549 cells. Overexpression of HPV16 E6 increased proliferation, invasion, proliferation cytokine secretion, and TLR3 expression of A549 cells, while TLR3 silence inhibited HPV16 E6-induced tumor bioactivities of A549 cells. Down-regulation of TLR3 suppressed HPV16 E6-induced phosphorylation of Src, but did not affect TRIF expression. Moreover, inhibition of Src pathway also suppressed proliferation and invasion of A549 cells. In conclusion, HPV16 E6 oncoprotein promoted the bioactivities of NSCLC cells. TLR3-Src signaling pathway might be involved in this procession by up-regulation of cytokine production. The interaction between HPV16 E6 protein and TLR3 might contribute to the poor prognosis of NSCLC.

Hepatitis B Surface Antigen Promotes the Invasion of Hepatitis B Virus-Related Hepatocellular Carcinoma Cells by Upregulation of Toll-Like Receptor 2.

Hepatitis B virus (HBV) infection is one of the major risk factors leading to the development of hepatocellular carcinoma (HCC). Hepatitis B surface antigen (HBsAg) plays a pivotal role in HBV-related HCC pathogenesis, and Toll-like receptor (TLR) 2 is also considered to mediate tumor progression. However, the interaction between HBsAg and TLR2 in HCC progression remains unclear. Thus, the aim of the study was to explore the effect of HBsAg-TLR2 pathway on growth and invasion of HBV-related HCC cells and examine the potential mechanisms been involved. The expression of TLR2 was measured in two different HCC cell lines (HepG2 and HepG2.2.15) with or without recombinant HBsAg by real-time reverse polymerase chain reaction and Western blot. Cellular proliferation, invasion, cytokine productions, and downstream signaling pathways were also measured in TLR2-silencing HepG2.2.15 cells in response to HBsAg stimulation. The mRNA and protein levels of TLR2 were significantly elevated in HepG2.2.15 cells than those in HepG2 cells. HBsAg simulation increased proinflammatory cytokine production and invasion of HepG2.2.15 cells, while this process was inhibited by TLR2 silence. However, TLR2 siRNA transfection alone did not affect the bioactivities of tumor cells. Moreover, HBsAg increased expression of MyD88 and phosphorylation of NF-κB p50 and p38MAPK. Downregulation of TLR2 inhibited HBsAg-induced MyD88 and p-NF-κB, but not p-p38MAPK in HepG2.2.15 cells. In conclusion, HBsAg stimulation promotes the invasion of HBV-related HCC cells. TLR2/MyD88/NF-κB signaling pathway may be involved in this procession by upregulation of cytokine production. The interaction between TLR2 and HBsAg may contribute to the poor prognosis of HBV-related HCC.

Increased CD226 Expression on CD8+ T Cells Is Associated with Upregulated Cytokine Production and Endothelial Cell Injury in Patients with Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular damage and fibrosis of the skin and internal organs. Because activated and oligoclonally expanded CD8(+) T cells can be detected in peripheral blood and lungs of SSc patients, effector memory CD8(+) T cells may play a critical role for organ involvement in SSc; however, the pathogenic functions of effector memory CD8(+) T cells remain incompletely understood. In this study, we performed DNA microarray analysis of the sort-purified effector memory CD8(+) T cells from SSc patients and healthy controls, and showed that the expression of genes related to immune response and cell adhesion, including CD226 (also known as DNAX accessory molecule-1 [DNAM-1]), was significantly altered. Moreover, detailed analysis of CD226 revealed that CD226(high)CD8(+) T cells were increased in SSc patients (mean, 50.7%) compared with healthy controls (32.9%) and were appreciably associated with the severity of skin sclerosis and interstitial lung disease. Furthermore, CD226(+)CD8(+) T cells produced higher amount of various cytokines than CD226(-) ones, and CD226(high)CD8(+) T cells from SSc patients showed upregulated IL-13 production and positive correlation with the cytotoxic capacity of CD8(+) T cells against HUVECs. Finally, the neutralization of CD226 in CD8(+) T cells impaired costimulation, cytokine productions, and cytolysis against HUVECs. These findings indicate that upregulated CD226 expression on CD8(+) T cells reflects disease severity and is involved in SSc pathogenesis via the production of various cytokines, including profibrotic IL-13 and endothelial cell injury, and that CD226 may be a useful target in the treatment of SSc.

MiR-146a suppresses cellular immune response during Japanese encephalitis virus JaOArS982 strain infection in human microglial cells.

BackgroundJapanese encephalitis virus (JEV) is the causative agent of Japanese encephalitis which is more prevalent in South and Southeast Asia. JEV is a neurotropic virus which infiltrates into the brain through vascular endothelial cells. JEV infects neurons and microglial cells which causes neuronal damage and inflammation. However, JEV also evades the cellular immune response to survive in host cells. Viruses are known to modulate the expression of microRNAs, which in turn modulate cellular immune response by targeting expression of antiviral genes. The aim of this study is to understand the anti-inflammatory role of miR-146a during JEV infection, which facilitates immune evasion.MethodsHuman brain microglial cells (CHME3) were infected by JEV: JaOArS982 and P20778 strain, and expression of miR-146a were analyzed. Overexpression and knockdown studies of miR-146a were done to see the effect on NF-κB pathway and antiviral Jak-STAT pathway. Regulatory role of miR-146a on expression of interferon-stimulated genes was determined by real-time PCR and luciferase assays.ResultsJEV infection elevated the expression of miR-146a in JaOArS982 strain which caused downregulation of TRAF6, IRAK1, IRAK2, and STAT1 genes. Exogenous overexpression of miR-146a led to suppression of NF-κB activation and abrogation of Jak-STAT pathway upon JEV infection which led to downregulation of interferon-stimulated genes (IFIT-1 and IFIT-2) and facilitated viral replication. JEV infection initially upregulated cytokine production and activated STAT1 activity but STAT1 levels reduced at later time point, which led to the downregulation of interferon-stimulated genes.ConclusionUpregulation of miR-146a by JEV JaOArS982 strain leads to suppression of NF-κB activity and disruption of antiviral Jak-STAT signaling which helps the virus to evade the cellular immune response. This effect of JEV infection on miR-146a expression was found to be strain specific.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0249-0) contains supplementary material, which is available to authorized users.

Human dendritic cells transfected with a human papilloma virus-18 construct display decreased mobility and upregulated cytokine production

The marked depletion of dendritic cells (DCs) in skin cancers, as well as preneoplastic and neoplastic cervical epithelium, suggests a central role for DCs in productive human papillomavirus (HPV) infection and cancer promotion. It has been suggested that HPV may facilitate tumor development by reducing DC density, contributing to a decrease in local immune surveillance. In this study, we have examined the response of human DCs transfected with a construct containing the HPV18 genome and their subsequent expression of papilloma virus proteins. Transfected cells expressed the L1 major capsid protein and upregulated E6 and E7 oncoprotein transcripts as detected by RT-PCR. Transfection of DCs also resulted in a significant increase in cytokine production. Finally, we observed that HPV18 transfection decreased the migratory activity of DCs. Our data indicate that HPV transfection of DCs leads to changes in migratory activity and cytokine production, which potentially can suppress or delay immune responses to viral antigens. Additionally, changes in cytokine production by HPV-transformed human fibroblasts and human cervical epithelial cells revealed that the migratory and antigen-presenting functions of DCs may be impaired by the suppressive effects of cytokines produced by HPV-infected epithelial and stromal cells.

IgE‐mediated basophil tumour necrosis factor alpha induces matrix metalloproteinase‐9 from monocytes

IgE-mediated activation of mast cells has been reported to induce the release of tumour necrosis alpha (TNF-α), which may display autocrine effects on these cells by inducing the generation of the tissue remodelling protease matrix metalloproteinase-9 (MMP-9). While mast cells and basophils have been shown to express complementary and partially overlapping roles, it is not clear whether a similar IgE/TNF-α/MMP-9 axis exists in the human basophil. The purpose of this study was thus to investigate whether IgE-mediated activation of human basophils induces TNF-α and MMP-9 release. Human peripheral blood mononuclear cells (PBMC), isolated basophils and monocytes were stimulated up to 21h with anti-IgE. Mediator releases were assessed by ELISA, and surface expressions of mediators were detected by flow cytometry. Upregulation of cytokine production was detected by Western blot and polymerase chain reaction (PCR). IgE-mediated activation of basophils induced the synthesis and release of both TNF-α and MMP-9 from PBMC. In contrast, IgE-mediated activation of purified basophils induced the release and cellular expression of TNF-α but not MMP-9. Isolated monocytes did not release MMP-9 upon anti-IgE stimulation, but MMP-9 release was induced by stimulating monocytes with supernatants from activated basophils, and this release was inhibited by anti-TNF-α neutralizing antibodies. Our results strongly indicate that human basophils release TNF-α following IgE-dependent activation and that this cytokine subsequently stimulates MMP-9 release from monocytes. These findings support a direct involvement of basophils in inflammation as well as suggesting a role for the basophil in tissue remodelling.