Reversing immunosuppression in the tumor microenvironment of fibrolamellar carcinoma via PD-1 and IL-10 blockade.

Fibrolamellar carcinoma (FLC) is a rare liver tumor driven by the DNAJ-PKAc fusion protein that affects healthy young patients. Little is known about the immune response to FLC, limiting rational design of immunotherapy. Multiplex immunohistochemistry and gene expression profiling were performed to characterize the FLC tumor immune microenvironment and adjacent non-tumor liver (NTL). Flow cytometry and T cell receptor (TCR) sequencing were performed to determine the phenotype of tumor-infiltrating immune cells and the extent of T cell clonal expansion. Fresh human FLC tumor slice cultures (TSCs) were treated with antibodies blocking programmed cell death protein-1 (PD-1) and interleukin-10 (IL-10), with results measured by cleaved caspase-3 immunohistochemistry. Immune cells were concentrated in fibrous stromal bands, rather than in the carcinoma cell compartment. In FLC, T cells demonstrated decreased activation and regulatory T cells in FLC had more frequent expression of PD-1 and CTLA-4 than in NTL. Furthermore, T cells had relatively low levels of clonal expansion despite high TCR conservation across individuals. Combination PD-1 and IL-10 blockade signficantly increased cell death in human FLC TSCs. Immunosuppresion in the FLC tumor microenvironment is characterized by T cell exclusion and exhaustion, which may be reversible with combination immunotherapy.

Production of an interleukin-10 blocking antibody by genetically engineered macrophages increases cancer cell death in human gastrointestinal tumor slice cultures.

Although it can promote effector T-cell function, the summative effect of interleukin-10 (IL-10) in the tumor microenvironment (TME) appears to be suppressive; therefore, blocking this critical regulatory cytokine has therapeutic potential to enhance antitumor immune function. As macrophages efficiently localize to the TME, we hypothesized that they could be used as a delivery vehicle for drugs designed to block this pathway. To test our hypothesis, we created and evaluated genetically engineered macrophages (GEMs) that produce an IL-10-blocking antibody (αIL-10). Healthy donor human peripheral blood mononuclear cells were differentiated and transduced with a novel lentivirus (LV) encoding BT-063, a humanized αIL-10 antibody. The efficacy of αIL-10 GEMs was assessed in human gastrointestinal tumor slice culture models developed from resected specimens of pancreatic ductal adenocarcinoma primary tumors and colorectal cancer liver metastases. LV transduction led to sustained production of BT-063 by αIL-10 GEMs for at least 21 days. Transduction did not alter GEM phenotype as evaluated by flow cytometry, but αIL-10 GEMs produced measurable quantities of BT-063 in the TME that was associated with an ~5-fold higher rate of tumor cell apoptosis than control.

Blockade of interleukin 10 potentiates antitumour immune function in human colorectal cancer liver metastases.

OBJECTIVE: Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures. DESIGN: We created organotypic slice cultures from human CRLM (n=38 patients' tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy. RESULTS: αIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8+ T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function. CONCLUSION: Neutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.

LN Monocytes Limit DC-Poly I:C Induced Cytotoxic T Cell Response via IL-10 and Induction of Suppressor CD4 T Cells.

Every immune response has accelerators and brakes. Depending on the pathogen or injury, monocytes can play either role, promoting or resolving immunity. Poly I:C, a potent TLR3 ligand, licenses cross-presenting dendritic cells (DC1) to accelerate a robust cytotoxic T cells response against a foreign antigen. Poly I:C thus has promise as an adjuvant in cancer immunotherapy and viral subunit vaccines. Like DC1s, monocytes are also abundant in the LNs. They may act as either immune accelerators or brakes, depending on the inflammatory mediator they encounter. However, little is known about their contribution to adaptive immunity in the context of antigen and Poly I:C. Using monocyte-deficient and chimeric mice, we demonstrate that LN monocytes indirectly dampen a Poly I:C induced antigen-specific cytotoxic T cell response, exerting a "braking" function. This effect is mediated by IL-10 production and induction of suppressor CD4+ T cells. In a metastatic melanoma model, we show that a triple-combination prophylactic treatment consisting of anti-IL-10, tumor peptides and Poly I:C works because removing IL-10 counteracts the monocytic brake, resulting in significantly fewer tumors compared to mice treated with tumor peptides and Poly I:C alone. Finally, in human LN tissue, we observed that monocytes (unlike DCs) express high levels of IL-10, suggesting that anti-IL-10 may be an important addition to treatments. Overall, our data demonstrates that LN monocytes regulate the induction of a robust DC1-mediated immune response. Neutralization of either IL-10 or monocytes can augment Poly I:C-based treatments and enhance T cell cytotoxicity.

Taraxacum mongolicum extract inhibited malignant phenotype of triple-negative breast cancer cells in tumor-associated macrophages microenvironment through suppressing IL-10 / STAT3 / PD-L1 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Triple-negative breast cancer (TNBC) is the most aggressive and the worst prognosis breast cancer with limited treatment options. Taraxacum mongolicum (also called dandelion) is a traditional Chinese medicine has been used to treat mastitis, breast abscess, and hyperplasia of mammary glands since ancient times. In modern pharmacological research, dandelion has been proven with anti-breast cancer activities. We previously reported that dandelion extract could induce apoptosis in TNBC cells. However, its anti-tumor effects and mechanisms in the tumor microenvironment have not yet been elucidated. AIM OF THE STUDY: Tumor-associated macrophages (TAMs) play an important role in regulating the interaction between tumor cells and the immune system. The present study aimed to investigate the effects and mechanisms of dandelion extract on TNBC cells under the microenvironment of TAMs, as well as its influence on the polarization of M2 macrophages. MATERIALS AND METHODS: M2 macrophages were induced by phorbol-12-myristate 13-acetate (PMA) and interleukin 4 (IL-4), and verified by flow cytometry, quantitative RT-PCR (qRT-PCR), Western blotting, and ELISA. MDA-MB-231 and MDA-MB-468 TNBC cells were co-cultured with the supernatant of M2 macrophage which providing the TAMs microenvironment. The antitumor activity of dandelion extract in TNBC cells was evaluated by MTT assay. The invasive and migratory capacity of TNBC cells was measured by transwell assays. The expression of protein and gene was assessed by Western blotting and qRT-PCR, respectively. RESULTS: TAMs microenvironment promoted the proliferation, migration, and invasion of TNBC cells. However, dandelion extract inhibited the malignant property of MDA-MB-231 and MDA-MB-468 cells induced by TAMs. Both of TAMs and IL-10 caused STAT3 activation and PD-L1 higher expression, the immunosuppressive molecules in TNBC cells, and this effect can be attenuated by IL-10 neutralizing antibody. Dandelion extract exerted inhibition on STAT3 and PD-L1 in TNBC cells under TAMs microenvironment. Furthermore, in M2 macrophages, dandelion extract remarkably promoted the expression of M1-like marker TNF-α, IL-8, and iNOS, but reduced M2-like marker IL-10, CD206, Arginase-1, and TGF-ß. CONCLUSION: Dandelion extract inhibited the proliferation, migration and invasion of TNBC cells in TAMs microenvironment through suppressing IL-10/STAT3/PD-L1 immunosuppressive signaling pathway. Furthermore, dandelion extract promoted the polarization of macrophages from M2 to M1 phenotype. Thus, our results indicated that dandelion may serve as a promising therapeutic strategy for TNBC by modulating tumor immune microenvironment.

Interleukin-10 suppression enhances T-cell antitumor immunity and responses to checkpoint blockade in chronic lymphocytic leukemia.

T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL), where CLL cells downregulate T-cell responses through regulatory molecules including programmed death ligand-1 (PD-L1) and Interleukin-10 (IL-10). Immune checkpoint blockade (ICB) aims to restore T-cell function by preventing the ligation of inhibitory receptors like PD-1. However, most CLL patients do not respond well to this therapy. Thus, we investigated whether IL-10 suppression could enhance antitumor T-cell activity and responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated analogue of the Sp1 inhibitor mithramycin (MTMox32E) to suppress CLL IL-10. MTMox32E treatment inhibited mouse and human CLL IL-10 production and maintained T-cell effector function in vitro. In the Eµ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden and increased CD8+ T-cell proliferation, effector and memory cell prevalence, and interferon-γ production. When combined with ICB, suppression of IL-10 improved responses to anti-PD-L1 as shown by a 4.5-fold decrease in CLL cell burden compared to anti-PD-L1 alone. Combination therapy also produced more interferon-γ+, cytotoxic effector KLRG1+, and memory CD8+ T-cells, and fewer exhausted T-cells. Since current therapies for CLL do not target IL-10, this provides a novel strategy to improve immunotherapies.

Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10.

The microRNA let-7d has been reported to be a tumor suppressor in renal cell carcinoma (RCC). Tumor-associated macrophages (TAM) are M2-polarized macrophages that can enhance tumor growth and angiogenesis in many human cancers. However, the role of let-7d in TAM-associated RCC progression remains elusive. First, we observed a strongly inverse correlation between let-7d expression and microvessel density in RCC tissues. Furthermore, the proliferation, migration, and tube formation of HUVECs were significantly inhibited by conditioned medium from a coculture system of the phorbol myristate acetate pretreated human THP-1 macrophages and let-7d-overexpressing RCC cells. Moreover, the proportion of M2 macrophages was significantly lower in the group that was cocultured with let-7d-overexpressing RCC cells. Subcutaneous xenografts formed by the injection of let-7d-overexpressing RCC cells together with THP-1 cells resulted in a significant decrease in the M2 macrophage ratio and microvessel density compared with those formed by the injection of control RCC cells with THP-1 cells. In silico and experimental analysis revealed interleukin-10 (IL-10) and IL-13 as let-7d target genes. Importantly, the addition of IL-10 and IL-13 counteracted the inhibitory effects of the conditioned medium from the coculture system with let-7d-overexpressing RCC cells in vitro. Additionally, overexpression of IL-10 and IL-13 reversed the effects of let-7d on macrophage M2 polarization and tumor angiogenesis in vivo. Finally, the expression of IL-10 and IL-13 were inversely correlated with the expression of let-7d in RCC clinical specimens. These results suggest that let-7d may inhibit intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-10 and IL-13.

An Immunosuppressive Effect of Melanoma-derived Exosomes on NY-ESO-1 Antigen-specific Human CD8+ T Cells is Dependent on IL-10 and Independent of BRAFV600E Mutation in Melanoma Cell Lines.

Exosomes, including human melanoma-derived exosomes (HMEX), are known to suppress the function of immune effector cells, which for HMEX has been associated with the surface presence of the immune checkpoint ligand PD-L1. This study investigated the relationship between the BRAF mutational status of melanoma cells and the inhibition of secreted HMEX exosomes on antigen-specific human T cells. Exosomes were isolated from two melanoma cell lines, 2183-Her4 and 888-mel, which are genetically wild-type BRAFWT and BRAFV600E, respectively. HMEX were isolated using a modified, size-exclusion chromatography (SEC) method shown to reduce co-isolation of non-exosome-associated cytokines compared to ultracentrifugation isolation. The immunoinhibitory effect of the exosomes was tested in vitro on patient-derived NY-ESO-1-specific CD8+ T cells challenged with NY-ESO-1 antigen. HMEX from both cell lines inhibited the immune response of antigen-specific T cells comparably, as evidenced by the reduction of IFN-γ and TNF-α in NY-ESO-1 tetramer-positive cells. This inhibition could be partially reversed by the presence of anti-PD-L1 and anti-IL-10 antibodies. IL-10 has been demonstrated to be a critical pathway for sustaining enhanced tumorigenesis in BRAFV600E mutant cells compared to BRAFWT melanoma cells. Thus, we demonstrate that HMEX inhibit antigen-specific T cell responses independent of the BRAF mutational status of the parent cells. In addition, PD-L1 and IL-10 contribute to the HMEX-mediated immunosuppression of antigen-specific human T cells. The inhibitory capacity of exosomes should be taken into consideration when developing therapies that are reliant upon the potency of customized, antigen-specific effector T cells.

Immunomodulatory Mechanism of Acyclic Nucleoside Phosphates in Treatment of Hepatitis B Virus Infection.

BACKGROUND AND AIMS: Current treatment with nucleos(t)ide analogs (NUCs) safely controls the replication of hepatitis B virus (HBV) and improves prognosis in patients with HBV. However, the inability to completely clear HBV is problematic, and novel therapies are desired. It has been believed that all NUCs have similar functions to inhibit HBV reverse transcriptase. However, our recent findings that only acyclic nucleoside phosphonates (ANPs; adefovir dipivoxil and tenofovir disoproxil fumarate) had an additional effect of inducing interferon (IFN)-λ3 in the gastrointestinal tract suggests that ANPs are not only distinct from nucleoside analogs (lamivudine and entecavir) in their structures but also in their functions. Because enteric lipopolysaccharide (LPS) can cross the intestine and affect peripheral blood mononuclear cells (PBMCs), we hypothesized that orally administered ANPs could have further additional effects to modulate LPS-mediated cytokine profile in PBMCs. APPROACH AND RESULTS: This study showed that pretreatment of PBMCs, from either healthy volunteers or patients with HBV, with ANPs inhibited LPS-mediated interleukin (IL)-10 production, which reciprocally induced IL-12p70 and tumor necrosis factor-α production in a dose-dependent manner. Furthermore, the combination of IFN-α and ANPs synergistically enhanced LPS-mediated IL-12p70 production in PBMCs. Mechanistic analyses revealed that cellular metabolites of ANPs directly bound the Akt protein, inhibiting its translocation to the plasma membrane, thereby impairing Akt phosphorylation. Therefore, pretreatment of PBMCs with ANPs impairs LPS-mediated IL-10 production. CONCLUSIONS: Among NUCs, only ANPs have an additional pharmacological effect modulating LPS-mediated cytokine production, which is expected to produce favorable immune responses toward HBV elimination. This additional immunomodulation by ANPs in PBMCs, as well as IFN-λ3 induction in the gastrointestinal tract, provides insights into HBV treatment.

Evaluating the effects of vanadyl sulfate on biomarkers of oxidative stress and inflammation in renal tissue of rats with diabetes type 2

Vanadyl sulfate (VS) is an ingredient in some food supplements and experimental drugs. This study was designed to assay the effects of VS on biomarkers of oxidative stress and inflammation in renal tissue of rats with diabetes type 2. 30 male Wistar rats were divided into three equal groups as follow: non-diabetics, non-treated diabetics and VS-treated diabetics. Diabetes type 2 has been induced through high fat diet and fructose in the animals. Diabetic rats were treated with 25 mg/kgBW of VS in water for 12 weeks. At the end of study, glucose and insulin were measured using commercially available kits in serum and biomarkers of oxidative stress and inflammation in renal homogenates of animals were measured by related methods. Compared to controls, glucose and insulin were increased significantly in non-treated diabetic rats (p-value <0.05) that showed the induction of diabetes type 2 in rats. The results showed that in VS-treated diabetic rats compared to the non-treated diabetic group, vanadyl sulfate significantly reduced the glucose and insulin secretion and changed renal inflammatory and oxidative markers, except protein carbonyl so that we couldn't find any significant changes. Our study showed that vanadyl supplementation had positive effects on oxidative stress and inflammation biomarkers in kidney of diabetic rats

Combination therapy targeting both innate and adaptive immunity improves survival in a pre-clinical model of ovarian cancer.

BACKGROUND: Despite major advancements in immunotherapy among a number of solid tumors, response rates among ovarian cancer patients remain modest. Standard treatment for ovarian cancer is still surgery followed by taxane- and platinum-based chemotherapy. Thus, there is an urgent need to develop novel treatment options for clinical translation. METHODS: Our approach was to analyze the effects of standard chemotherapy in the tumor microenvironment of mice harboring orthotopic, syngeneic ID8-Vegf-Defb29 ovarian tumors in order to mechanistically determine a complementary immunotherapy combination. Specifically, we interrogated the molecular and cellular consequences of chemotherapy by analyzing gene expression and flow cytometry data. RESULTS: These data show that there is an immunosuppressive shift in the myeloid compartment, with increased expression of IL-10 and ARG1, but no activation of CD3+ T cells shortly after chemotherapy treatment. We therefore selected immunotherapies that target both the innate and adaptive arms of the immune system. Survival studies revealed that standard chemotherapy was complemented most effectively by a combination of anti-IL-10, 2'3'-cGAMP, and anti-PD-L1. Immunotherapy dramatically decreased the immunosuppressive myeloid population while chemotherapy effectively activated dendritic cells. Together, combination treatment increased the number of activated T and dendritic cells as well as expression of cytotoxic factors. It was also determined that the immunotherapy had to be administered concurrently with the chemotherapy to reverse the acute immunosuppression caused by chemotherapy. Mechanistic studies revealed that antitumor immunity in this context was driven by CD4+ T cells, which acquired a highly activated phenotype. Our data suggest that these CD4+ T cells can kill cancer cells directly via granzyme B-mediated cytotoxicity. Finally, we showed that this combination therapy is also effective at delaying tumor growth substantially in an aggressive model of lung cancer, which is also treated clinically with taxane- and platinum-based chemotherapy. CONCLUSIONS: This work highlights the importance of CD4+ T cells in tumor immunology. Furthermore, the data support the initiation of clinical trials in ovarian cancer that target both innate and adaptive immunity, with a focus on optimizing dosing schedules.

Interleukin 10 promotes growth and invasion of glioma cells by up-regulating KPNA 2 in vitro.

OBJECTIVE: Glioma is one of the leading causes of death worldwide with high incidence, recurrence, and mortality. Interleukin-10 (IL-10) is a cytokine with dual function in many types of tumors. Although IL-10 is overexpressed and promotes tumor progression in human primary brain tumor, the mechanisms are largely unknown. MATERIALS AND METHODS: Glioma cells were treated with different dosages of IL-10. The cell growth was detected by CCK-8, and the invasion was measured by Transwell. The relative expression of messenger RNAs was detected by quantitative real-time polymerase chain reaction. RESULTS: We found that IL-10 treatment significantly enhanced glioma cell growth and invasion. Moreover, KPNA2 was significantly upregulated after treatment with IL-10. By performing knockdown experiments, we found that the glioma cell growth and invasion were significantly declined. CONCLUSIONS: The results indicated that knockdown of KPNA2 significantly inhibited the growth and invasion of glioma cells. Moreover, IL-10 promotes glioma progression via upregulation of KPNA2. This study will be of important significance and provides a potential target for treatment of patients with glioma.

IL-10 knockdown with siRNA enhances the efficacy of Doxorubicin chemotherapy in EBV-positive tumors by inducing lytic cycle via PI3K/p38 MAPK/NF-kB pathway.

High levels of IL-10 expression in Epstein-Barr virus (EBV) associated tumors have been reported and it is likely to be important for maintaining EBV latency and EBV-associated tumors. The switch from the latent form of EBV to the lytic form in tumor cells can lead to tumor cell lysis. Here, we found that knockdown of IL-10 induced EBV lytic replication. Subsequently, we demonstrated that IL-10 knockdown activated BZLF1 promoter through PI3K-p38 MAPK-NF-κB signaling pathway. Interestingly, we verified that VEGF-A was required for IL-10 knockdown to activate PI3K signaling and the accompanying EBV lytic induction. Exogenous recombinant human VEGF-A induced PI3K activation and EBV lytic infection, and inhibition of VEGF-A signaling prevented the PI3K/AKT phosphorylation and EBV reactivation responded to IL-10 knockdown. Most importantly, IL-10 knockdown synergized with chemotherapeutic agent Doxorubicin to kill EBV associated tumor cells in vitro and repress EBV-positive tumor growth in vivo. Our results suggest that inhibition of IL-10 has the potential to serve as a new supplemental strategy for the treatment of EBV-associated tumors.

Synthesized natural peptides from amphibian skin secretions increase the efficacy of a therapeutic vaccine by recruiting more T cells to the tumour site.

BACKGROUND: Therapeutic vaccines against cervical cancer remain ineffective. Previously, we demonstrated that blocking the signalling of a cytokine, interleukin 10, at the time of immunisation elicited significantly higher numbers of antigen specific T cells and inhibited tumour growth in mice. RESULTS: In the current paper, we demonstrate, in a HPV16 E6/E7 transformed TC-1 tumour mouse model, that despite increased antigen specific T cell numbers, blocking IL-10 signalling at the time of immunisation does not increase the survival time of the TC-1 tumour bearing mice compared to mice receiving the same immunisation with no IL-10 signalling blockade. Moreover, the function of tumour infiltrating T cells isolated 3 weeks post TC-1 transplantation is more suppressed than those isolated 2 weeks after tumour inoculation. We demonstrate that synthesized caerin peptides, derived from amphibian skin secretions, 1) were able to inhibit TC-1 tumour growth both in vitro and in vivo; 2) are environmentally stable; and 3) promote the secretion of pro-inflammatory interlukine-6 by TC-1 cells. Notably caerin peptides were able to increase the survival time of TC-1 tumour bearing mice after therapeutic vaccination with a HPV16E7 peptide-based vaccine containing IL-10 inhibitor, via recruiting increased levels of T cells to the tumour site. CONCLUSION: Caerin peptides increase the efficacy of a therapeutic vaccine by recruiting more T cells to the tumour site.

Comparative proteomic study reveals the enhanced immune response with the blockade of interleukin 10 with anti-IL-10 and anti-IL-10 receptor antibodies in human U937 cells.

Blocking cytokine interleukin 10 (IL-10) at the time of immunisation enhances vaccine induced T cell responses and improves control of tumour cell growth in vivo. However, the effect of an IL-10 blockade on the biological function of macrophages has not been explored. In the current paper, a macrophage precursor cell line, U937 cells, was selected to investigate the differential expression of proteins and relevant cell signalling pathway changes, when stimulated with lipopolysaccharide (LPS) in the presence of antibodies to IL-10 or IL-10 receptor. We used a quantitative proteomic strategy to investigate variations in protein profiles of U937 cells following the treatments with LPS, LPS plus human anti-IL10 antibody and anti-IL10R antibody in 24hrs, respectively. The LPS treatment significantly activated actin-related cell matrix formation and immune response pathways. The addition of anti-IL10 and anti-IL10R antibody further promoted the immune response and potentially effect macrophage survival through PI3K/AKT signalling; however, the latter appeared to also upregulated oncogene XRCC5 and Cajal body associated processes.

Inhibition of tumor necrosis factor alpha and increased of interleukin 10 by Lactobacillus: a molecular mechanism protection against TNBS-induced ulcerative colitis in chicks.

The purpose of this study was to evaluate the effects and mechanism of Lactobacillus on ameliorating ulcerative colitis chicks induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). There are three groups in this study, control, Lactobacillus and ulcerative colitis groups. 1-day-old chicks were fed with microcapsules containing Lactobacillus LA-5 daily for Lactobacillus group and clustered with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to make the model of ulcerative colitis at ten-day-old. Chicks in control and ulcerative colitis groups were fed with empty microcapsules daily at 1-day-old and then chicks in ulcerative colitis group were induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) for preparation of ulcerative colitis model at 10-day-old. We detected the changes of mRNA and protein expression of TNF-α and IL-10 in the colon by Real-Time PCR and Western Blot. Histopathology evaluation on colon was conducted. Results showed that chicks pretreated with Lactobacillus had striking injury improvement compared with ulcerative colitis group in histopathology. Compared with ulcerative colitis group, down-regulation of TNF-α and up-regulation of IL-10 were observed in Lactobacillus group chicks. Therefore, Lactobacillus could improve the injury of intestinal mucosa and reduce inflammatory response by regulating mRNA and protein expression levels of TNF-α and IL-10, respectively. In conclusion, Lactobacillus could ameliorate the effects on chicks of TNBS-induced ulcerative colitis by reducing the inflammation and regulating the expression of TNF-α and IL-10, respectively.

Differential effect of LPS and paclitaxel on microglial functional phenotypes and circulating cytokines: the possible role of CX3CR1 and IL-4/10 in blocking persistent inflammation.

Neuroinflammation plays a role in cancer chemotherapy-induced chronic pain. Thus far, most studies have focused on neuroinflammation suppression. However, there are limited reports of which factor is involved in the transition from acute inflammation to chronic inflammation, resulting in neuroinflammation and chronic pain. Here, we compared the inflammatory reaction and pain response induced by LPS and paclitaxel. LPS (0.5 mg/kg) or paclitaxel (2 mg/kg/day for 5 days) was administered intraperitoneally to mice, and mechanical allodynia was examined by von Frey test. LPS induced transient mechanical allodynia, whereas paclitaxel induced persistent mechanical allodynia. The CD86/CX3CR1 ratio remained unchanged due to CX3CR1 elevation following LPS injection, whereas the ratio was increased on day 1 after paclitaxel injection. LPS also increased CD45, CCL2, and CCL5 mRNA in the spinal cord and circulating pro- and anti-inflammatory cytokines 1 day after injection; however, the pattern was not consistent. Paclitaxel gradually increased inflammatory cytokines in the spinal cord. CX3CR1 might be involved in blocking the transition from acute pain to persistent pain in the LPS group. In addition, serum IL-4 and IL-10 elevation in the LPS group may be associated with chronic pain prevention. Therefore, targeting CX3CR1, IL-4, and IL-10 might be an alternative therapeutic strategy.

Lipoteichoic Acid Isolated from Lactobacillus plantarum Maintains Inflammatory Homeostasis through Regulation of Th1- and Th2-Induced Cytokines.

Lipoteichoic acid isolated from Lactobacillus plantarum K8 (pLTA) alleviates lipopolysaccharide (LPS)-induced excessive inflammation through inhibition of TNF-α and interleukin (IL)-6. In addition, pLTA increases the survival rate of mice in a septic shock model. In the current study, we have found that pLTA contributes to homeostasis through regulation of pro- and anti-inflammatory cytokine production. In detail, pLTA decreased the production of IL-10 by phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells stimulated with prostaglandin E2 (PGE-2) and LPS. However, TNF-α production which was inhibited by PGE-2+LPS increased by pLTA treatment. The regulatory effects of IL-10 and TNF-α induced by PGE-2 and LPS in PMA-differentiated THP-1 cells were mediated by pLTA, but not by other LTAs isolated from either Staphylococcus aureus (aLTA) or L. sakei (sLTA). Further studies revealed that pLTA-mediated IL-10 inhibition and TNF-α induction in PGE-2+LPS-stimulated PMA-differentiated THP-1 cells were mediated by dephosphorylation of p38 and phosphorylation of c-Jun N-terminal kinase (JNK), respectively. Reduction of pLTA-mediated IL-10 inhibited the metastasis of breast cancer cells (MDA-MB-231), which was induced by IL-10 or conditioned media prepared from PGE-2+LPS-stimulated PMA-differentiated THP-1 cells. Taken together, our data suggest that pLTA contributes to inflammatory homeostasis through induction of repressed pro-inflammatory cytokines as well as inhibition of excessive anti-inflammatory cytokines.

Real-time monitoring of IL-6 and IL-10 reporter expression for anti-inflammation activity in live RAW 264.7 cells.

In previous study, we suggested that the interleukin (IL)-6 and IL-10 could serve as a good biomarker for anti-inflammation that related to chronic inflammatory disease. Recently, we are finding new anti-inflammation compounds from natural products by screening of IL-6 and IL-10 levels. Although, we could measure IL-6 and IL-10 levels by several methods. However, all methods could not measure continuous kinetic of IL-6 and IL-10 levels. Most methods have multiple steps and take a long time. Therefore, there is no a suitable method for screening. To this end, we established IL-6 and IL-10 promoter assay which can monitor with reference gene as Glyceraldehyde 3-phosphate dehydrogenase (gapdh) promoter in living single cell. It could determine IL-6 and IL-10 levels continuously in real-time within two steps. We evaluated IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells with well-known anti-inflammatory compounds such as quercetin, xanthones, ß-D-glucan and dexamethasone. As the results, the expression of IL-6 and IL-10 reporters were strongly induced by LPS. The expression of IL-6 reporter was inhibited by all anti-inflammation compounds in LPS-induced RAW 264.7 cells. The expression of IL-10 reporter was inhibited by quercetin, xanthones and dexamethasone in LPS-induced RAW 264.7 cells. While, expression of IL-10 reporter was induced by ß-D-glucan. These results indicated that this assay could use for determination of IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells for anti-inflammation activity. Moreover, the results showed that natural compounds have an effect on the time course of IL-6 and IL-10 expressions. Therefore, real-time monitoring has a merit for natural compounds screening. We suggested that this assay could serve as a compound screening assay for anti-inflammation activity.

Cytokine-Modulated Natural Killer Cells Differentially Regulate the Activity of the Hepatitis C Virus.

HCV genotype 2a strain JFH-1 replicates and produces viral particles efficiently in human hepatocellular carcinoma (huh) 7.5 cells, which provide a stable in vitro cell infection system for the hepatitis C virus (HCVcc system). Natural killer (NK) cells are large lymphoid cells that recognize and kill virus-infected cells. In this study, we investigated the interaction between NK cells and the HCVcc system. IL-10 is a typical immune regulatory cytokine that is produced mostly by NK cells and macrophages. IL-21 is one of the main cytokines that stimulate the activation of NK cells. First, we used anti-IL-10 to neutralize IL-10 in a coculture of NK cells and HCVcc. Anti-IL-10 treatment increased the maturation of NK cells by enhancing the frequency of the CD56+dim population in NK-92 cells. However, with anti-IL-10 treatment of NK cells in coculture with J6/JFH-1-huh 7.5 cells, there was a significant decrease in the expression of STAT1 and STAT5 proteins in NK-92 cells and an increase in the HCV Core and NS3 proteins. In addition, rIL-21 treatment increased the frequency of the CD56+dim population in NK-92 cells, Also, there was a dramatic increase in the expression of STAT1 and STAT5 proteins in rIL-21 pre-stimulated NK cells and a decrease in the expression of HCV Core protein in coculture with J6/JFH-1-huh 7.5 cells. In summary, we found that the functional activation of NK cells can be modulated by anti-IL-10 or rIL-21, which controls the expression of HCV proteins as well as HCV RNA replication.