Immune Regulatory Factors Research Articles

How Autophagy Shapes the Tumor Microenvironment in Ovarian Cancer.

Ovarian cancer (OC) is characterized by a high mortality rate due to the late diagnosis and the elevated metastatic potential. Autophagy, a lysosomal-driven catabolic process, contributes to the macromolecular turnover, cell homeostasis, and survival, and as such, it represents a pathway targetable for anti-cancer therapies. It is now recognized that the vascularization and the cellular composition of the tumor microenvironment influence the development and progression of OC by controlling the availability of nutrients, oxygen, growth factors, and inflammatory and immune-regulatory soluble factors that ultimately impinge on autophagy regulation in cancer cells. An increasing body of evidence indicates that OC carcinogenesis is associated, at least in the early stages, to insufficient autophagy. On the other hand, when the tumor is already established, autophagy activation provides a survival advantage to the cancer cells that face metabolic stress and protects from the macromolecules and organelles damages induced by chemo- and radiotherapy. Additionally, upregulation of autophagy may lead cancer cells to a non-proliferative dormant state that protects the cells from toxic injuries while preserving their stem-like properties. Further to complicate the picture, autophagy is deregulated also in stromal cells. Thus, changes in the tumor microenvironment reflect on the metabolic crosstalk between cancer and stromal cells impacting on their autophagy levels and, consequently, on cancer progression. Here, we present a brief overview of the role of autophagy in OC hallmarks, including tumor dormancy, chemoresistance, metastasis, and cell metabolism, with an emphasis on the bidirectional metabolic crosstalk between cancer cells and stromal cells in shaping the OC microenvironment.

Transcriptome and proteome profiling reveal complementary scavenger and immune features of rat liver sinusoidal endothelial cells and liver macrophages

BackgroundLiver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs; liver resident macrophages) form the body’s most effective scavenger cell system for the removal of harmful blood-borne substances, ranging from modified self-proteins to pathogens and xenobiotics. Controversies in the literature regarding the LSEC phenotype pose a challenge when determining distinct functionalities of KCs and LSECs. This may be due to overlapping functions of the two cells, insufficient purification and/or identification of the cells, rapid dedifferentiation of LSECs in vitro, or species differences. We therefore characterized and quantitatively compared expressed gene products of freshly isolated, highly pure LSECs (fenestrated SE-1/FcγRIIb2+) and KCs (CD11b/c+) from Sprague Dawley, Crl:CD (SD), male rats using high throughput mRNA-sequencing and label-free proteomics.ResultsWe observed a robust correlation between the proteomes and transcriptomes of the two cell types. Integrative analysis of the global molecular profile demonstrated the immunological aspects of LSECs. The constitutive expression of several immune genes and corresponding proteins of LSECs bore some resemblance with the expression in macrophages. LSECs and KCs both expressed high levels of scavenger receptors (SR) and C-type lectins. Equivalent expression of SR-A1 (Msr1), mannose receptor (Mrc1), SR-B1 (Scarb1), and SR-B3 (Scarb2) suggested functional similarity between the two cell types, while functional distinction between the cells was evidenced by LSEC-specific expression of the SRs stabilin-1 (Stab1) and stabilin-2 (Stab2), and the C-type lectins LSECtin (Clec4g) and DC-SIGNR (Clec4m). Many immune regulatory factors were differentially expressed in LSECs and KCs, with one cell predominantly expressing a specific cytokine/chemokine and the other cell the cognate receptor, illustrating the complex cytokine milieu of the sinusoids. Both cells expressed genes and proteins involved in antigen processing and presentation, and lymphocyte co-stimulation.ConclusionsOur findings support complementary and partly overlapping scavenging and immune functions of LSECs and KCs. This highlights the importance of including LSECs in studies of liver immunity, and liver clearance and toxicity of large molecule drugs and nano-formulations.

Research progress of interleukin-33 and its receptor ST2 in sepsis

Sepsis is a life-threatening organ dysfunction caused by an imbalance in the response to infection. Clinically the effects of anti-infection and fluid resuscitation are limited, and the morbidity and mortality of sepsis are still high. Interleukin-33 (IL-33), a member of the IL-1 family, binds to various cell types through the ST2-IL-1 receptor helper protein complex. IL-33 and its receptor ST2 play an important role as immune regulatory factors in sepsis. This article reviews the pathophysiological characteristics of sepsis, the biological characteristics of IL-33 and its receptor ST2, and the relationship between IL-33/ST2 and sepsis, so as to provide new ideas for the diagnosis and treatment of sepsis.

The Functional Impact of Alternative Splicing and Single Nucleotide Polymorphisms in Rheumatoid Arthritis.

Advances in genomics and proteomics aid the identification of genes associated with various diseases. Genome-Wide Association Studies (GWAS) have identified multiple loci as risk alleles for susceptibility to Rheumatoid Arthritis (RA). A bisection of RA risk can be attributed to genetic factors. Over 100 associated genetic loci that encompass immune regulatory factors have been found to be linked with RA. Aberrant Single Nucleotide Polymorphisms (SNPs) and alternative splicing mechanisms in such loci induce RA. These aberrations are viewed as potential therapeutic targets due to their association with a multitude of diseases. This review presents a few imperious genes whose alterations can cause severe bone deformities culminating in RA.

Matrisome Provides a Supportive Microenvironment for Skin Functions of Diverse Species.

A biomaterial scaffold is a promising tool employed to drive tissue regeneration. This technology has been successfully applied in human tissue rebuilding, particularly for the skin. Meanwhile, there is still room for further improvement, such as maintaining sufficient functionality of a biomaterial scaffold. Here, we developed a new decellularization method to generate a complete anatomical skin biomatrix scaffold with a preserved extracellular matrix (ECM) architecture. We performed proteomic and bioinformatic analyses of the skin scaffold maps of humans, pigs, and rats and systematically analyzed the interaction between ECM proteins and different cell types in the skin microenvironment. These interactions served to quantify the structure and function of the skin's Matrisome comprising core ECM components and ECM-associated soluble signaling molecules required for the regulation of epidermal development. We primarily found that the properties of the skin ECM were species-specific. For example, the composition and function of the ECM of the human skin were more similar to those of pigs compared with those of rats. However, the skin ECM of the pig was significantly deficient in its enzyme systems and immune regulatory factors compared with that of humans. These findings provide a new understanding of the role of the skin ECM niche as well as an attractive strategy that can apply tissue engineering principles to skin biomatrix scaffold materials, which promises to accelerate and enhance tissue regeneration.

Localized Immunomodulation with PD-L1 Results in Sustained Survival and Function of Allogeneic Islets without Chronic Immunosuppression

Allogeneic islet transplantation is limited by adverse effects of chronic immunosuppression used to control rejection. The programmed cell death 1 pathway as an important immune checkpoint has the potential to obviate the need for chronic immunosuppression. We generated an oligomeric form of programmed cell death 1 ligand chimeric with core streptavidin (SA-PDL1) that inhibited the T effector cell response to alloantigens and converted T conventional cells into CD4+Foxp3+ T regulatory cells. The SA-PDL1 protein was effectively displayed on the surface of biotinylated mouse islets without a negative impact islet viability and insulin secretion. Transplantation of SA-PDL1-engineered islet grafts with a short course of rapamycin regimen resulted in sustained graft survival and function in >90% of allogeneic recipients over a 100-d observation period. Long-term survival was associated with increased levels of intragraft transcripts for innate and adaptive immune regulatory factors, including IDO-1, arginase-1, Foxp3, TGF-β, IL-10, and decreased levels of proinflammatory T-bet, IL-1β, TNF-α, and IFN-γ as assessed on day 3 posttransplantation. T cells of long-term graft recipients generated a proliferative response to donor Ags at a similar magnitude to T cells of naive animals, suggestive of the localized nature of tolerance. Immunohistochemical analyses showed intense peri-islet infiltration of T regulatory cells in long-term grafts and systemic depletion of this cell population resulted in prompt rejection. The transient display of SA-PDL1 protein on the surface of islets serves as a practical means of localized immunomodulation that accomplishes sustained graft survival in the absence of chronic immunosuppression with potential clinical implications.

Abstract B85: Anticancer efficacy of a novel immune-stimulating oncolytic virus: VG161 in gastrointestinal cancers

Abstract Oncolytic viruses (OVs) are the wild-type or genomic modified viruses, which can specifically replicate in cancer cells but not in normal cells. They cause cancer cell lysis and more importantly, their replication in tumor cells induces an antitumor immune response from the host. As synergistic stimulation of antitumor immune response requires multiple immune-regulatory factors, OVs armed with multiple immune-modulating factors are of high potential in cancer immunotherapy. We have constructed a novel herpes simplex virus type-1 virus (HSV-1), VG161, that is armed with IL12, IL15, and a unique PDL-1 peptide blocker. The aim of this study is to investigate the anticancer efficacy of VG161 in gastrointestinal (GI) cancers including gastric, hepatic, and colorectal cancers. The immune-stimulating activities in tumor-bearing immune competent mice were also demonstrated. VG161 was given to the tumor-bearing animals by intratumor injection (5 × 106-5 × 107 PFU/animal, once a day for 3-5 days). Antitumor activity of VG161 was tested in both immune-deficient mice as well as in immune-competent mice. Oncolytic activity of VG161 was demonstrated in human gastric (SGC-7901), hepatic (patient-derived xenograft, PDX), and colorectal (LS174T) cancer-bearing nude mice as the human tumors are susceptible to HSV infection and the immune system is compromised in those models. To determine the antitumor immune response activated by VG161, colorectal cancer (CT26) syngeneic model was used in immune competent animals and T cell subpopulation analysis, ELISPOT, and tumor rechallenge assay was applied to measure specific antitumor immunity. VG161 caused complete tumor eradication in PDX and CT26 tumor models and significantly inhibited tumor growth in SGC-7901 and LS174T models. All animals treated with VG161 survived for many months till sacrificed. In the CT26 model, no tumor could be found after rechallenging with the same tumor cells. The number of CD8+ tumor infiltrated T cells was significantly increased, and systemic specific anticancer immunity was confirmed by ELISPOT assay on T cells isolated from spleen. VG161 is a novel oncolytic virus that is both strong in oncolysis and stimulating antitumor immunity for GI cancers. Intratumorally expressing multiple immune-regulatory factors by an oncolytic virus may significantly change the GI tumor immune microenvironment and stimulate anticancer immunity, to further enhance the efficacy of the oncolytic virus. Citation Format: Ronghua Zhao, Jun Ding, Tianchi Liu, Shuyun Liu, Dmitry Chouljenko, Yanal Murad, Erica Lee, Guoyu Liu, Luke Bu, William Jia. Anticancer efficacy of a novel immune-stimulating oncolytic virus: VG161 in gastrointestinal cancers [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B85.

Analysis of Immunological Treatment Effects of Virotherapy in Tumor Tissue.

In addition of being directly tumoricidal, oncolytic viruses have emerged as potent partners for established and investigational immunotherapies due to their immune-stimulatory effects. The shifting focus on virus-mediated immune modulation calls for a comprehensive analysis of the tumor microenvironment (TME) and the factors orchestrating the antiviral and antitumor immune response. The oncolytic VSV-GP studied in our lab is a safe and potent antitumor agent with a fast replication cycle and killing of a broad range of different cancer types. It induces a robust local inflammatory conversion of the TME and drives a strong adaptive immune response toward the tumor. Here we present our multidisciplinary approach to study VSV-GP treatment effects in tumors by assessing both immune cells (tumor-infiltrating lymphocytes and tumor-associated macrophages) and immune-regulatory factors (cytokines) as well as characterizing immune signatures using an immune-targeted NanoString gene expression system.

Abstract B059: Synergistic activation of antitumor immunity by an oncolytic virus VG161 armed with multiple immune-stimulating genes

Abstract Oncolytic viruses (OVs) are among the most powerful approaches in cancer immunotherapy. OVs not only cause cancer cell lysis but more importantly, their infection in tumors induces anti-tumor immune response from the host, resulting in lasting anti-tumor immunity. It has been recognized that anti-tumor immune response requires multiple immune regulatory factors that act synergistically and tumor microenvironment is critical for tumor to grow. Herpes simplex virus type-1 (HSV-1) has been approved by FDA as an oncolytic viral drug to treat melanoma. One advantage of HSV-1 is its large genomic capacity for carrying multiple exogenous genes.A HSV-1 oncolytic viral vector (VG161) was constructed to simultaneously express IL12, IL15 with its receptor alpha unit and a PDL-1 blocking peptide. Antitumor activity of VG161 was tested in both immune competent mice (CT26 and A20 tumor models) and nude mice for human tumor models (LNCaP and U87). Since CT26 and A20 are poorly permissive for HSV-1 replication, the mouse tumor models were able to demonstrate the antitumor immune response induced by VG161 while oncolytic activity of VG161 was demonstrated in LNCaP and U87 models since the immune system is compromised in those models.VG161 completely inhibited tumors in all the models tested and the animals survived tumor-free for many months till sacrificed. VG161 induced tumor oncolysis in both LNCaP and U87 tumors. In the CT26 model, animals were protected from the second challenging with CT26 cells following previous virally induced tumor regression. Furthermore, in a A20 double tumor model, intratumoral injection into the tumor on one side caused tumor regression on both sides. Transcriptome analysis showed significant change in tumor microenvironment. Finally, tumor specific memory T-cells were evident in the treated animals. The anti-tumor immune response by VG161 was significantly stronger than similar viruses that did not express any immune stimulating gene or only express GM-CSF. These results showed that intratumorally expressed multiple immune regulatory factors by an oncolytic virus may significantly change the tumor immune microenvironment to enhance efficacy of the oncolytic virus. Citation Format: Ronghua Zhao, Jun Ding, Dmitry Choujenko, Yanal Murad, Erica Lee, Guoyu Liu, Luke Bu, William Jia. Synergistic activation of antitumor immunity by an oncolytic virus VG161 armed with multiple immune-stimulating genes [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B059.

Abstract 5641: Synergy of immunomodulators delivered by an HSV-1 oncolytic virus can enhance its therapeutic effects in vivo

Abstract Oncolytic HSV-1 (oHSV-1) are among the most promising therapies for cancer. Besides the oncolytic activity delivered by the virus against tumor cells, this treatment can also induce a potent immune response against tumor antigens (whether classic TAA, or neoantigens specific to the tumor being targeted). We have constructed an oncolytic HSV-1 virus (VG161) to deliver 2 immunomodulator cytokines, IL12 and IL15, to the tumor micro-environment. These two cytokines can work synergistically to trigger an efficient anti-tumor immune response. A PD-L1 mimic peptide capable of blocking PD-1/PD-L1 interaction is also delivered as a Fc fusion peptide (TF-Fc) to help in maintaining an effective immune response against the tumor. For the construction of the recombinant virus, the exogenous components were cloned under CMV promoter control. The ICP34.5 gene was deleted as a safety measure for the recombinant virus. The expression of Il12, IL15 and TF-Fc were confirmed, in vitro and in vivo, by qRT-PCR, ELISA and Western blot. Upon intra-tumoral injection of VG161, expressed IL12, IL15 and TF-Fc were detected only in the injected tumor mass, but not in the animals' blood or other organs. The bio-distribution of the virus was tested by qPCR after injecting VG161 intratumorally, and most of the injected virus were concentrated in the tumor mass. Minimal amounts of the virus were detected in the liver and spleen for short periods after virus injection. We have demonstrated the superior activity of the VG161 virus, compared to the back-bone virus (with no payloads) using immune-competent mouse models (CT26) and nude mice implanted with human tumor cells lines (LNCaP and U87). VG161 induced tumor oncolysis resulting in complete tumor regression in nude mice. In the CT26 model, the tumor regressed to undetectable limits upon intra-tumoral injection with VG161. When the treated mice were challenged with the same tumor, the tumor cells did not grow. The added efficacy in CT26 tumor model can be attributed to the immune response generated by the modified virus, as demonstrated by a higher number of tumor-infiltrating CD8 T cells. The specific activity of T cells against the tumor cells was also demonstrated by ELISpot assay. Finally, memory T-cells were evident in the treated animals demonstrated by multiple assays. Conclusions This work has demonstrated the safety and efficacy of VG161, a novel oncolytic virus which can induce a strong anti-tumor immunity and oncolytic activity. We have also demonstrated that the Intra-tumoral expression of multiple immune regulatory factors may significantly change the tumor immune microenvironment to enhance the efficacy of the oncolytic virus. Citation Format: Yanal M. Murad, Jun Ding, Dmitry Chouljenko , Erica Lee, Luke Bu, Guoyu Liu, Ronghua Zhao, Zahid Delwar, Will Liu, William Jia. Synergy of immunomodulators delivered by an HSV-1 oncolytic virus can enhance its therapeutic effects in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5641.

Expression of immune regulatory genes in early, mid and late stages of pig (Sus scrofa domesticus) gestation

Semi-allogeneic foetus and placenta exploit various mechanisms to avoid immune-mediated maternal rejection. Several factors and cytokines are attributed for production of immune tolerance during gestation and very little information on expression of these immune-regulatory genes is available in pig. Chorioallantoic membrane (CAM) from early, mid and late gestational stages (n=4) were analysed for expression of immune regulatory genes, viz. Fas ligand (FasL), transporter for antigen processing-1 (TAP-1), transforming growth factor β1 (TGF-β1) and macrophage migration inhibitory factor (MIF) whereas Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as housekeeping gene. FasL gene expression was significantly higher in mid (13.46 fold) and late (28.77 fold) gestation than the early (1 fold) stage.TAP-1 mRNA expression was enhanced by 4.95 fold and 2.69 fold during mid and late gestation respectively in comparison to the early (1 fold) stage. TGF-β1 gene expression was up regulated in mid (2.43 fold) and late (2.29 fold) gestation than the early (1 fold) stage. MIF mRNA expression was enhanced in mid (3.04 fold) and late (1.59 fold) gestation in relation to the early (1 fold) stage. Placenta of pig remains entirely epitheliochorial which may minimise immune recognition and is supposed to diminish potent immune-regulatory mechanisms. However, our present study revealed consistent expression for immune regulatory factors which suggests immune modulation does exist in pig and may impart a role in pregnancy success.

Canonical Wnt Signaling in CD11c+ APCs Regulates Microbiota-Induced Inflammation and Immune Cell Homeostasis in the Colon.

Aberrant Wnt/β-catenin signaling occurs in several inflammatory diseases, including inflammatory bowel disease and inflammatory bowel disease-associated colon carcinogenesis. However, its role in shaping mucosal immune responses to commensals in the gut remains unknown. In this study, we investigated the importance of canonical Wnt signaling in CD11c+ APCs in controlling intestinal inflammation. Using a mouse model of ulcerative colitis, we demonstrated that canonical Wnt signaling in intestinal CD11c+ APCs controls intestinal inflammation by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt coreceptors, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) in CD11c+ APCs in LRP5/6ΔCD11c mice, resulted in enhanced intestinal inflammation with increased histopathological severity of colonic tissue. This was due to microbiota-dependent increased production of proinflammatory cytokines and decreased expression of immune-regulatory factors such as IL-10, retinoic acid, and IDO. Mechanistically, loss of LRP5/6-mediated signaling in CD11c+ APCs resulted in altered microflora and T cell homeostasis. Furthermore, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice resulted in reduced intestinal inflammation with decreased histopathological severity of colonic tissue. These results reveal a mechanism by which intestinal APCs control intestinal inflammation and immune homeostasis via the canonical Wnt-signaling pathway.

HIV-1 Tat-induced diarrhea is improved by the PPARalpha agonist, palmitoylethanolamide, by suppressing the activation of enteric glia

BackgroundDiarrhea is a severe complication in HIV-1-infected patients with Trans-activator of transcription (HIV-1 Tat) protein being recognized as a major underlying cause. Beside its direct enterotoxic effects, Tat protein has been recently shown to affect enteric glial cell (EGC) activity. EGCs regulate intestinal inflammatory responses by secreting pro-inflammatory molecules; nonetheless, they might also release immune-regulatory factors, as palmytoilethanolamide (PEA), which exerts anti-inflammatory effects by activating PPARα receptors. We aimed at clarifying whether EGCs are involved in HIV-1 Tat-induced diarrhea and if PEA exerts antidiarrheal activity.MethodsDiarrhea was induced by intracolonic administration of HIV-1 Tat protein in rats at day 1. PEA alone or in the presence of peroxisome proliferator-activated receptor (PPAR) antagonists was given intraperitoneally from day 2 to day 7. S100B, iNOS, NF-kappaB, TLR4 and GFAP expression were evaluated in submucosal plexi, while S100B and NO levels were measured in EGC submucosal plexi lysates, respectively. To verify whether PEA effects were PPARα-mediated, PPARα−/− mice were also used. After 7 days from diarrhea induction, endogenous PEA levels were measured in submucosal plexi homogenates deriving from rats and PPARα−/− mice.ResultsHIV-1 Tat protein induced rapid onset diarrhea alongside with a significant activation of EGCs. Tat administration significantly increased all hallmarks of neuroinflammation by triggering TLR4 and NF-kappaB activation and S100B and iNOS expression. Endogenous PEA levels were increased following HIV-1 Tat exposure in both wildtype and knockout animals. In PPARα−/− mice, PEA displayed no effects. In wildtype rats, PEA, via PPARα-dependent mechanism, resulted in a significant antidiarrheal activity in parallel with marked reduction of EGC-sustained neuroinflammation.ConclusionsEGCs mediate HIV-1 Tat-induced diarrhea by sustaining the intestinal neuroinflammatory response. These effects are regulated by PEA through a selective PPARα-dependent mechanism. PEA might be considered as an adjuvant therapy in HIV-1-induced diarrhea.

GPR81, a Cell-Surface Receptor for Lactate, Regulates Intestinal Homeostasis and Protects Mice from Experimental Colitis.

At mucosal sites such as the intestine, the immune system launches robust immunity against invading pathogens while maintaining a state of tolerance to commensal flora and ingested food Ags. The molecular mechanisms underlying this phenomenon remain poorly understood. In this study, we report that signaling by GPR81, a receptor for lactate, in colonic dendritic cells and macrophages plays an important role in suppressing colonic inflammation and restoring colonic homeostasis. Genetic deletion of GPR81 in mice led to increased Th1/Th17 cell differentiation and reduced regulatory T cell differentiation, resulting in enhanced susceptibility to colonic inflammation. This was due to increased production of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and decreased expression of immune regulatory factors (IL-10, retinoic acid, and IDO) by intestinal APCs lacking GPR81. Consistent with these findings, pharmacological activation of GPR81 decreased inflammatory cytokine expression and ameliorated colonic inflammation. Taken together, these findings identify a new and important role for the GPR81 signaling pathway in regulating immune tolerance and colonic inflammation. Thus, manipulation of the GPR81 pathway could provide novel opportunities for enhancing regulatory responses and treating colonic inflammation.

Dynamic expression of cytokine and transcription factor genes during experimental Fasciola gigantica infection in buffaloes

BackgroundDetermining the mechanisms involved in the immune-pathogenesis of the tropical liver fluke, Fasciola gigantica, is crucial to the development of any effective therapeutic intervention. Here, we examined the differential gene expression of cytokines and transcription factors in the liver of F. gigantica-infected buffaloes, over the course of infection.MethodsWater buffaloes (swamp type) were infected orally with 500 F. gigantica encysted metacercariae. Liver tissue samples were collected 3, 10, 28, 42, 70 and 98 days post-infection (dpi). Levels of gene expression of nine cytokines (IFN-γ, TGF-β, IL-1β, IL-4, IL-6, IL-10, IL-12B, IL-13 and IL-17A) and four transcription factors (T-bet, GATA-3, Foxp3 and ROR-γτ) were determined using quantitative real-time PCR (qRT-PCR). We evaluated any correlation between gene expression of these immune-regulatory factors and the severity of liver pathology.ResultsHistopathological examination revealed that cellular infiltration, hemorrhage and fibrosis without calcification in the liver parenchyma of infected buffaloes, increased over the course of infection. This progressive pathology was attributed to dysregulated and excessive inflammatory responses induced by infection. The early infection phase (3–10 dpi) was marked by a generalized immunosuppression and elevated TGF-β expression in order to facilitate parasite colonization. A mixed Th1/Th2 immune response was dominant from 28 to 70 dpi, to promote parasite survival while minimizing host tissue damage. During late infection (98 dpi), the response was biased towards Th1/Treg in order to inhibit the host’s Th2 protective response and promote chronic infection. Both IL-10 and IL-17A and the Th17/Treg balance, played key roles in mediating the inflammatory and immunoregulatory mechanisms in the liver during chronic fasciolosis.ConclusionsOur data showed distinct CD4+ T helper (Th) polarization and cytokine dysregulation in response to F. gigantica infection in water buffaloes over the course of infection. Characterizing the temporal expression profiles for host immune genes during infection should provide important information for defining how F. gigantica adapts and survives in the liver of buffaloes and how host immune responses influence F. gigantica pathogenicity.

Self-Assembled Injectable Peptide Hydrogels Capable of Triggering Antitumor Immune Response.

Self-assembled peptide hydrogels are particularly appealing for drug delivery, tissue engineering, and antitumor therapy due to various advantageous features including excellent biocompatibility and biodegradability, defined molecular and higher organized structures, and easy availability. However, the poor mechanical and rheological properties of assembled peptide hydrogels cause difficulties in injection, thus limiting further applications. Herein, injectable peptide-based hydrogels with tunable mechanical and rheological properties were obtained by combination with a positively charged poly peptide (poly-l-lysine, PLL). Electrostatic coupling between PLL and a self-assembling dipeptide (Fmoc-FF) provides a smart switch to enable the fibrous hydrogels to be shear-thinning and self-healing, thus leading to the formation of supramolecular hydrogels with rheological properties suitable for injection. The latter can be flexibly adjusted by merely varying the concentration or the molecular weight of the polypeptide to satisfy a variety of requirements in biological applications. The hydrogels, consisting of helical nanofibers stabilized with disulfide bonds, are prepared and further injected for antitumor therapy. The results demonstrate that the helical fibrous hydrogel, without the addition of antigens, immune regulatory factors, and adjuvants, can activate T cell response and efficiently suppress tumor growth. Therefore, injectable hydrogels self-assembled by a combination of small peptides and biomacromolecules present a great potential for biomedical applications, especially for development of a new type of immuno-responsive materials toward antitumor therapy.

Canonical Wnt Signaling in CD11c+ antigen presenting cells controls inflammation-associated colon carcinogenesis and commensal homeostasis

Abstract The intestine contains high levels of the Wnt family of ligands, and the Wnt-signaling pathway is essential for intestinal homeostasis. Thus, aberrant Wnt/β-catenin-signaling may result in inflammatory bowel disease (IBD) and IBD-associated colon carcinogenesis. Although past studies have focused on this signaling pathway’s role in intestinal stem cell proliferation and epithelial cell maintenance, its role in shaping mucosal immune responses and commensal homeostasis in the gut remains unknown. In the present study, using a mouse model of colitis-associated colorectal cancer, we demonstrated that canonical Wnt-signaling in intestinal CD11c+ antigen presenting cells (APCs) controls intestinal inflammation and suppresses inflammation-driven colon carcinogenesis by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt co-receptors, low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6), in CD11c+ APCs in mice (LRP5/6ΔAPC mice) caused enhanced intestinal inflammation with increased tumor numbers in the colon due to increased production of pro-inflammatory cytokines and decreased expression of immune regulatory factors resulting from commensal dysbiosis. Mechanistically, loss of LRP5/6-mediated signaling in intestinal APCs resulted in enhanced effector T cell differentiation and decreased regulatory T cell differentiation. Furthermore, our study demonstrates that in LRP5/6ΔAPC mice conditional activation of β-catenin in intestinal APCs or in vivo blockade of IL-6 results in reduced intestinal inflammation with decreased colon tumor burden. These results reveal a mechanism by which intestinal APCs control gut inflammation and commensal homeostasis via canonical Wnt signaling.

Genetics and Molecular Biology of Epstein-Barr Virus-Encoded BART MicroRNA: A Paradigm for Viral Modulation of Host Immune Response Genes and Genome Stability.

Epstein-Barr virus, a ubiquitous human herpesvirus, is associated through epidemiologic evidence with common autoimmune syndromes and cancers. However, specific genetic mechanisms of pathogenesis have been difficult to identify. In this review, the author summarizes evidence that recently discovered noncoding RNAs termed microRNA encoded by Epstein-Barr virus BARF (BamHI A right frame) termed BART (BamHI A right transcripts) are modulators of human immune response genes and genome stability in infected and bystander cells. BART expression is apparently regulated by complex feedback loops with the host immune response regulatory NF-κB transcription factors. EBV-encoded BZLF-1 (ZEBRA) protein could also regulate BART since ZEBRA contains a terminal region similar to ankyrin proteins such as IκBα that regulate host NF-κB. BALF-2 (BamHI A left frame transcript), a viral homologue of the immunoglobulin and T cell receptor gene recombinase RAG-1 (recombination-activating gene-1), may also be coregulated with BART since BALF-2 regulatory sequences are located near the BART locus. Viral-encoded microRNA and viral mRNA transferred to bystander cells through vesicles, defective viral particles, or other mechanisms suggest a new paradigm in which bystander or hit-and-run mechanisms enable the virus to transiently or chronically alter human immune response genes as well as the stability of the human genome.

Myeloma Exosomes Prime the Microenvironment to Support Survival and Growth of Myeloma Cells

IntroductionMyeloma bone marrow serum primes healthy donor (HD) bone marrow cells to support the survival and growth of myeloma plasma cells (MMPC) in co-culture. Following interaction with the MMPC, the phenotype of the HD bone marrow stroma changes and the expression of immune regulatory factors is increased while expression of genes associated with osteoblastogenesis and WNT signaling is suppressed. The aim of this study was to determine whether exosomes in the myeloma bone marrow serum have a role in priming the microenvironment.MethodsSerum was separated from clotted bone marrow aspirates from myeloma patients obtained during scheduled clinic visits, and from healthy donors of similar ages.Exosomes were prepared from 1-2 ml of serum using Invitrogen’s Total Exosome Isolation kit according to manufacturer’s instruction. Exosomes were 30-130 nm in diameter as determined by Dynamic Light Scatter on a Brookhaven Instruments Corp. ZetaPlus Particle Sizing Software, and visualized by scanning electron microscopy. Small RNA (≤200 nucleotides) was isolated using Qiagen’s miRNeasy kits, quality and quantity assessed with Qubit 2. 96 microRNAs (miRs) were analyzed by qRT-PCR on Fluidigm’s BioMark platform using 96x96 multiplex chips and normalized to spiked in C. elegance miR mimetic.Primary CD138-expressing cells were isolated from EDTA-anticoagulated bone marrow aspirates. Luciferase-expressing stroma-dependent myeloma cells were described previously (Bam, BMC Cancer 2015:864). Healthy donor mesenchymal stem cells (MSC) were prepared from bone particles obtained during orthopedic surgery, and cultured in a-MEM supplemented with 10% FBS.Primary myeloma cells or luciferase-expressing stroma dependent myeloma cells were added to wells containing monolayers of MSC in α-MEM supplemented with 10% FBS in 96 well culture plates. For each experiment 3 conditions were used: A control group, a group to which 10% myeloma bone marrow serum was added, and a third group to which exosomes isolated from a matching bone marrow serum aliquot were added. The number of primary MMPC dictated that these experiments were carried out in duplicates, while the stroma dependent cells were plated in 5 replicates. Viable primary cells counts or luciferase bioluminescence were evaluated after 4 days of co-culture.ResultsExosomes supported survival and growth of primary MMPC in co-culture with HD MSC, but had no effect on MMPC cultured alone, indicating that the myeloma bone marrow serum exosomes prime the MSC to support the MMPC. Exosomes from 5 of the 7 patients supported growth of stroma-dependent myeloma cells co-cultured with HD MSC. In a representative experiment, the bioluminescence of stroma-dependent myeloma cells was 46% higher in co-cultures supplemented with bone marrow serum (p=7.3E-6) and 23% higher (p=2.0E-6) when exosomes were used. The effect was reproduced when primary MMPC were used: in a representative experiment, the number of primary MPC recovered from the co-cultures supplemented with bone marrow serum was 41% higher, and when supplemented with exosomes 28% higher than in controls. Primary MMPC did not survive in the absence of MSCs and bone marrow serum or exosomes did not enhance their survival.ConclusionsExosome from bone marrow serum of myeloma patients prime the bone marrow microenvironment to support survival and growth of primary MMPC. Bone marrow serum is more effective, indicating that other factors in the serum are also important. The differences in microRNA contents between healthy donor and myeloma exosomes may provide an insight of the regulatory mechanisms molding the myeloma bone marrow microenvironment. DisclosuresMorgan:Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding.

The Inflammatory Role of Platelets: Translational Insights from Experimental Studies of Autoimmune Disorders.

Beyond their indispensable role in hemostasis, platelets have shown to affect the development of inflammatory disorders, as they have been epidemiologically and mechanistically linked to diseases featuring an inflammatory reaction in inflammatory diseases like multiple sclerosis, rheumatoid arthritis and inflammatory bowel disorders. The identification of novel molecular mechanisms linking inflammation and to platelets has highlighted them as new targets for therapeutic interventions. In particular, genetic and pharmacological studies have identified an important role for platelets in neuroinflammation. This review summarizes the main molecular links between platelets and inflammation, focusing on immune regulatory factors, receptors, cellular targets and signaling pathways by which they can amplify inflammatory reactions and that make them potential therapeutic targets.