Th2-type Cytokines Research Articles

Ameliorating effect of CpG-ODN (oligodeoxynucleotide) against radiation-induced lung injury in mice.

While radiation-induced lung injury (RILI) is known to be progressed by Th2 skewed, pro-inflammatory immune response, there have been few therapeutic attempts through Th1 immune modulation. We investigated whether the immunostimulant CpG-oligodeoxynucleotide (CpG-ODN) would be effective against RILI by way of measuring reactive oxygen species (ROS) and nitric oxides (NO), histopathology, micro-three-dimensional computer tomography (CT), and cytokine profiling. We found that KSK CpG-ODN (K-CpG) significantly reduced histopathological fibrosis when compared to the positive control (PC) group (p < 0.01). The levels of ROS production in serum and splenocyte of PC group were significantly higher than that of K-CpG group (p < 0.01). The production of nitric oxide (NO) in CpG-ODNs group was higher than that of PC group. Last, cytokine profiling illustrated that the protein concentrations of Th1-type cytokines such as IL-12 and TNF-α as well as Th2-type cytokine IL-5 in K-CpG group inclined to be significantly (p < 0.001 or p < 0.01) higher than those of in PC group. Collectively, our study clearly indicates that K-CpG is effective against RILI in mice by modulating the innate immune response. To our knowledge, this is the first note on anti-RILI effect of human type, K-CpG, clinically implying the potential of immunotherapy for RILI control.

Evaluation of Anti-Colitis Effect of KM1608 and Biodistribution of Dehydrocostus Lactone in Mice Using Bioimaging Analysis.

Inflammatory bowel disease (IBD) is a chronic relapsing disorder modulated by numerous factors. Recent failures of drugs targeting single factors suggest that multitargeting drugs could be useful for the treatment of IBD. Natural medicines may be an alternative option for the treatment of IBD, owing to the complex nature of the disease. However, most natural medicines have poor in vitro and in vivo translational potential because of inadequate pharmacokinetic study. KM1608, a mixture of the medicinal plants Aucklandia lappa, Terminalia chebula, and Zingiber officinale, was examined for its anti-colitis effects and biodistribution using bioimaging. Dehydrocostus lactone, as a marker compound, was analyzed to assess the biodistribution of KM1608. KM1608 significantly attenuated the disease activity of dextran sodium sulfate-induced colitis in mice and suppressed inflammatory mediators such as myeloperoxidase, proinflammatory cytokines (TNF-α and IL-6), and the Th2-type cytokine IL-4 in the colon. Optical fluorescence imaging revealed that KM1608 was distributed in the intestinal area as a target organ. Collectively, our findings suggest that KM1608 is a potential therapeutic formulation for IBD.

Lactobacillus johnsonii-activated chicken bone marrow-derived dendritic cells exhibit maturation and increased expression of cytokines and chemokines in vitro

Lactobacillus species are typical members of gut microflora that immunomodulatory effects and can regulate a variety of immune cells, such as dendritic cells (DCs). Notably, DCs possess the unique ability to initiate primary immune responses. Notably, DCs possess the unique ability to initiate primary immune responses. In this study, we investigated the effects of Lactobacillus johnsonii (L. johnsonii) on the maturation and activation of chicken bone marrow-derived dendritic cells (chBM-DCs). The chBM-DCs generated from chicken bone marrow monocytes were stimulated using lethally irradiated L. johnsonii. L. johnsonii-stimulated chBM-DCs upregulated the expression of major histocompatibility complex class II (MHC-II), CD40, and CD86, decreased phagocytosis, and increased the ability to induce the proliferation of allogeneic T cells, which displayed a mature phenotype and function. Upon maturation with L. johnsonii, the expression of Th1-type cytokines [interleukin (IL)-12, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α)], a Th2-type cytokine (IL-10), pro-inflammatory cytokines (IL-1β and IL-6), and chemokines (CXCLi1 and CXCLi2) greatly increased; however, a high expression of IL-10 was only observed at mid-late time points for chBM-DCs stimulated with high doses of L. johnsonii. Moreover, L. johnsonii upregulated the mRNA levels of TLR2 and TLR5. These results reveal that L. johnsonii plays a potentially important role in modulating the immunological functions of chBM-DCs, suggesting that it influences and mediates immune responses in vitro.

Blocking the Notch signal transduction pathway promotes tumor growth in osteosarcoma by affecting polarization of TAM to M2 phenotype.

BackgroundOsteosarcoma is a primary malignant tumor that seriously affects the health and life of patients. It is of great clinical significance to explore the molecular mechanism of osteosarcoma development and develop the corresponding therapeutic targets. Th1/Th2 cytokines in the normal human body are in a state of dynamic balance. When this balance is destroyed, it is related to many diseases such as a tumor, autoimmune disease, microbial infection, transplant rejection, among many others.MethodThe model of mouse tumor-associated macrophage (TAM) was induced by being co-cultured with inducer granulocyte-macrophage colony stimulating factor (GM-CSF) and osteosarcoma S180 cells. The Notch1 knockout mice were obtained by gene targeting technology. The distribution of M1- and M2-type TAMs in the tumor was visualized by immunofluorescence staining. And the western-blot testing was used to detect and quantified the protein level of Notch1 and Th1/Th2-type cytokines.ResultsIn this study, the polarization of TAMs to the M2 phenotype occurred after coculture with osteosarcoma S180 cells and secretion level Th1/Th2-type cytokines changed. Also, the expression level of Notch1 reduced significantly. Further, the critical transcription factor Notch1 of the Notch signaling pathway was knocked out in mice. The tumor volume of Notch1 knockout mice was significantly more extensive than of the control mice. The results of microstructural observation on tumor showed that M2-type TAMs infiltrated into tumor increased with increased expression of Th2-type cytokines, but M1-type TAMs reduced with reduced expression of Th1-type cytokines.ConclusionsAccording to our results, the Notch signal transduction pathway participates in tumor occurrence and growth with a negative role by maintaining Th1/Th2 balance.

1537P Phase I/II study with CXCL12 inhibitor NOX-A12 and pembrolizumab in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer

NOX-A12 is a CXCL12 inhibitor that prevents activation of receptors CXCR4 and CXCR7 and blocks formation of CXCL12 concentration gradients. The Opera study is a phase I/II study to investigate pharmacodynamics of NOX-A12 monotherapy and safety and efficacy of a combination with pembrolizumab in metastatic MSS colorectal (CRC) and pancreatic (PaC) cancer where PD-1 inhibition alone has no clinical benefit. Patients received NOX-A12 twice weekly for 2 weeks. Biopsies were taken from liver metastases before and after treatment to analyze immune cells and cytokines. Subsequently, patients received repeated 21-day cycles of NOX-A12 and pembrolizumab. 11 patients had CRC and 9 PaC. All patients were heavily pretreated with 5 (CRC) and 3 mean lines (PaC) of prior treatment. Best response to last prior treatment was progressive disease in 95% of cases. The AE profile was comparable with the pembrolizumab profile and typical for the underlying diseases. 2 weeks of NOX-A12 monotherapy induced changes in the tumor microenvironment: an upregulated cassette of Th1 cytokines in approx. 50% of patients, being associated with better clinical stabilization. Biopsy analysis before and after NOX-A12 monotherapy showed agglomeration of T cells and migration of T cells towards cancer cells in patients where NOX-A12 induced a Th1-type response. These trends suggest that CXCL12 inhibition increased effector immune cell infiltration of the tumor, resulting in a more effective immune response. 25% of patients achieved stable disease (SD) and long term disease control. Remarkably, 7 patients showed prolonged time on treatment vs. prior therapy. Median PFS was 1.87 months, OS was 42% at 6 and 22% at 12 months. Agglomeration of T cells within tumors was seen in ∼half of the patients where NOX-A12 had induced a Th1-type cytokine response. This was accompanied by reduced distances between T and cancer cells, i.e. more engaged T cells. Increased infiltration of effector immune cells into tumor was associated with SD in 25% of patients and prolonged time on treatment vs. prior therapy for 35% of pts. Safety profile of the combination was consistent with that of pembrolizumab in advanced cancer patients.

IFI27 as a potential indicator for severe Enterovirus 71-infected hand foot and mouth disease

The pathogenesis of Enterovirus 71 (EV71)-induced severe hand foot and mouth disease (HFMD) has not yet been clearly established. Further study into the mechanisms underlying host immune responses to EV71 infection and identifying important predictors will be crucial to antiviral treatment and early recognition of severe HFMD. The present study establishes that T help (Th)1 type, Th2 type, and Th17 type cytokine levels in serum of peripheral blood from patients with severe HFMD is higher than in peripheral blood from healthy subjects. The most significant increase occurred as the IL-6. In order to identify the important molecules in peripheral blood mononuclear cells (PBMCs) from severe HFMD patients, we performed transcriptome sequencing analysis of PBMC from severe HFMD patients and compared them to healthy controls. Interferon α-inducible protein 27 (IFI27) and cluster of differentiation 27 (CD27) were found to be the most significant differentially expressed gene. Finally, IFI27 was proved to be present at higher levels in patients with severe HFMD than in patients with mild HFMD. Our results suggest that IFI27 may be an indicator of the severity of cases EV71-induced HFMD.

Effects of Maillard reaction on structural modification and potential allergenicity of peanut 7S globulin (Ara h 1).

Ara h 1 is a major food allergen in peanuts. Recently, many studies have revealed that the Maillard reaction (MR) affects the allergenicity of food proteins. To investigate the influence of the MR on the allergenicity of Ara h 1, R-Ara h 1 was processed with glucose in dry heating conditions for different periods. The extent of the MR was assessed by four methods. The changes in secondary and tertiary structures were characterized through spectroscopy assays. Advanced glycation end products (AGE) structures were identified by protein sample dry heating for 60 min, indicating the formation of AGE-Ara h 1. Simulated gastric fluid (SGF) digestion analysis showed that AGE-Ara h 1 has higher resistance to peptic digestion than R-Ara h 1. The BALB/c mouse model was also utilized to explore the effect of the MR on the allergenicity of Ara h 1, and the results showed that the Th2-type cytokines, antibodies, and histamine content increased, and there was a greater degree of degranulation of rat basophilic leukemia (RBL) cells in the AGE-Ara h 1 group compared with the R-Ara h 1 group. During the process of dry heating, proteins participated in the MR with changes in secondary and tertiary structures. The condition applying a temperature of 100 °C for 60 min caused the formation of AGE-Ara h 1. Simulated gastric fluid digestion analysis showed that AGE-Ara h 1 had a greater resistance to peptic digestion than R-Ara h 1. The BALB/c mouse model showed that AGE-Ara h 1 had more allergenicity, indicating that the MR could enhance the allergenicity of Ara h 1. © 2020 Society of Chemical Industry.

Abstract CT117: Phase 1/2 study with CXCL12 inhibitor NOX-A12 and pembrolizumab in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer

Abstract Background: NOX-A12 is an inhibitor of the chemokine CXCL12 which prevents engagement with its receptors CXCR4 and CXCR7 and blocks the formation of chemotactic CXCL12 concentration gradients in tissue. The Opera study (NCT03168139) is a Phase 1/2 study to evaluate pharmacodynamic effects and safety of monotherapy with NOX-A12 as well as safety and efficacy of a combination with pembrolizumab in metastatic microsatellite-stable colorectal and pancreatic cancer where PD-1 inhibition alone has not shown clinical benefit. Methods: Patients received 300 mg NOX-A12 twice weekly during a 2-week monotherapy phase. Biopsies were taken from liver metastases before and after NOX-A12 monotherapy for analysis of immune cell infiltration and cytokine signature. In a combination phase, patients received repeated 21-day cycles of 300 mg NOX-A12 and 200 mg pembrolizumab until progression or intolerable toxicity. Results: 11 of the patients had CRC and 9 PaC. All patients were heavily pretreated with 5 (colorectal cancer) and 3 median lines (pancreatic cancer) of prior treatment. Best responses to last prior treatment was progressive disease for 95% of patients. The AE profile was comparable with the pembrolizumab profile or typical for the underlying diseases. Two weeks of NOX-A12 monotherapy induced Th1 cytokines (IFNγ, IL-2, IL-16) in approx. half of the patients. Comparison of tumor biopsies from before and after NOX-A12 monotherapy shows trends towards (i) agglomeration of T cells and (ii) migration of T cells towards cancer cells in the patient sub-group where NOX-A12 induced a Th1-type cytokine response. These trends are consistent with preclinical data (Feig 2013, PNAS 110:20212; Zboralski 2017, Cancer Immunol Res 5:950), suggesting that neutralization of CXCL12 signaling - either through one or both receptors - increased infiltration of effector immune cells into tumor tissue and resulted in a more effective immune response. Although there were no objective responses, 25% of patients achieved stable disease; 7 patients showed prolonged time on treatment vs. the prior line of therapy. Median progression-free survival was 1.87 months, overall survival was 42% at 6 months and 22% at 12 months. Conclusions: A trend towards agglomeration of T cells within tumors was observed in about half of the patients where NOX-A12 had induced a Th1-type cytokine response. This agglomeration was accompanied by reduced distances between T cells and cancer cells, suggesting increased infiltration of effector immune cells into tumor tissue. Treatment with NOX-A12 plus pembrolizumab in the combination therapy part of the study resulted in stable disease in 25% of patients, and prolonged time on treatment vs. prior therapy for 35% of patients. The safety profile of the combination therapy was consistent with that of pembrolizumab in advanced cancer patients. Citation Format: Niels Halama, Anja Williams, Ulrike Prüfer, Anna Frömming, Diana Beyer, Dirk Eulberg, Jarl Ulf Jungnelius, Aram Mangasarian. Phase 1/2 study with CXCL12 inhibitor NOX-A12 and pembrolizumab in patients with microsatellite-stable, metastatic colorectal or pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT117.

Interleukin 25, thymic stromal lymphopoietin and house dust mites in pathogenesis of atopic dermatitis.

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in the world. It is characterized by recurrent eczematous skin lesions, fluctuating course and chronic pruritus. Increasing evidence suggest that AD is more common in adults than previously thought. The disease is characterized by an impaired skin barrier, aberrant Th2-type cytokine production and intensive pruritus. Epithelial keratinocytes constitute the first physical, chemical and immunological barrier, classified as a part of the innate defense system. These keratinocytes secrete various factors, e.g. alarmins such as thymic stromal lymphopoietin (TSLP) and interleukin 25 (IL-25). Serum levels of substance P (SP) have been reported to be increased in patients with AD and correlated with itch intensity. Several previous studies reported a positive association between AD severity and house dust mites (HDM) sensitization. The aim of the study was to analyze IL-25, TSLP and SP concentrations in blood serum of adult patients with severe AD, depending on the degree of allergy to HDM. There were 31 adult AD patients enrolled into the study and a control group that consisted of 20 healthy subjects. AD was diagnosed on the basis of Hanifin and Rajka criteria. SCORing Atopic Dermatitis (SCORAD) and visual analogue (VAS) scores were used to assess the intensity of pruritus and blood content of specific IgE to HDM, as well as TSLP, IL-25 cytokines and SP was measured. Our study presents the evidence that IL-25 serum concentration is increased in patients with atopic dermatitis and this cytokine plays an important role in pathogenesis of this disease. HDM could stimulate the release of IL-25 which aggravates the disease severity. Our results corroborate previous findings on the role of TSLP in atopic dermatitis.

Potent T-cell mediated immune response against Legionella pneumophila in mice following vaccination with detoxified lipopolysaccharide non-covalently combined with recombinant flagellin A and peptidoglycan-associated lipoprotein

BackgroundLegionella pneumophila is a Gram-negative intracellular bacterium and the cause of an atypical pneumonia in humans – legionnaire's disease. Immunological assessment of bacterial antigens clarifies the way that host may develop protection against the pathogen. Lipopolysaccharide (LPS) is the main antigen of Gram-negative bacteria but is less studied because of its carbohydrate nature. Here, we immunized mice with detoxified LPS in combination with immunogenic proteins and looked into the result of bacterial challenge. MethodsLPS of L. pneumophila was extracted by hot phenol-water method. Purified LPS was detoxified by sodium hydroxide alkaline procedure. BALB/c mice were immunized mainly with non-covalent combination of detoxified LPS (dLPS) and either of recombinant FlaA or PAL separately. Afterwards, specific serum IgG was assessed by ELISA. Mice were challenged intravenously with sublethal dose of L. pneumpphila then splenocytes were cultured. Cytokine responses of splenocytes were analyzed by ELISA. ResultsPolysaccharide antigen did not elicit significant serum IgG. Combination of the dLPS with recombinant FlaA and PAL led to risen IgG and its subclasses (IgG1, IgG2a and IgG2b) against polysaccharide. Mice immunized with combination of the dLPS and recombinant proteins showed significant elevation of cytokine responses in splenocyte culture after being challenged with L. pneumophila. ConclusionsOur results suggest that combination of polysaccharide antigen derived from Legionella LPS may confer raised cell-mediated responses against the pathogen when combined with Th-1 stimulating protein antigens. Although not covalently bond, Legionella detoxified LPS combination with recombinant FlaA and PAL effectively elicited Th-1 type cytokines and humoral responses against L. pneumophila in BALB/c mice.

Costello syndrome model mice with a Hras G12S mutation are susceptible to develop house dust mite-induced atopic dermatitis

Costello syndrome is an autosomal dominant disorder that is caused by germline HRAS mutations. Patients with Costello syndrome present craniofacial abnormalities, cardiac defects, and cancer predisposition, as well as skin abnormalities, including papillomas, keratosis pilaris, and eczematous dermatitis. However, the mechanisms underlying the dermatological abnormalities remain unclear. Here, we demonstrated that knock-in mice expressing an Hras G12S mutation (HrasG12S/+ mice) are susceptible to develop atopic dermatitis (AD)-like skin lesions, including eczema, pruritus, elevated serum IgE levels, acanthosis, and the infiltration of mast cells, basophils, and type-2 innate lymphoid cells in the dermis, after stimulation with house dust mite allergens (Dermatophagoides farinae, Dfb). Reduced skin barrier function, increased proliferation of phosphorylated ERK (p-ERK)-positive epidermal cells, and increased Th2-type cytokines as well as epithelial cell-derived cytokines, including IL-33, were observed in the skin tissue of HrasG12S/+ mice compared with Hras+/+ mice. Cultured HrasG12S/+ keratinocytes exhibited increased IL-33 expression after Dfb stimulation. PD0325901, an MEK inhibitor, ameliorated AD-like symptoms in HrasG12S/+ mice, showing decreased proliferation of p-ERK-positive epidermal cells and decreased expression of IL-33. Our findings indicate that the epidermis of HrasG12S/+ mice stimulated by Dfb strongly induced IL-33 expression and type-2 innate lymphoid cells, resulting in AD-like skin lesions. These results suggest that the epidermis of HrasG12S/+ mice are prone to development of eczematous dermatitis stimulated with house dust mite allergens.

Modulation of Th1/Th2 Cytokine Balance by Quercetin In Vitro.

Background: Allergic rhinitis (AR) is well known to be an IgE-mediated chronic inflammatory disease in the nasal wall, which is primarily mediated by Th2-type cytokines such as IL-4, IL-5, and IL-13. Although quercetin is also accepted to attenuate the development of allergic diseases such as AR, the influence of quercetin on Th2-type cytokine production is not well understood. The present study was designed to examine whether quercetin could attenuate the development of AR via the modulation of Th2-type cytokine production using an in vitro cell culture technique. Methods: Human peripheral-blood CD4+ T cells (1 × 106 cells/mL) were cultured with 10.0 ng/mL IL-4 in the presence or absence of quercetin. The levels of IL-5, IL-13, and INF-γ in 24 h culture supernatants were examined by ELISA. The influence of quercetin on the phosphorylation of transcription factors NF-κB and STAT6, and mRNA expression for cytokines were also examined by ELISA and RT-PCR, respectively. Results: Treatment of cells with quercetin at more than 5.0 μM inhibited the production of IL-5 and IL-13 from CD4+ T cells induced by IL-4 stimulation through the suppression of transcription factor activation and cytokine mRNA expression. On the other hand, quercetin at more than 5.0 μM abrogated the inhibitory action of IL-4 on INF-γ production from CD4+ T cells in vitro. Conclusions: The immunomodulatory effects of quercetin, especially on cytokine production, may be responsible, in part, for the mode of therapeutic action of quercetin on allergic diseases, including AR.

Investigating the role of L. pnuemophila LPS derivatives in formation of specific cell-mediated immune responses against the pathogen

Legionella pneumophila is a Gram-negative intracellular bacterium and causes legionnaire's disease an –atypical pneumonia in humans. Lipopolysaccharide (LPS) is the main antigen of Gram-negative bacteria but is less studied because of its carbohydrate nature. Here, we immunized mice with detoxified LPS and O-antigen polysaccharide in combination with bovine serum albumin (BSA) and explored the immunological responses of mice to the bacterial infection.LPS of L. pneumophila was extracted by hot phenol-water method. Purified LPS was detoxified by sodium hydroxide alkaline procedure. O-polysaccharide antigen (OPS) obtained by acetic acid treatment of LPS. BALB/c mice were immunized mainly with non-covalent combination of detoxified LPS (dLPS) or OPS with BSA separately. Pure polysaccharide antigens did not elicit significant serum IgG against LPS. Combination of the dLPS and OPS with BSA resulted in risen IgG and its subclasses (IgG1 and IgG2a) against lipopolysaccharide. Mice were challenged intravenously with sublethal dose of L. pneumpphila. Then, splenocytes were cultured and cytokine responses of splenocytes to pathogenic Legionella was studied by ELISA. Mice immunized with combination of the dLPS or OPS and BSA showed significant elevation of cytokine responses to pathogenic L. pneumophila.Our results suggest that combination of the polysaccharide antigen derived from Legionella LPS may confer raised cell-mediated responses against the pathogen when combined with a protein antigen which is capable of eliciting cell-mediated responses. Although not covalently bond, Legionella polysaccharides combined with BSA effectively elicited Th-1 type cytokines and humoral responses against L. pneumophila in BALB/c mice.

Bulleyaconitine A inhibits the lung inflammation and airway remodeling through restoring Th1/Th2 balance in asthmatic model mice.

The current study aimed to study the effects of Bulleyaconitine A (BLA) on asthma. Asthmatic mice model was established by ovalbumin (OVA) stimulation, and the model mice were treated by BLA. After BLA treatment, the changes in lung and airway resistances, total and differential leukocytes in the bronchoalveolar lavage fluid (BALF) were detected, and the changes in lung inflammation and airway remodeling were observed. Moreover, the secretion of IgE, Th1/Th2-type and IL-17A cytokines in BALF and serum of the asthmatic mice were determined. The resuts showed that BLA attenuated OVA-induced lung and airway resistances, inhibited the inflammatory cell recruitment in BALF and the inflammation and airway remodeling of the asthmatic mice. In addition, BLA suppressed the secretion of IgE, Th2-type cytokines, and IL-17A, but enhanced secretions of Th1-type cytokines in BALF and serum. The current study discovered that BLA inhibited the lung inflammation and airway remodeling via restoring the Th1/Th2 balance in asthmatic mice.

Effects of intrauterine exposure to maternal-derived HBeAg on T cell immunity in cord blood.

Immature immune system and immune tolerance induced by exposure to HBeAg in utero and/or shortly after infection in newborns were reportedly the causes of chronic HBV infection. To investigate the effect of maternal-derived HBeAg on neonatal T cell immunity, we analysed and compared T cell phenotypes and functions among neonates born to HBsAg+ /HBeAg+ mothers (HBeAg+ neonates), HBsAg+ /HBeAg- mothers (HBeAg- neonates) and healthy control mothers (HC neonates), using flow cytometry. The results showed that neonatal T cell phenotypes were similar regardless of HBeAg exposure. Upon anti-CD3 and anti-CD28 stimulation in HBeAg+ neonates, CD4+ T cell production of IFN-γ (P<.05) was significantly enhanced, while CD8+ T cells secreted significantly more IL-2 compared with those in HBeAg- and HC groups (P<.05). Moreover, similar levels of IFN-γ and IL-10 were observed in the culture supernatant after stimulation with rHBsAg, rHBcAg or rHBeAg among HBeAg+ , HBeAg- and HC neonates, whereas HBeAg+ neonates produced more TNF-α than HBeAg- neonates upon stimulation with rHBcAg. In conclusion, the results indicated that the HBsAg+ /HBeAg+ maternal environment did not influence the phenotypes of cord blood T cells but boosted neonatal non-specific Th1-type cytokine production.

Ganoderma lucidum polysaccharide (GLP) enhances antitumor immune response by regulating differentiation and inhibition of MDSCs via a CARD9-NF-κB-IDO pathway.

A homogeneous polysaccharide (GLP), with an average molecular weight of 4.44 × 104 Da, was isolated and purified from the fruiting bodies of Ganoderma lucidum. In this work, we examined the antitumor activities of GLP using a mouse Lewis lung cancer (LLC) model and explored possible molecular pathways involved in its immunomodulatory mechanism on tumor–host interaction. GLP administration (25 and 100 mg/kg) significantly inhibited tumor growth, as evidenced by the decreased tumor volume and tumor weight, as well as histological features of tumor tissues with concomitant down-regulation of proliferating cell nuclear antigen (PCNA) proliferative marker. Less myeloid-derived suppressor cells (MDSCs) were accumulated in both spleen and tumor tissues from GLP-treated mice. In contrast, the percentage of CD4+ and CD8+ T cells together with the production of Th1-type cytokines (IFN-γ and IL-12) was increased in the spleen of LLC-bearing mice following GLP administration. Furthermore, GLP administration reversed the attenuated expression of CARD9, p-Syk and p-p65, and increased indoleamine 2,3-dioxygenase (IDO) protein expression in MDSCs of LLC-bearing mice. Collectively, our data demonstrated the first time that GLP induced the differentiation of MDSCs and inhibited the accumulation of MDSCs via CARD9-NF-κB-IDO pathway, thus prevented lung cancer development.

Characterization of exosome-like vesicles derived from Taenia pisiformis cysticercus and their immunoregulatory role on macrophages

BackgroundTaenia pisiformis is one of the most common intestinal parasites in canines, and leads to serious economic losses in the rabbit breeding industry. Exosome-like vesicles from parasites play crucial roles in host-parasite interactions by transferring cargo from parasites to host cells and by modulating host immunological response through inducing production of host-derived cytokines. Nevertheless, the mechanism by which exosome-like vesicles from T. pisiformis cysticercus regulate the macrophage immune response remains unknown.MethodsUsing ultracentrifugation, we isolated exosome-like vesicles from excretory/secretory products (ESP) of T. pisiformis cysticercus. The morphology and size of purified vesicles were confirmed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The components of proteins and miRNAs within these vesicles were identified by proteomic analysis and high-throughput small RNA sequencing. The biological function of targets of exosomal miRNAs was predicted by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Moreover, the expression of Th1- and Th2-type immune response associated cytokines in RAW264.7 macrophages were evaluated by qPCR and ELISA. We found that exosome-like vesicles were typical cup-shaped vesicles with diameters from 30 to 150 nm. A total of 87 proteins were identified by proteomic analysis, including proteins prominently associated with exosome-like vesicles biogenesis and vesicle trafficking. 41 known miRNAs and 18 novel miRNAs were identified in the exosome-like vesicles. Eleven selected miRNAs, including 7 known miRNAs (miR-71-5p, miR-10a-5p, miR-let-7-5p, miR-745-3p, miR-219-5p, miR-124-3p and miR-4989-3p) and 4 novel miRNAs (novel-mir-3, novel-mir-7, novel-mir-8 and novel-mir-11) were validated to exist in metacestiodes and exosome-like vesicles of T. pisiformis cysticercus by qPCR. The functions of most targets of exosomal miRNAs were mainly associated with signal transduction and the immune system. Additionally, T. pisiformis cysticercus-derived vesicles induced the production of IL-4, IL-6, IL-10, IL-13 and Arg-1, but downregulated the expression of IL-12, IFN-γ and iNOS in RAW264.7 macrophages.ConclusionsWe demonstrated that proteins and miRNAs enclosed within exosome-like vesicles from T. pisiformis cysticercus have immunomodulatory functions. Furthermore, exosome-like vesicles were shown to induce the macrophage Th2-type immune response in vitro. Our study suggests that exosome-like vesicles play an important role in the interaction between cysticerci and their hosts.

Enkephalins as a therapeutic intervention for visceral leishmaniasis

Visceral leishmaniasis (VL) is a neglected tropical disease with high incidence of mortality and morbidity. The emergence of drug resistant parasites, severe toxic effects of existing anti-leishmanials, and nonexistence of an effective vector control measures and human vaccine(s) against VL poses a serious problem to VL treatment and control. In VL, the disease pathogenicity is correlated with the up-regulation of Th2 cytokines (IL-4, IL-10 and TGF-β), which can deactivate macrophages and favors the growth of intracellular parasite and disease clearance is expedited through the increased levels of Th1 mediated cytokines (IL-12) which can activate macrophages to release IFN-γ; stimulates inducible NOS to release NO and kills the leishmania parasite. Enkephalins (ENKs), are endogenous neuropeptides with immune stimulatory properties. ENKs and its fragmented peptides at lower concentrations activates Th1 type cytokines and inhibits Th2 type cytokines, which may be helpful in parasite clearance. ENKs in combination with currently available anti-leishmanial drugs may be helpful in reducing the toxicity and duration of therapy. Therefore, we hypothesized that the ENKs alone or in combination with current recommended anti-leishmanial agents might be effective in the treatment of VL with enhanced efficacy and safety profile.

AST-008: A novel approach to TLR9 agonism with PD-1 blockade for anti-PD-1 refractory Merkel cell carcinoma (MCC) and cutaneous squamous cell carcinoma (CSCC).

TPS3164 Background: AST-008 is a potent spherical nucleic acid configuration of a toll-like receptor (TLR) 9 agonist oligonucleotide being developed for the treatment of MCC and CSCC in patients (pts) progressing on immune checkpoint inhibitor (CPI) monotherapy. Previously presented data suggests AST-008 elicits a Th1-type cytokine response and immune cell activation. This parallel-arm phase II design will permit assessment of efficacy in two advanced skin cancers with high unmet medical need. Methods: The study has an open label, multicenter phase Ib/II dose escalation/expansion design. The phase 1b dose escalation used a 3+3 design with increasing doses of intratumoral (IT) AST-008 plus standard flat dose pembrolizumab. The phase II dose expansion is using the recommended regimen (RP2D) of IT AST-008 plus flat dose pembrolizumab or cemiplimab to treat two cohorts of pts with advanced/metastatic MCC or CSCC, respectively. The key objective of the phase II expansion is to provide an estimate of preliminary efficacy of IT AST-008 with pembrolizumab or cemiplimab in pts whose disease progressed on a single-agent CPI therapy. Endpoints also include safety, pharmacokinetic and pharmacodynamic assessments. Pts must have at least two evaluable lesions by RECIST v1.1. One or more lesions may be injected with AST-008, but one lesion (the “witness” lesion) must remain un-injected throughout the study to differentially assess effects of AST-008 on the injected and witness lesions. The RP2D of AST-008 was derived from the dose escalation phase together with pembrolizumab. That RP2D is being used not only in the MCC cohort where AST-008 is combined with pembrolizumab, but also in the CSCC cohort where AST-008 is combined with cemiplimab. To ensure safety of the AST-008/cemiplimab combination, the first 6 pts in the CSCC cohort will be monitored for dose limiting toxicities through the first 5 weeks of dosing, and then the data review committee will assess safety before approving additional patient enrollment to that cohort. Each expansion cohort will enroll up to 29 pts with a Simon two-stage optimal design. In the first stage, 10 pts will be accrued. If there are 0 responses in these 10 pts, enrollment in that arm will be stopped. Otherwise, 19 additional pts will be accrued for a total of 29. The null hypothesis will be rejected if 4 or more responses are observed in 29 pts. This design yields a type I error rate of 0.05 and power of 80% when the true response rate is 20%. The planned phase II enrollment is 58 pts across about 15 US-based sites. Clinical trial information: NCT03684785 .

ESX5 associated PE proteins of Mycobacterium tuberculosis drives Th1 type polarization: an immunomodulatory paradigm

Abstract Mycobacterium tuberculosis (M.tb) harbors various effector molecules that affect human immune system. There exists a varied difference between the slow growing pathogenic mycobacteria compared to the fast growing non-pathogenic strains. One marked difference is the presence of ESX5, a type VII secretion system that is found to be most recently evolved and has been acquired by the pathogenic M.tb. ESX5 is involved in the secretion of an enigmatic PE/PPE family of proteins that are exclusive to the genus mycobacteriacea. PE/PPE are the effector virulent molecules that play vivid role in modulating host immune system. ESX5 locus has 2 pe genes and 3 ppe genes. Of the translated products, two PE proteins are paralogs, but contrastingly found to have different PPE binding partners as these proteins function as heterodimers, homodimers and individual proteins. THP1 macrophages (differentiated human monocytes) when treated with different micro-concentrations of PE 1X and PE 1Y induce differential Th1 type cytokine responses. PE 1X and PE 1Y induced stronger TNF-α response at 24hrs of protein exposure that increased markedly at 48hrs. IL-12 p70 was found to decrease from 24hrs to 48hrs during PE 1X treatment whereas PE 1Y exposure led to the secretion of IL-12 p70 which was constantly maintained from 24hrs to 48hrs of exposure time. PE 1X specifically mediated TLR2 dependent activation of macrophages that was responsible for the secretion of pro-inflammatory cytokines. On the contrary PE 1Y activated macrophages through TLR4 dependent cascade. These results therefore, conclude that PE 1X and PE 1Y contribute profoundly towards Th1 polarization of the immune response.