To Toll-like Receptor Research Articles

Novel Protein-Based Pneumococcal Vaccines: Assessing the Use of Distinct Protein Fragments Instead of Full-Length Proteins as Vaccine Antigens.

Non-serotype-specific protein-based pneumococcal vaccines have received extensive research focus due to the limitations of polysaccharide-based vaccines. Pneumococcal proteins (PnPs), universally expressed among serotypes, may induce broader immune responses, stimulating humoral and cellular immunity, while being easier to manufacture and less expensive. Such an approach has raised issues mainly associated with sequence/level of expression variability, chemical instability, as well as possible undesirable reactogenicity and autoimmune properties. A step forward employs the identification of highly-conserved antigenic regions within PnPs with the potential to retain the benefits of protein antigens. Besides, their low-cost and stable construction facilitates the combination of several antigenic regions or peptides that may impair different stages of pneumococcal disease offering even wider serotype coverage and more efficient protection. This review discusses the up-to-date progress on PnPs that are currently under clinical evaluation and the challenges for their licensure. Focus is given on the progress on the identification of antigenic regions/peptides within PnPs and their evaluation as vaccine candidates, accessing their potential to overcome the issues associated with full-length protein antigens. Particular mention is given of the use of newer delivery system technologies including conjugation to Toll-like receptors (TLRs) and reformulation into nanoparticles to enhance the poor immunogenicity of such antigens.

Maturing Human CD127+ CCR7+ PDL1+ Dendritic Cells Express AIRE in the Absence of Tissue Restricted Antigens.

Expression of the Autoimmune regulator (AIRE) outside of the thymus has long been suggested in both humans and mice, but the cellular source in humans has remained undefined. Here we identify AIRE expression in human tonsils and extensively analyzed these “extra-thymic AIRE expressing cells” (eTACs) using combinations of flow cytometry, CyTOF and single cell RNA-sequencing. We identified AIRE+ cells as dendritic cells (DCs) with a mature and migratory phenotype including high levels of antigen presenting molecules and costimulatory molecules, and specific expression of CD127, CCR7, and PDL1. These cells also possessed the ability to stimulate and re-stimulate T cells and displayed reduced responses to toll-like receptor (TLR) agonists compared to conventional DCs. While expression of AIRE was enriched within CCR7+CD127+ DCs, single-cell RNA sequencing revealed expression of AIRE to be transient, rather than stable, and associated with the differentiation to a mature phenotype. The role of AIRE in central tolerance induction within the thymus is well-established, however our study shows that AIRE expression within the periphery is not associated with an enriched expression of tissue-restricted antigens (TRAs). This unexpected finding, suggestive of wider functions of AIRE, may provide an explanation for the non-autoimmune symptoms of APECED patients who lack functional AIRE.

Glucose as a DAMP, Danger Associated Molecular Pattern: A New Proposition of Glucose Molecule in Inflammation-Associated Diabetes

Inflammation is the human body’s defense mechanism to protect from foreign invaders- yet is also the causal agent of an array of diseases that immensely burden our society today. The innate immune response is a nonspecific mechanism through which inflammatory cells (e.g. neutrophils, macrophages, etc.), destroy pathogens such as bacteria, fungi, and viruses, and also respond to internal tissue injury. The death of local tissues through necrosis can lead to the introduction of molecular sequences normally found on the inside of the cell – to the extracellular environment. These sequences are termed damage associated molecular patterns (DAMPs), and can bind to toll like receptors (TLRs) on inflammatory cells to propagate a pro-inflammatory response through the release of cytokines and chemoattractants. It is established that intracellular molecules such as DNA, histones, and ATP act as DAMPs upon extracellular release.1 However, the potential of glucose as a DAMP is a research target that requires further investigation.

Mechanisms and Therapeutic Targets of Depression After Intracerebral Hemorrhage.

The relationship between depression and intracerebral hemorrhage (ICH) is complicated. One of the most common neuropsychiatric comorbidities of hemorrhagic stroke is Post-ICH depression. Depression, as a neuropsychiatric symptom, also negatively impacts the outcome of ICH by enhancing morbidity, disability, and mortality. However, the ICH outcome can be improved by antidepressants such as the frequently-used selective serotonin reuptake inhibitors. This review therefore presents the mechanisms of post-ICH depression, we grouped the mechanisms according to inflammation, oxidative stress (OS), apoptosis and autophagy, and explained them through their several associated signaling pathways. Inflammation is mainly related to Toll-like receptors (TLRs), the NF-kB mediated signal pathway, the PPAR-γ-dependent pathway, as well as other signaling pathways. OS is associated to nuclear factor erythroid-2 related factor 2 (Nrf2), the PI3K/Akt pathway and the MAPK/P38 pathway. Moreover, autophagy is associated with the mTOR signaling cascade and the NF-kB mediated signal pathway, while apoptosis is correlated with the death receptor-mediated apoptosis pathway, mitochondrial apoptosis pathway, caspase-independent pathways and others. Furthermore, we found that neuroinflammation, oxidative stress, autophagy, and apoptosis experience interactions with one another. Additionally, it may provide several potential therapeutic targets for patients that might suffer from depression after ICH.

Myeloma Cells Have the Ability to Suppress Th1-Inducing Capacity but Enhance Th2-Mediated Response of Dendritic Cells By Producing Factors Other Than Soluble SLAMF7

Background: Lenalidomide (LEN), as a backbone drug for multiple myeloma (MM), has both direct tumoricidal and immunomodulatory effects in MM. LEN displays immunopotentiating activity, including augmentation of T and natural killer cell function and to induce selective reduction of regulatory T cells. Thus, the immune-based cancer treatments are currently important in MM therapy. Since myeloma cells together with immune cells are immersed in the pathogenic micro-environment, understanding the influence of micro-environment against myeloma cells and immune system may lead to the development of novel treatment strategies for MM. Series of analyses have clarified a functional plasticity of dendritic cells (DCs) to induce Th1 or Th2 response and DCs are pivotal in orchestrating both innate and acquire immune responses also in the anti-tumor immunity. Here, we focused on the effects of myeloma environment to modulate the human myeloid DCs (mDCs), which are the major regulators to induce Th1 or Th2 responses. Soluble form of SLAMF7 (sSLAMF7) is a pivotal environmental component of MM and functions as a self-ligand of myeloma cells. sSLAMF7 has been detected in the serum of patients with MM at higher levels than healthy individuals and therefore may presumably play some roles in pathophysiological condition of MM. To evaluate the functions of soluble factors/components derived from myeloma cells, we analyzed whether supernatants of myeloma cell lines could modulate mDC functions. Methods: Using cell sorting, blood human CD11+ mDCs were isolated from healthy adult volunteers. The DC functions were analyzed after culture for 24 h with supernatant of myeloma cell lines, NCI-H929 (H929) or MM.1R. This study was approved by the Institutional Review Board of Kansai Medical University.Results: We here found that both supernatants of myeloma cell lines, H929 and MM.1R did not affect CD86 expression on mDCs in response to toll-like receptor (TLR)-ligand (R848), but upregulated their CD86 expression in response to Th2-inducing cytokine, thymic stromal lymphopoietin (TSLP). Of note, the supernatants inhibited mDC-derived IL-12 production in response to R848 (p < 0.05: H929; 25-30% of cont. and MM1.R; less than 50 % of cont.), while in response to TSLP, dose-dependently enhanced the Th2-recruiting chemokine CCL17/TARC production (p < 0.05: H929; 130- 210 % of cont. and MM1.R; 120-185 % of cont.), which functions as chemoattractant for memory Th2 cells and contribute to promote humoral response. In addition, we revealed that these capacities were further enhanced by the addition of LEN at clinical in vivo plasma concentration of 1 µM. Thus, MM micro-environment could have the potential to shift DC-mediated response from Th1 to Th2. To clarify whether sSLAMF7 in the supernatant of myeloma cell lines play a role in the Th1/Th2-modulating effect, we calculated the sSLAMF7 concentration in the supernatants and analyzed an inhibitory effect of elotuzumab for the DC function induced by the supernatants. sSLAMF7 could not be detected at all in the supernatants of both myeloma cell lines, even in any concentration of cells. Furthermore, addition of 30 to 100 ug/ml (clinical trough serum concentration) elotuzumab into the culture of mDCs and the supernatants did not affect the CD86 expression and IL-12/CCL17 production. Hence, factors other than sSLAMF7 from myeloma cells can trigger DCs to suppress Th1 response and to promote Th2 response.Conclusion: Our data suggest that the MM micro-environment, while suppress the Th1-inducing capacity, rather lead to enhance Th2 response at DC phase. As sSLAMF7 is detected in the serum of patients with MM but not in the supernatants of MM cell lines in the experimental setting, there is a limitation that our data does not consider the action of sSLAMF7. However, the present result provides an important clue for understanding the biological basis for the MM micro-environment against host immune defense at onset. DisclosuresIto:Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria; Novartis Pharma: Honoraria; Pfizer: Honoraria; Mundipharma: Honoraria; Celgene: Honoraria.

TLR9 Signaling Suppresses the Canonical Plasma Cell Differentiation Program in Follicular B Cells.

The relative potency and quality of mouse B cell response to Toll-like receptors (TLRs) signaling varies significantly depending on the B cell subset and on the TLR member being engaged. Although it has been shown that marginal zone cells respond faster than follicular (FO) splenic B cells to TLR4 stimulus, FO B cells retain full capacity to proliferate and generate plasmablasts and plasma cells (PBs/PCs) with 2–3 days delayed kinetics. It is not clear whether this scenario could be extended to other members of the TLR family. Here, using quantitative cell culture conditions optimized for B cell growth and differentiation, we show that TLR9 signaling by CpG, while promoting vigorous proliferation, completely fails to induce differentiation of FO B cells into PBs/PCs. Little or absent Ig secretion following TLR9 stimulus was accompanied by lack of expression of cell surface markers and canonical transcription factors involved in PB/PC differentiation. Moreover, not only TLR9 did not induce plasmocyte differentiation, but it also strongly inhibited the massive PB/PC differentiation of FO B cells triggered by LPS/TLR4. Our study reveals unexpected opposite roles for TLR4 and TLR9 in the control of plasma cell differentiation program and disagrees with previous conclusions obtained in high-density cultures conditions on the generation of plasmocytes by TRL9 signaling. The potential implications of these findings on the role of TLR9 in controlling self-tolerance, clonal sizes and regulation of humoral responses are discussed.

Leukemia inhibitory factor via the Toll-like receptor 5 signaling pathway involves aggravation of cachexia induced by human gastric cancer-derived 85As2 cells in rats.

Cancer cachexia is highly prevalent in gastric cancer patients and characterized by decreased food consumption and body weight. We previously created a rat model of cancer cachexia using MKN45cl85 and 85As2 cells derived from human gastric cancer. The 85As2 cells induced cachexia more potently compared to MKN45cl85 cells. To clarify the mechanism underlying the difference in the cachexia-inducing ability of these cells, we conducted DNA microarray analysis, focusing on cell proliferation and the production of leukemia inhibitory factor (LIF), a cachexia-inducing factor. The plasma human LIF levels of 85As2-induced cachexic rats increased as symptoms worsened, whereas the plasma levels of MKNcl85 were low. 85As2 cells displayed more genetic changes compared to MKN45cl85 cells, which were related to Toll-like receptor (TLR) 4/5 signaling. Stimulation of both cells with TLR4 (lipopolysaccharide) or TLR5 (flagellin) agonists did not affect proliferation. However, in 82As2 cells, LIF production was significantly increased by stimulation with TLR5, which was suppressed by an inhibitor of interleukin-1 receptor-associated kinase-1/4, which are important factors in the TLR5 signaling pathway. The increase in LIF production resulting from activation of the TLR5 signaling pathway may contribute to the cachexia-inducing ability of 85As2 cells.

LPS-опосредованное воспаление при сердечно-сосудистых заболеваниях

Возникновение и развитие сердечной недостаточности как проявление атеросклеротического процесса или первичного поражения кардиомиоцитов при кардиомиопатиях может быть связано с накоплением в крови эндотоксинов - компонентов бактериальных клеток, в частности липополисахарида (ЛПС) клеточной стенки грамотрицательных бактерий. ЛПС при связывании с TOLL-подобными рецепторами (TLR) запускают сигнальные пути, вследствие чего происходит синтез провоспалительных цитокинов, выработка активных форм кислорода и азота, развитие окислительного стресса и воспаления в месте атеросклеротического поражения. Активация ЛПС-TLR4-сигнальных путей в тромбоцитах способствует развитию тромбоза. При ишемии миокарда и сердечной недостаточности наблюдается дисбаланс между окислительным стрессом и антиоксидантными механизмами. Миокард имеет эндогенные восстановительные механизмы, в том числе системы тиоредоксина (Trx) и глутатиона (GSH), которые направлены на удаление активных форм кислорода и восстановление окисленных белков, часть из которых участвует в ЛПС-TLR4-сигнальных путях. Например, GSH способен модифицировать адапторный белок MyD88, а Trx является прямым ингибитором протеинкиназы ASK1, способствуя подавлению проапоптотических сигнальных путей во время острого воспаления в кардиомиоцитах. Кроме того, Trx предотвращает дисфункцию митохондрий, увеличивает производство АТФ, ингибирует апоптоз и тем самым оказывает кардиопротекторное действие. Emergence and development of heart failure as a manifestation of atherosclerotic process or primary lesion to cardiomyocytes in cardiomyopathies can be caused by blood accumulation of endotoxins, components of bacterial cells, such as lipopolysaccharide (LPS) of gram-negative bacterial cell wall. LPS binding to TOLL-like receptors (TLR) triggers signaling pathways to induce synthesis of pro-inflammatory cytokines, production of reactive oxygen and nitrogen species, and development of oxidative stress and inflammation at the site of atherosclerotic lesion. Activation of LPS-TLR4-signaling pathways in platelets contributes to development of thrombosis. An imbalance between oxidative stress and antioxidant mechanisms is observed in myocardial ischemia and heart failure. The myocardium has endogenous restorative mechanisms, including the systems of thioredoxin (Trx) and glutathione (GSH) designed for removing active oxygen species and reducing oxidized proteins, some of which are involved in LPS-TLR4 signaling pathways. For example, GSH is able to modify the adaptor protein MyD88 while Trx is a direct inhibitor of protein kinase ASK1, to facilitate suppression of proapoptotic signaling pathways during acute inflammation in cardiomyocytes. In addition, Trx prevents mitochondrial dysfunction, increases ATP production, inhibits apoptosis and, thereby, exerts a cardioprotective effect.

Treatment with lecinoxoids attenuates focal and segmental glomerulosclerosis development in nephrectomized rats.

Focal segmental glomerulosclerosis (FSGS) is a scarring process associated with chronic low‐grade inflammation ascribed to toll‐like receptor (TLR) activation and monocyte migration. We developed synthetic, small‐molecule lecinoxoids, VB‐201 and VB‐703, that differentially inhibit TLR‐2‐ and TLR‐4‐mediated activation and monocyte migration. The efficacy of anti‐inflammatory lecinoxoid treatment on FSGS development was explored using a 5/6 nephrectomy rat model. Five‐sixths of nephrectomized rats were treated with lecinoxoids VB‐201, VB‐703 or PBS, for 7 weeks. Upon sacrifice, albumin/creatinine ratio, glomerulosclerosis, fibrosis‐related gene expression and the number of glomerular and interstitial monocyte were evaluated. Treatment of nephrectomized rats with lecinoxoids ameliorated glomerulosclerosis. The percentage of damaged glomeruli, glomerular sclerosis and glomeruli fibrotic score was significantly reduced following VB‐201 and VB‐703 treatment. VB‐703 attenuated the expression of fibrosis hallmark genes collagen, fibronectin (FN) and transforming growth factor β (TGF‐β) in kidneys and improved albumin/creatinine ratio with higher efficacy than did VB‐201, but only VB‐201 significantly reduced the number of glomerular and interstitial monocytes. These results indicate that treatment with TLR‐2, and more prominently, TLR‐4 antagonizing lecinoxioids, is sufficient to significantly inhibit FSGS. Moreover, inhibiting monocyte migration can also contribute to treatment of FSGS. Our data demonstrate that targeting TLR‐2‐TLR‐4 and/or monocyte migration directly affects the priming phase of fibrosis and may consequently perturb disease parthogenesis.

TLR2 Expression on Leukemic B Cells from Patients with Chronic Lymphocytic Leukemia

Antigenic stimulation is considered as a possible trigger of neoplastic transformation in chronic lymphocytic leukemia (CLL). B-cell receptor plays a key role in the interactions between the microenvironment and leukemic cells; however, an important role has also been attributed to Toll-like receptors (TLRs). It is believed that disorders of TLR expression may play a part in the pathogenesis of CLL. In this study, we investigated the potential role of TLR2 in CLL by analyzing its expression on leukemic B cells in correlation with clinical and laboratory parameters characterizing disease activity and patients’ immune status. We assessed the frequencies of TLR2+/CD19+ cells by the flow cytometry method in peripheral blood of 119 patients with CLL. The percentage of TLR2+/CD19+ cells was significantly lower in patients with CLL as compared to the healthy volunteers. There was also a lower percentage of TLR2+/CD19+ cells in CLL patients with poor prognostic factors, such as ZAP70 and/or CD38 expression, 17p and/or 11q deletion. On the other hand, among patients with del(13q14) associated with favorable prognosis, the percentage of TLR2+/CD19+ cells was higher than among those with del(11q22) and/or del(17p13) as well as in the control group. We found an association between low percentage of CD19+/CD5+/TLR2+ cells and shorter time to treatment. We also demonstrated the relationship between low percentage of CD19+/CD5+ TLR2-positive and overall survival (OS) of CLL patients. CLL patients with a proportion of 1.6% TLR2-positive B CD5+ cells (according to the receiver operating characteristic curve analysis) or more had a longer time to treatment and longer OS than the group with a lower percentage of TLR2 positive cells. To sum up, the results of the study suggest that low TLR2 expression is associated with poor prognosis in CLL patients. The monitoring of CD19+/CD5+/TLR2+ cells number may provide useful information on disease activity. Level of TLR2 expression on leukemic B cells may be an important factor of immunological dysfunction for patients with CLL. Our study suggests that TLR2 could becomes potential biological markers for the clinical outcome in patients with CLL.

Secreted products of oral bacteria and biofilms impede mineralization of apical papilla stem cells in TLR-, species-, and culture-dependent fashion

Regenerative endodontics exploits the mineralization potential of stem cells from the apical papilla (SCAPs) in order to promote root maturation of permanent immature teeth. SCAPs may encounter post-disinfection residual bacteria either in planktonic or in biofilm growth mode. Bacterial components bind to Toll-like receptors (TLRs) and trigger pro-inflammatory responses. We hypothesized that biofilm-triggered TLR activation affects the mineralization potential of human SCAPs. SCAPs were challenged with conditioned media derived from standardized dual-species biofilms and planktonic bacterial cultures and their inflammatory status and mineralization capacity were studied. Bacterial products from both growth modes (planktonic vs. biofilm) compromised cell viability, proliferation and mineralization capacity of SCAPs, but in a species- and growth mode-dependent fashion. While TLR4 expression remained unaffected, TLR2 expression was upregulated coinciding with a pro-inflammatory activation of SCAPs. Moreover, TLR and its downstream TGF-β-associated kinase (TAK1) appeared to be blocking mineralization, as inhibition of these factors restored it. In conclusion, bacterial products promoted the pro-inflammatory status and inhibited mineralization of human SCAPs in a TLR-, species-, and culture-dependent fashion. TLR2 emerged as the pivotal mediator of these responses and further research is warranted towards the judicious manipulation of SCAPs in order to modify the untoward events of TLR-priming and signaling.

PRDX6 Inhibits Neurogenesis through Downregulation of WDFY1-Mediated TLR4 Signal

Impaired neurogenesis has been associated with several brain disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). The role of peroxiredoxin 6 (PRDX6) in neurodegenerative diseases is very controversial. To demonstrate the role of PRDX6 in neurogenesis, we compared the neurogenesis ability of PRDX6-overexpressing transgenic (Tg) mice and wild-type mice and studied the involved molecular mechanisms. We showed that the neurogenesis of neural stem cells (NSCs) and the expression of the marker protein were lower in PRDX6 Tg-mice than in wild-type mice. To determine the factors involved in PRDX6-related neural stem cell impairment, we performed a microarray experiment. We showed that the expression of WDFY1 was dramatically decreased in PRDX6-Tg mice. Moreover, WDFY1 siRNA decreases the differentiation ability of primary neural stem cells. Interestingly, WDFY1 reportedly recruits the signaling adaptor TIR-domain-containing adapter-inducing interferon-β (TRIF) to toll-like receptors (TLRs); thus, we showed the relationship among TLRs, PRDX6, and WDFY1. We showed that TLR4 was dramatically reduced in PRDX6 Tg mice, and reduced TLR4 expression and neurogenesis was reversed by the introduction of WDFY1 plasmid in the neural stem cells from PRDX6 Tg mice. This study indicated that PRDX6 inhibits the neurogenesis of neural precursor cells through TLR4-dependent downregulation of WDFY1 and suggested that the inhibitory effect of PRDX6 on neurogenesis play a role in the development of neurodegenerative diseases in the PRDX6 overexpressing transgenic mice.

Decreased CD4+CD25+CD127dim/- Regulatory T Cells and T Helper 17 Cell Responsiveness to Toll-Like Receptor 2 in Chronic Hepatitis C Patients with Daclatasvir Plus Asunaprevir Therapy.

Direct-acting antivirals (DAAs) not only rapidly inhibited hepatitis C virus (HCV) replication but also modulated innate and adaptive immune response in chronic hepatitis C patients. However, the regulatory activity of DAAs to Toll-like receptor 2 (TLR2) stimulation on CD4+CD25+CD127dim/- regulatory T cells (Tregs) and T helper (Th) 17 cells was not completely understood. In the present study, a total of 23 patients with chronic HCV genotype 1b infection were enrolled, and blood samples were collected at baseline (treatment naive), end of therapy (EOT), and 12 weeks after EOT (SVR12) with daclatasvir plus asunaprevir therapy. TLR2 expression on Tregs and Th17 cells was measured by flow cytometry. Cellular proliferation, cytokine production, and suppressive activity were also tested in purified CD4+CD25+CD127dim/- Tregs in response to the stimulation of Pam3Csk4, an agonist of TLR2. Inhibition of HCV RNA by daclatasvir and asunaprevir did not affect either percentage of Tregs/Th17 cells or TLR2 expression on Tregs/Th17 cells. Pam3Csk4 stimulation also did not influence either cellular proliferation or Tregs/Th17 proportion at each time point. Stimulation with Pam3Csk4 only enhanced the suppressive function and interleukin (IL)-35 production by Tregs purified from baseline, but not those from EOT or SVR12. Similarly, Pam3Csk4 stimulation only elevated Th17 cell frequency of CD4+ T cells from baseline, but not those from EOT or SVR12. Moreover, daclatasvir and asunaprevir therapy did not promote TLR2-induced shift of Tregs toward Th17-like phenotype and function. These data suggested that daclatasvir plus asunaprevir therapy resulted in the decreased responsiveness of Tregs/Th17 cells to TLR2 stimulation in chronic hepatitis C patients, which might provide a novel mechanism underlying DAA-induced immunoregulation.

Innate immune responses to toll-like receptor stimulation are altered during the course of pregnancy

During pregnancy the maternal immune system has to develop tolerance towards the developing fetus. These changes in maternal immunity can result in increased severity of certain infections, but also in amelioration of autoimmune diseases. Pregnancy-related hormones have been suggested to play a central role in the adaptation of the maternal immune system, but their specific effects on innate immune function is not well understood. In a longitudinal study of pregnant women, we investigated innate immune cell function in response to toll-like receptors (TLR) 4 and 7 stimulation, two TLR pathways playing a critical role in early innate immune recognition of bacteria and viruses. IFNα production by TLR7-stimulated pDCs was decreased in early pregnancy, and increased towards the end of pregnancy. In contrast, pro-inflammatory TLR4-induced TNFα production by monocytes was increased during early pregnancy, but declined after the first trimester. Changes in cytokine production were associated with changes in pregnancy-related hormones and monocyte subpopulations over the course of pregnancy. These data demonstrating a significant association between pregnancy-related hormones and modulation of innate immune responses mediated by TLRs provide novel insights into the immunological adaptations occurring during pregnancy.

Elucidating synergistic and antagonistic effects of B cell receptor and toll-like receptors 3, 4, and 9 in B cell activation

Abstract B cells are key components of the humoral immune response. Two forms of B cell activation come from engagement of the B cell receptor (BCR) with foreign antigens and from pathogen-associated molecular patters (PAMPs) binding to toll-like receptors (TLRs). For TLRs, MyD88 and TRIF are the major adaptors that lead to NFkB and IRF3 activation respectively. Among various TLRs that are present in B cells, TLR9 is only able to use MyD88 and TLR3 only signals through TRIF, while TLR4 uses both pathways. Therefore, these TLRs provide excellent tools to investigate the roles of TRIF and MyD88 pathways in B cell function and the interplay between them. Here, using stimulation through these three different TLRs either individually or in combination with each other and with/or without anti-IgM, we observed that there is synergy and antagonism between these pathways in terms of activation induced metabolic boost, cytokine production, IgM secretion, expression of various cell surface markers, and antigen presentation. We identified the TRIF pathway as an inducer of Type-I interferon secretion by B cells, which eventually favors the expression of costimulatory molecules such as CD86. On the other hand, MyD88 stimulation antagonized the expression of costimulatory molecules and dramatically induced rapid proliferation and production of IgM as well as proinflammatory cytokines such as IL-6 and TNF. Using B cells obtained from MyD88 or TRIF KO mice, we showed that the interplay between pathways is of limited scale; however, for TLR4 signaling MyD88 has a largely permissive effect while TRIF determines the magnitude of response. These results further our understanding of the complex TLR signaling machinery in B lymphocytes.

Pellino 1 is a novel regulator of TNF and TLR signalling in human myometrial and amnion cells

Preterm birth is the primary cause of neonatal deaths and morbidities. Pathological processes causally linked to preterm birth are inflammation and infection. Pellino-1 (Peli1) has previously been found to regulate the inflammatory response in non-gestational tissues in response to toll-like receptor (TLR) ligands and pro-inflammatory cytokines. The aims of this study were to determine the effect of labor on Peli1 expression in myometrium and fetal membranes, and the effect of Peli1 silencing by siRNA (siPELI1) on the production of pro-inflammatory and pro-labor mediators. The expression of Peli1 was found to be higher in myometrium and fetal membranes with term labor, compared to non-laboring samples. Peli1 mRNA and protein expression was also higher in amnion from women with preterm histological chorioamnionitis. In human primary myometrial cells, siPELI1 transfected cells showed a decrease in pro-inflammatory cytokine IL6, chemokines (CXCL8, CCL2) and adhesion molecule ICAM1 when in the presence of pro-inflammatory cytokine TNF, TLR2/6 ligand fsl-1, TLR5 ligand flagellin, and TLR3 ligand poly(I:C). Similarly in primary amnion cells, siPELI1 transfected cells decreased IL1B-induced expression and secretion of IL6 and CXCL8. In siPELI1 transfected myometrial cells, there was a decrease in prostaglandin PGF2α and its receptor, PTGFR mRNA expression when treated with TNF. There was a decrease in NF-κB RELA transcriptional activity in siPELI1 transfected cells in the presence of TNF, fsl-1 and flagellin, but not poly(I:C). Our study suggests a novel role for Peli1 in regulating pro-inflammatory and pro-labor mediators through TNF and TLR signalling.

Novel tumour antigens and the development of optimal vaccine design.

The interplay between tumours and the immune system has long been known to involve complex interactions between tumour cells, immune cells and the tumour microenvironment. The progress of checkpoint inhibitors in the clinic in the last decade has highlighted again the role of the immune system in the fight against cancer. Numerous efforts have been undertaken to develop ways of stimulating the cellular immune response to eradicate tumours. These interventions include the identification of appropriate tumour antigens as targets for therapy. In this review, we summarize progress in selection of target tumour antigen. Targeting self antigens has the problem of thymic deletion of high-affinity T-cell responses leaving a diminished repertoire of low-affinity T cells that fail to kill tumour cells. Thymic regulation appears to be less stringent for differentiation of cancer-testis antigens, as many tumour rejection antigens fall into this category. More recently, targeting neo-epitopes or post-translational modifications such as a phosphorylation or stress-induced citrullination has shown great promise in preclinical studies. Of particular interest is that the responses can be mediated by both CD4 and CD8 T cells. Previous vaccines have targeted CD8 T-cell responses but more recently, the central role of CD4 T cells in orchestrating inflammation within tumours and also differentiating into potent killer cells has been recognized. The design of vaccines to induce such immune responses is discussed herein. Liposomally encoded ribonucleic acid (RNA), targeted deoxyribonucleic acid (DNA) or long peptides linked to toll-like receptor (TLR) adjuvants are the most promising new vaccine approaches. These exciting new approaches suggest that the 'Holy Grail' of a simple nontoxic cancer vaccine may be on the horizon. A major hurdle in tumour therapy is also to overcome the suppressive tumour environment. We address current progress in combination therapies and suggest that these are likely to show the most promise for the future.

Ecrg4 peptide is the ligand of multiple scavenger receptors

Esophageal cancer-related gene 4 (Ecrg4) encodes a hormone-like peptide that is believed to be involved in a variety of physiological phenomena, including tumour suppression. Recent progress in the study of Ecrg4 has shown that Ecrg4 is a proinflammatory factor and induces the expression of several cytokines and chemokines in macrophages/microglia. However, the detailed molecular mechanisms of Ecrg4 signalling, especially the Ecrg4 receptors, remain poorly understood. Here, using retrovirus-mediated expression cloning, we identified lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1) as a membrane protein that binds amino acid residues 71–132 of Ecrg4 (Ecrg4(71–132)). Moreover, in addition to LOX-1, several scavenger receptors, such as Scarf1, Cd36 and Stabilin-1, facilitated the efficient internalisation of Ecrg4(71–132) into cells. A broad competitive inhibitor of scavenger receptors, polyinosinic acid, reduced both the binding of Ecrg4(71–132) and the activation of NF-κB in microglia. This activation was dependent on MyD88, an adaptor protein that recruits signalling proteins to Toll-like receptors (TLRs), with the consequent induction of various immune responses. These data suggest that multiple scavenger receptors recognise Ecrg4(71–132) and transduce its signals, together with TLRs, in microglia.

Polyethylenimine-Mediated CpG Oligodeoxynucleotide Delivery Stimulates Bifurcated Cytokine Induction.

CpG oligodeoxynucleotides (CpG ODNs) bind to toll-like receptor 9 (TLR9) and activate the immune system. Thus, CpG ODNs have attracted considerable interest as immunotherapeutic agents or adjuvants for many diseases. Herein we report that polyethylenimine (PEI) functions as a multifunctional vector for both enhancing and regulating the immunostimulatory activity of CpG ODNs. PEI and CpG ODNs formed nanopolyplexes (NPs), which possessed good biocompatibility. PEI-CpG ODN NPs facilitated the internalization of CpG ODNs and induced increasing amounts of cytokines. Most importantly, PEI-CpG ODN NPs induced interleukin-6 (IL-6) and interferon-α (IFN-α) simultaneously, while class B CpG ODNs induced only IL-6. In contrast to class C CpG ODNs, which also simultaneously induce IFN-α and IL-6, the ratio of IFN-α and IL-6 induced by PEI-CpG ODN NPs could be regulated by changing the N/P ratio. This flexible bifurcated cytokine is promising for treating diseases such as cancer that require IL-6 and IFN-α simultaneously. Our findings propose a regulatory effect of PEI on the immunostimulatory activity of CpG ODNs, which will shed light on the development of highly efficient nonviral vectors for CpG ODN-based immunotherapy.

Abstract PD6-06: Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients

Abstract Cells within the tumor microenvironment, including but not limited to macrophages and mesenchymal stem cells (MSCs), can promote the phenotype and aggressiveness of inflammatory breast cancer (IBC). For example, co-injection of MSCs with SUM149 IBC cells significantly increased the clinical features of IBC such as skin invasion and metastasis. Our preliminary work showed that MSCs can be educated by co-culture with M1 polarized (anti-tumor) or M2 polarized (pro-tumor) mouse Raw macrophages. Such education of MSCs by M2 Raw macrophages leads to increased IL6 secretion by MSCs, relative to M1-educated or uneducated MSCs. M2-educated MSCs also have increased migration toward IBC cell lines SUM149 and IBC3, effects that can be blocked by an anti-IL6 antibody. Co-culture with M2-educated MSCs also enhances migration and mammosphere formation of IBC cells. Radiation response of IBC cells upon interactions with cells from the tumor microenvironment was also analyzed. Preliminary work shows that co-culture of IBC cells (SUM149 and KPL4) with M2-polarized human THP1 macrophages, prior to ionizing radiation, mediates radiation resistance of IBC cells, and this effect can be decreased by either adding HDL lipoproteins during co-culture period or by STAT6 inhibitors that block IL4/IL13-mediated phosphorylation of STAT6 and M2-polarization in THP1 macrophages. MSCs can also be polarized into either a MSC1 phenotype or a MSC2 phenotype by exposure to toll-like receptor (TLR) ligands TLR4 or TLR3, respectively. Indoleamine-pyrrole 2,3-dioxygenase (IDO) expression in MSCs is a marker of MSC2 polarization that is induced after exposure with TLR3 ligand (PolyIC) relative to MSC1 (TLR4 stimulated; LPS-treated) or parental MSCs. Similar to macrophage polarization, while MSC1 mediates anti-tumor effects, MSC2 are immunosuppressive and thus contribute to tumor growth. Preliminary work also shows that co-culture of IBC cells with MSC2 mediates radioresistance and this can be decreased as well by exposure to HDL during co-culture period prior to radiation. HDL protective effects, in part, can be explained by decreased expression of TLR3-induced IDO mRNA levels in MSC2. In the present work, we extended the above mentioned observations regarding the crosstalk between mouse Raw macrophages and MSCs by analyzing the effect of co-culture of human THP1 macrophages (parental designated as M0, M1- or M2-polarized) with MSCs on the IDO mRNA expression in MSCs, a marker of MSC2 polarization. Surprisingly, co-culture of M1-polarized THP1 with MSCs resulted in a robust increased expression of IDO mRNA in MSC relative to parental MSC (uneducated) or MSCs co-cultured with M2-THP1. Further studies are needed to determine the effects of increased IDO expression in MSC, upon M1-THP1 co-culture, on the aggressive behavior of IBC cells and whether this could be altered with IDO inhibitors. Our results suggest that there could be inter-species differences between mouse and human macrophages on the education of human MSCs. Based on our findings we propose testing a combination of STAT6 inhibitors that reverse M2-polarization of macrophages and IDO inhibitors that can decrease MSC2 phenotype mediated by TLR3 exposure and/or M1-THP1 education. Citation Format: Rahal OM, Wolfe AR, Mandal PK, Larson R, Tin S, Reuben JM, McMurray JS, Woodward WA. Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-06.