Production Of GM-CSF Research Articles

Abstract 1575: Intratumoral injection of interleukin-17 inhibits distant metastasis of radiation-induced recurrent tumor

Abstract Interleukin-17 (IL-17) is a pro-inflammatory cytokine that leads to the production of GM-CSF, G-CSF, IL-1, IL-6, TNF-α. It can be secreted by various cells, including T cells, NKT cells, fibroblasts, and even tumor cells. In addition, reports have shown that IL-17 could also promote tumor growth by recruiting and expanding granulocytic myeloid suppressor cells (G-MDSCs). In our preliminary study, we found that Tramp-C1, a murine prostate cancer cell line, had remarkable IL-17 level when it grew in pre-irradiated (PreIR) stroma. In contrast, B16F0, a murine melanoma cell line, had much less IL-17 and stronger tumor bed effect, in terms of tumor growth delay, but lung metastasis were formed gradually as B16F0 developed. When the recombinant IL-17 was directly injected into the B16F0 tumors growing in PreIR stroma, we found that the probability of lung metastasis was decreased despite a slight increase of tumor growth at primary site. This indicates that IL-17 may have different effects on irradiated tumor bed and distant metastatic site. The influence of IL-17 on tumor microenvironments of distant metastatic sites and irradiated tumor bed is currently under investigation. In conclusion, we propose a new therapeutic approach of reducing distant metastasis following radiation therapy.. This work was kindly supported by MOST-104-2314-B-182 -024 grant to Fang-Hsin Chen. Citation Format: Yi-Nan Li, Fang-Hsin Chen, Ji-Hong Hong, Chi-Shiun Chiang. Intratumoral injection of interleukin-17 inhibits distant metastasis of radiation-induced recurrent tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1575.

Abstract 2234: Exploring the oncolytic potential of Newcastle Disease Virus

Abstract Oncolytic viruses are live, replication-competent viruses that infect and/or replicate selectively in tumour cells leading to the destruction of the infected cell. Cell death occurs as a natural consequence of the viral life cycle and newly produced virions can infect and kill neighbouring tumour cells leading to an amplified therapeutic effect. Oncolysis has the added benefit of potentially producing novel tumour antigens that can induce an immune-mediated therapeutic response. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has proven safety and demonstrated efficacy against a variety of mammalian cancers in PhI clinical studies. Using reverse genetics, we generated a recombinant strain of NDV that overcomes environmental and regulatory concerns uncoupling oncolytic potency and avian pathogenicity. We have additionally inserted the GM-CSF gene in order to maximize anti-tumour immune response and provide an in situ, patient/tumour- specific vaccine, combined with potent oncolysis. We have evaluated the biological characteristics of recNDVGM-CSF in vivo and in vitro. In vitro recNDVGM-CSF selectively replicates in and kills a wide variety of tumour cell types. In vivo, using a fibrosarcoma model that supports viral replication, a single administration (intra-tumoural or systemic) was able to cure 80% of tumour bearing mice. Additionally, following IV administration recNDVGM-CSF localises to and replicates within the tumour leading to a localised GM-CSF production within the tumour. The inherent properties of NDV (self-propagation, tumour-selective replication, immune activation) coupled with the ability to genetically engineer NDV to express therapeutic transgenes may provide a multi-modal attack on the tumour, delivering greater benefit to patients. Citation Format: James A. Harper, Jon Travers, Ruth Franks, Shannon Burke, Christel Navarro, Cheng Xing, Weijia Wang, Qi Xu, Robert W. Wilkinson, Hong Jin, Danielle Carroll. Exploring the oncolytic potential of Newcastle Disease Virus. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2234.

Specific effect of the HLDF differentiation factor on the cytokine production potential of immunocompetent blood cells in stomach adenocarcinoma.

The cytokine production potential of immunocompetent cells from the blood of stomach adenocarcinoma patients was analyzed after the pretreatment of cells with the HLDF differentiation factor with subsequent exposure to polyclonal activators (HLDF+PA). IL-1β, IL-1Ra, TNFα, IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-18BPa, IFNγ, G-CSF, and GM-CSF were quantified in the supernatants after precipitation of the cells. Specific effects of HLDF+PA were manifested as an increase in the production of IL-8, IL-17, and GM-CSF due to suppression of Th1-dependent immune reactions in a Th17-mediated mechanism that is a part of a broader functional antagonism of Th1 and Th17 lymphocyte subpopulations.

The Effect of a BSA Conjugate of a Synthetic Hexasaccharide Related to the Fragment of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 on the Activation of Innate and Adaptive Immune Responses.

We report the effect of a bovine serum albumin (BSA) conjugate of a synthetic hexasaccharide (HS) related to the fragment of the capsular polysaccharide (PS) of Streptococcus pneumoniae type 14 on the stimulation of innate immune system and the subsequent development of a PS-specific antibody response. Glycoconjugate (GC) in the presence (GC + AL) or absence of aluminum hydroxide was administered to mice twice. GC increased the number of TLR2-expressing cells and induced the maturation of dendritic cells (CD11c+, CD80+ and, MHCII+), which secreted IL-1β, IL-6, and TNFα into the culture medium. The level of IL-1β, IL-10, IFNγ, and TNFα in the blood increased within 24 h after the single GC administration to mice. On day 7, the numbers of splenic CD4+ and CD8+ T lymphocytes and B lymphocytes increased. After the second immunization, the levels of CD4+ and CD8+ T lymphocytes were lower than in the control, whereas the B cell, NK cell, and MHC class II-expressing cell numbers remained enhanced. However, of the presence of anti-PS, IgG antibodies were not detected. The addition of aluminum hydroxide to GC stimulated the production of GM-CSF, IL-1β, IL-5, IL-6, IL-10, IL-17, IFNγ, and TNFα. Anti-PS IgG1 antibody titers 7 days after the second immunization were high. During that period, normal levels of splenic CD4+ T lymphocytes were maintained, whereas reduced CD8+ T lymphocyte numbers and increased levels of B lymphocytes, NK cells, and MHC class II-expressing cell numbers were observed. Anti-PS IgG levels diminished until day 92. A booster immunization with GC + AL stimulated the production of anti-PS IgG memory antibodies, which were determined within 97 days. The elucidation of specific features of the effect of the synthetic HS conjugate on the stimulation of innate, cell-mediated immunity, and antibody response can favor the optimization of GC vaccine design.

Maintenance of Mycobacterium tuberculosis-specific T cell responses in End Stage Renal Disease (ESRD) and implications for diagnostic efficacy

End-stage renal disease (ESRD) patients exhibit elevated risk of tuberculosis (TB) reactivation, but current diagnostics, including the interferon gamma release assay (IGRA), exhibit poor sensitivity in ESRD. We tested 80 ESRD patients and found an 18.75% prevalence of IGRA positivity. A subset of patients was assessed for Mtb-specific expression of 44 cytokines/chemokines, and CD4+ T cell phenotype and function. Similar to non-ESRD IGRA+ individuals, Mtb-specific IFNγ, IL-1RA, IP-10, MCP-3 and IL-2 responses were identified in the ESRD IGRA+ group. 27% of the ESRD IGRA- group exhibited MCP-3 or IL-2 Mtb-specific responses, which may identify cases of latent TB infection in ESRD. Stimulation of PBMC with PPD demonstrated similar CD4+ T cell production of IFNγ, TNFα and GM-CSF by ESRD patients. The reported low sensitivity of the IGRA in ESRD cohorts is therefore unlikely to be due to poor T cell cytokine secretion, and may instead reflect defects in antigen presentation.

Airway epithelial cells promote allergic sensitization to cockroach allergen through GM-CSF production

Abstract Allergic asthma is an inflammatory disease of the airways driven by an overactive immune response against allergens. Allergen-specific TH2 cells are critical to the pathogenesis of asthma but factors controlling the initiation of TH2 responses are not well understood. The emerging picture is one that points to a role for airway epithelial cells (AECs) and epithelial-derived cytokines in promoting TH2 immunity to allergens. However, allergens are structurally diverse and can elicit different epithelial-derived cytokines. Thus, there are multiple pathways that lead to TH2 immunity and mechanisms of sensitization that are unique to individual allergens remain to be elucidated. Cockroach allergen (CRA) is an environmental allergen that is strongly associated with the incidence of allergic asthma. Despite its clinical importance, very little is known about the molecular and cellular pathways involved in allergic sensitization to CRA. To determine the mechanisms that regulate allergic sensitization to CRA, we intranasally sensitized and challenged mice with CRA to induce allergic airway inflammation with hallmark features of asthma. Using this model, we demonstrate that allergic sensitization to CRA requires activation of AECs. MyD88 deletion in AECs reduced allergic airway response to CRA as well as production of GM-CSF during sensitization. Using a neutralizing antibody against GM-CSF, we showed that GM-CSF is essential for TH2 priming and allergic sensitization to CRA, likely through activation of dendritic cells. When GM-CSF was intranasally delivered during sensitization, TH2-mediated eosinophilia was restored in the absence of MyD88. Thus, AECs promote allergic sensitization to CRA through production of GM-CSF.

Cross-talk between NKT cells and tumor associated macrophages in the tumor microenvironment

Abstract CD1d-reactive Va24-invariant NKT cells (NKTs) control tumor growth via yet poorly understood interactions with tumor-associated macrophages (TAMs) and other myeloid cells. TAMs comprise M1- and M2-like subsets, but only CD163high M2-like TAMs are associated with poor outcome in neuroblastoma (NB) and other tumors. Here, we demonstrate that NKTs selectively target M2-like TAMs via contact-dependent and independent mechanisms. Upon direct contact with antigen-pulsed M1 or M2 macrophages, NKTs selectively killed the latter. Although the killing was strictly CD1d-dependent, CD1d expression between macrophage subsets was equivalent. In contrast, only M2-polarized macrophages expressed CD204, a scavenger receptor responsible for cellular uptake of CD1d ligands. Anti-CD204 blocking mAb inhibited NKT-cell cytotoxicity against M2 in vitro while lentiviral transduction with CD204 cDNA rendered M1 sensitive to NKT cytotoxicity. We also found that antigen-activated NKTs could reprogram M2- into functional M1-like macrophages via GM-CSF production. Furthermore, adoptive transfer of human NKTs resulted in a M1-like polarization of TAMs in metastatic NB xenografts in humanized NSG mice. However, tumors progressed even after NKT cell treatment, suggesting a tumor escape mechanism. Indeed, we found that exposure to activated NKTs resulted in rapid up-regulation of PD-L1 on both M2 and M1 subsets, which correlated with suppressed NKT proliferation and cytokine production. Thus, our results reveal a novel mechanism of immune regulation, in which NKTs control tumor-supportive inflammation via killing or reprogramming of M2-like TAMs. However NKT function is negatively regulated by TAMs at least in part in a PD-1-dependent manner.

GM-CSF is not required for EAE induction but contributes to both brain and spinal cord inflammation

Abstract Myelin-specific CD4+ T cells initiate inflammation in the central nervous system during induction of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. CD4+ T cell pathogenesis has been attributed to the secretion of various cytokines, including IL-17, GM-CSF, and IFN-γ. The relative importance of these CD4+ cytokines in EAE has been the subject of numerous studies, and in C57BL/6 and B10.PL mice, GM-CSF production by T cells is required to induce EAE, while IL-17 and IFN-γ are not required. However, our studies in C3Heb/Fej mice do not support an essential role for GM-CSF in the induction of EAE. Our model is unique in that C3Heb/Fej mice develop brain inflammation in addition to the spinal cord inflammation seen in most other EAE models. We have previously shown that IL-17 is important for (and IFN-γ inhibits) neutrophil recruitment and parenchymal inflammation in the brain of these mice, while IFN-γ promotes spinal cord inflammation. We found that GM-CSF−/− T cells were capable of inducing brain and spinal cord inflammation in wildtype recipient mice. In IL-17RA−/− recipients, wildtype T cells induce intermediate levels of brain inflammation and normal spinal cord inflammation. However, when GM-CSF−/− T cells were transferred into IL-17RA−/− recipient mice, there was a complete loss of clinical signs of brain inflammation, and spinal cord inflammation was significantly reduced, indicating that GM-CSF contributes to both brain and spinal cord inflammation. These data reveal that GM-CSF, IL-17, and IFN-γ all influence inflammation in the brain and spinal cord in C3Heb/Fej mice; while none are required, coordinately these cytokines shape CNS inflammation.

Bowman birk protease inhibitor suppresses GM-CSF, Th17 cells and CNS autoimmune inflammatory demyelination

Abstract Serine proteases play numerous roles in immune system and their inhibition modulates immune and autoimmune responses. Bowman Birk inhibitor (BBI), a soybean-derived serine protease inhibitor potently suppresses experimental autoimmune encephalitis (EAE) after oral administration. In this study, we show that BBI inhibits development of Th17 cells and reduces Th17 cell encephalitogenicity by suppressing their GM-CSF production and decreasing their numbers in the CNS of EAE mice. We also demonstrate that BBI induces IFN-γ production in Th17 cell culture, and that lack of IFN-γ or IFN-γR abrogates Th17 suppressive effect of BBI. In addition, BBI requires IL-27R, Stat-1, T-bet, and IL-10 to inhibit Th17 development. Taken together, our data demonstrate that BBI suppresses pathogenic Th17 cells through IFN- γ dependent pathway and ameliorates EAE. This suggests a novel immunomodulatory effect for serine protease inhibitors in immunity. In addition, BBI has potential to be safe oral therapy for autoimmune diseases, such as multiple sclerosis.

IL-1-induced Bhlhe40 identifies pathogenic TH cells in a model of autoimmune neuroinflammation

Abstract The features that define autoreactive TH cell pathogenicity remain obscure. We have previously shown that TH cells require the transcription factor Bhlhe40 to mediate experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Here, using Bhlhe40-EGFP reporter mice and analyzing both polyclonal and TCR transgenic CD4+ T cells, we found that Bhlhe40 expression was heterogeneous after EAE induction. Bhlhe40-expressing CD4+ T cells displayed marked production of IFN-γ, IL-17A, and GM-CSF, while exhibiting reduced expression of the anti-inflammatory cytokine IL-10 and the regulatory T cell transcription factor Foxp3. In adoptive transfer EAE models Bhlhe40-deficient TH1 and TH17 cells were both nonencephalitogenic. Pertussis toxin (PTX), a classical coadjuvant for actively induced EAE, promoted IL-1β production by myeloid cells in the draining lymph node and served as a strong stimulus for Bhlhe40 expression in TH cells. Furthermore, PTX coadjuvanticity was Bhlhe40 dependent. IL-1β induced Bhlhe40 expression in polarized TH17 cells, and Bhlhe40-expressing cells exhibited an encephalitogenic transcriptional signature. In vivo, IL-1R signaling was required for full Bhlhe40 expression by TH cells after immunization. Overall, we demonstrate that Bhlhe40 expression identifies encephalitogenic TH cells and define a PTX-IL-1-Bhlhe40 pathway active in EAE.

Myeloid cytokines distinctly and reversibly control LAIR-1 (CD305) expression on monocytes-mΦs/monocyte-DCs.

Abstract Cross-linking of the inhibitory immunoreceptor LAIR-1 (CD305) on the cell surface phosphorylates LAIR-1 inhibitory motifs (ITIM) and restricts the growth of monocyte-derived dendritic cells (mono-DCs). Despite the suggestion that LAIR-1’s ability to prevent unwarranted immunity is compromised in autoimmunity, much remains to be learned about the control of LAIR-1 expression. Here, we compared the ability of various myeloid cytokines to control LAIR-1 expression on the surface of monocytes/macrophages (mϕs) and mono-DCs. Treatment of freshly isolated blood monocytes from healthy donors with mono-DC growth factors (GM-CSF+IL-4/GM-CSF+IL-13) led to reduced levels of LAIR-1 at the critical monocyte-to-mono-DC juncture. In contrast, M-CSF (a steady state/M2 cytokine) sustained LAIR-1+CD14+CD33m/M+CD16+ mono-mϕs. M-CSF effects on LAIR-1 occurred under serum-supplemented and serum-free conditions. Addition of M-CSF to mono-DCs previously cultured in GM-CSF+IL-4 promptly reversed the down-regulatory effects of GM-CSF+IL-4 producing increases in LAIR-1+CD14+ cells. Conversely, addition of GM-CSF+IL-4 to M-CSF exposed cultures lowered levels of LAIR-1 and CD14. M-CSF treatment of SLE blood myeloid cells (featuring activated monocytes/mono-DCs lacking LAIR-1, CD14) produced high levels of LAIR-1 and CD14 vs GM-CSF/IL-4. Thus: 1. The expression of LAIR-1 on monocytes/mϕs and mono-DCs is distinctly and reversibly controlled by myeloid cytokines. 2. LAIR-1 expression on circulating myeloid cells in SLE may be regulated by M-CSF.

The minor house dust mite allergen Der p 13 is a fatty acid-binding protein and an activator of a TLR2-mediated innate immune response.

The house dust mite (HDM) allergen Der p 13 could be a lipid-binding protein able to activate key innate signaling pathways in the initiation of the allergic response. We investigated the IgE reactivity of recombinant Der p 13 (rDer p 13), its lipid-binding activities, and its capacity to stimulate airway epithelium cells. Purified rDer p 13 was characterized by mass spectrometry, circular dichroism, fluorescence-based lipid-binding assays, and in silico structural prediction. IgE-binding activity and allergenic potential of Der p 13 were examined by ELISA, basophil degranulation assays, and in vitro airway epithelial cell activation assays. Protein modeling and biophysical analysis indicated that Der p 13 adopts a β-barrel structure with a predominately apolar pocket representing a potential binding site for hydrophobic ligands. Fluorescent lipid-binding assays confirmed that the protein is highly selective for ligands and that it binds a fatty acid with a dissociation constant typical of lipid transporter proteins. The low IgE-binding frequency (7%, n = 224) in Thai HDM-allergic patients as well as the limited propensity to activate basophil degranulation classifies Der p 13 as a minor HDM allergen. Nevertheless, the protein with its presumptively associated lipid(s) triggered the production of IL-8 and GM-CSF in respiratory epithelial cells through a TLR2-, MyD88-, NF-kB-, and MAPK-dependent signaling pathway. Although a minor allergen, Der p 13 may, through its lipid-binding capacity, play a role in the initiation of the HDM-allergic response through TLR2 activation.

IL-17A synergistically stimulates TNF-α-induced IL-8 production in human airway epithelial cells: A potential role in amplifying airway inflammation

ABSTRACTBackground: Recent reports have suggested an involvement of neutrophilic inflammation driven by interleukin (IL)-17 from Th17 cells, especially in severe, refractory asthma. It remains unknown about the possible interactions of this cytokine and other proinflammatory cytokines to direct neutrophilic airway inflammation. Materials and Methods: We evaluated the effects of IL-17A, IL-17E, and IL-17F in combination with other stimuli such as tumor necrosis factor (TNF) –α on the production and expression of IL-8 in human bronchial epithelial cells. We also studied their effects on other cytokine production. The possible role of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways was evaluated by specific inhibitors. We examined the effects of anti-asthma drugs, such as steroids or salmeterol. Results: IL-17A alone induced only a minimal effect on IL-8 expression. IL-17A, but not IL-17E or IL-17F, in combination with TNF-α showed a synergistic effect on IL-8 expression. Similar findings were found when combination with IL-1β and IL-17A were used, but such was not the case with lipopolysaccharide (LPS). In addition, we further found such synergy on GM-CSF production. The synergy with TNF-α and IL-17A was significantly inhibited by MAPKs inhibitors. Corticosteroids such as fluticasone propionate and dexamethasone, but not salmeterol, partially suppressed the IL-17A and TNF-α-induced IL-8 production. Conclusions: IL-17A in the combination with TNF-α or IL-1β showed a synergistic augmenting effect on IL-8 and GM-CSF production in human airway epithelial cells.

Anti-Inflammatory Effect of Quercetin on RAW 264.7 Mouse Macrophages Induced with Polyinosinic-Polycytidylic Acid.

Quercetin (3,3′,4′,5,6-pentahydroxyflavone) is a well-known antioxidant and a flavonol found in many fruits, leaves, and vegetables. Quercetin also has known anti-inflammatory effects on lipopolysaccharide-induced macrophages. However, the effects of quercetin on virus-induced macrophages have not been fully reported. In this study, the anti-inflammatory effect of quercetin on double-stranded RNA (dsRNA)-induced macrophages was examined. Quercetin at concentrations up to 50 μM significantly inhibited the production of NO, IL-6, MCP-1, IP-10, RANTES, GM-CSF, G-CSF, TNF-α, LIF, LIX, and VEGF as well as calcium release in dsRNA (50 µg/mL of polyinosinic-polycytidylic acid)-induced RAW 264.7 mouse macrophages (p < 0.05). Quercetin at concentrations up to 50 μM also significantly inhibited mRNA expression of signal transducer and activated transcription 1 (STAT1) and STAT3 in dsRNA-induced RAW 264.7 cells (p < 0.05). In conclusion, quercetin had alleviating effects on viral inflammation based on inhibition of NO, cytokines, chemokines, and growth factors in dsRNA-induced macrophages via the calcium-STAT pathway.

Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKβ), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor.

Bjcul, a snake venom lectin, modulates monocyte-derived macrophages to a pro-inflammatory profile in vitro

Macrophages are cells of high plasticity and can act in different ways to ensure that the appropriate immune response remains controlled. This study shows the effects of the C-type Bothrops jararacussu venom lectin (BJcuL) on the activation of human macrophages derived from the U937 cell line. BJcuL binds on the cell surface, and this event is inhibited by its specific carbohydrate. It induced phagocytosis and production of H2O2, and expression of antigen presentation molecules. It also enhanced the production of TNF-α, GM-CSF and IL-6 by macrophages and indirectly induced T cells to an increased production of TNF-α, IFN-γ and IL-6 in the presence of LPS. Our results suggest that BJcuL can modulate macrophage functional activation towards an M1 state.

Differential expression of p38 MAPK α, β, γ, δ isoforms in nucleus pulposus modulates macrophage polarization in intervertebral disc degeneration.

P38MAPK mediates cytokine induced inflammation in nucleus pulposus (NP) cells and involves in multiple cellular processes which are related to intervertebral disc degeneration (IDD). The aim of this study was to investigate the expression, activation and function of p38 MAPK isoforms (α,β, γ and δ) in degenerative NP and the effect of p38 activation in NP cells on macrophage polarization. P38 α, β and δ isoforms are preferential expressed, whereas the p38γ isoform is absent in human NP tissue. LV-sh-p38α, sh-p38β transfection in NP cells significantly decreased the ADAMTS-4,-5, MMP-13,CCL3 expression and restored collagen-II and aggrecan expression upon IL-1β stimulation. As compared with p38α and p38β, p38δ exhibited an opposite effect on ADAMTS-4,-5, MMP-13 and aggrecan expression in NP cells. Furthermore, the production of GM-CSF and IFNγ which were trigged by p38α or p38β in NP cells induced macrophage polarization into M1 phenotype. Our finding indicates that p38 MAPK α, β and δ isoform are predominantly expressed and activated in IDD. P38 positive NP cells modulate macrophage polarization through the production of GM-CSF and IFNγ. Hence, Our study suggests that selectively targeting p38 isoforms could ameliorate the inflammation in IDD and regard IDD progression.

Abstract P4-04-16: Characterization of an autologous tumor cell vaccine against breast cancer in mice

Abstract The vast majority of breast cancer-related deaths are due to progressive recurrences of non-metastatic disease. Current adjuvant therapies aimed at preventing breast cancer recurrence are ineffective for tens of thousands of women each year. In addition, current adjuvant therapies carry significant co-morbidities. Active, specific immune-based strategies have the potential to train a patient's immune system to recognize and eliminate occult metastases. Here, we explore autologous tumor cell vaccines (ATCVs) as a strategy to prevent tumor recurrence and improve survival. Because &amp;gt;90% of breast cancer patients undergo resection, autologous tumor cells are readily available for the production of personalized vaccine. Patient-specific vaccination is particularly attractive for breast cancer which is highly heterogeneous. The major disadvantage of ATCVs, is poor immunogenicity. Thus, in order to develop an effective, immunogenic ATCV against breast cancer, we wanted to understand the features of immunogenicity. In this study, we evaluated the immunogenicities of two murine breast cancer cell lines, EMT6 and 4T1. For tumor protection studies, BALB/c female mice were given priming and booster vaccinations, ten days apart, with 1,000,000 irradiated (100Gy) cells. Ten days after the booster vaccination, mice were challenged with live tumor cells. Mice vaccinated with EMT6 cells were completely protected against a live EMT6 challenge in 80% of mice. However, mice vaccinated with irradiated 4T1 cells failed to provide any protection against a live 4T1 challenge. Most interestingly, when mice were vaccinated with a mixture of irradiated EMT6 and 4T1 cells, the protective response against EMT6 challenge was significantly diminished as 60% of mice developed tumors. This finding implied that non-immunogenic 4T1 cells released one or more immunosuppressive factors that inhibited anti-EMT6 immunity. Thus, we investigated the levels of different immunosuppressive cytokines, GM-CSF, IL-6, MCP-1, TGF-β and VEGF released by both 4T1 and EMT6 cells before and after irradiation. Of the different cytokines measured, we found that only GM-CSF is produced at significantly higher levels by 4T1 cells than EMT6 cells. Since at higher levels (&amp;gt;200pg/ml) GM-CSF can induce the accumulation of large amounts of myeloid derived suppressor cells (MDSCs) in the tumor site and lymphoid organs, we believe it to be the reason for the failure of the hybrid vaccine. Future studies will determine if blocking GM-CSF production by 4T1 cells will decrease the accumulation of MDSCs and subsequently increase anti-tumor immunity. Citation Format: Ravindranathan S, Kurtz SL, Smith SG, Koppolu B, Nguyen KG, Zaharoff DA. Characterization of an autologous tumor cell vaccine against breast cancer in mice. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-16.

Cross-protective efficacy from a immunogen firstly identified in Leishmania infantum against tegumentary leishmaniasis.

Experimental vaccine candidates have been evaluated to prevent leishmaniasis, but no commercial vaccine has been proved to be effective against more than one parasite species. LiHyT is a Leishmania-specific protein that was firstly identified as protective against Leishmania infantum. In this study, LiHyT was evaluated as a vaccine to against two Leishmania species causing tegumentary leishmaniasis (TL): Leishmania major and Leishmania braziliensis. BALB/c mice were immunized with rLiHyT plus saponin and lately challenged with promastigotes of the two parasite species. The immune response generated was evaluated before and 10 weeks after infection, as well as the parasite burden at this time after infection. The vaccination induced a Th1 response, which was characterized by the production of IFN-γ, IL-12 and GM-CSF, as well as by high levels of IgG2a antibodies, after in vitro stimulation using both the protein and parasite extracts. After challenge, vaccinated mice showed significant reductions in their infected footpads, as well as in the parasite burden in the tissue and organs evaluated, when compared to the control groups. The anti-Leishmania Th1 response was maintained after infection, being the IFN-γ production based mainly on CD4(+) T cells. We described one conserved Leishmania-specific protein that could compose a pan-Leishmania vaccine.

Selective ORAI1 Inhibition Ameliorates Autoimmune Central Nervous System Inflammation by Suppressing Effector but Not Regulatory T Cell Function.

The function of CD4(+) T cells is dependent on Ca(2+) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS of ORAI1-deficient animals were strongly reduced along with almost completely abolished production of IL-17A, IFN-γ, and GM-CSF despite only partially reduced Ca(2+) influx. In Th1 and Th17 cells differentiated in vitro, ORAI1 was required for cytokine production but not the expression of Th1- and Th17-specific transcription factors T-bet and RORγt. The differentiation and function of induced regulatory T cells, by contrast, was independent of ORAI1. Importantly, induced genetic deletion of Orai1 in adoptively transferred, MOG-specific T cells was able to halt EAE progression after disease onset. Likewise, treatment of wild-type mice with a selective CRAC channel inhibitor after EAE onset ameliorated disease. Genetic deletion of Orai1 and pharmacological ORAI1 inhibition reduced the leukocyte numbers in the CNS and attenuated Th1/Th17 cell-mediated cytokine production. In human CD4(+) T cells, CRAC channel inhibition reduced the expression of IL-17A, IFN-γ, and other cytokines in a dose-dependent manner. Taken together, these findings support the conclusion that Th1 and Th17 cell function is particularly dependent on CRAC channels, which could be exploited as a therapeutic approach to T cell-mediated autoimmune diseases.