Release Of Immunomodulatory Cytokines Research Articles

Biological properties of stem cells from the apical papilla exposed to lipopolysaccharides: An in vitro study

ObjectiveThe aim of this study was to analyze the effect of lipopolysaccharides (LPS) on the biological properties of stem cells from the apical papilla (SCAPs), such as viability, adhesion to dentin, odontoblast-like differentiation, mineralization, and release of immunomodulatory cytokines. DesignSCAPs were isolated from immature teeth of three donors (10 to 15 years old) and cultured in mineralizing media with or without 1 μg/mL lipopolysaccharide (LPS). Cells were seeded and cultured under standardized conditions; viability was assessed by MTT assay on days 1, 3, 5, and 7; adhesion to dentin was analyzed using an environmental scanning electron microscope after 2 days; the expression of odontogenic and mineralization genes (DSPP, DMP-1, OCN, Col1A1) was evaluated through qPCR after 14 days, mineralization was evaluated with alizarin red staining after 21 days; and the release of immunomodulatory cytokines (IL-6 and IL-10) was measured by ELISA after 1 and 7 days. The Kruskal-Wallis test was performed to detect the effect of LPS on SCAPs, followed by the Dunn-Sidak test. ResultsLPS presence in the culture media affected SCAPs viability on day 5 and increased IL-6 secretion by day 7, however, SCAPs retained the adhesion to dentin and mineralization capacities, as well as the differentiation capacity into a mineralizing phenotype. ConclusionIn conclusion, within the limitations of this in vitro study, and under the inflammatory microenvironment simulated in this study, stem cells from the apical papilla were found with retained adhesion capacity to dentin, differentiation into a mineralizing phenotype, mineralization, and release of IL-10.

Targeting MYC in combination with epigenetic regulators induces synergistic anti-leukemic effects in MLLr leukemia and simultaneously improves immunity

MLL rearranged (MLLr) leukemias are associated with a poor prognosis and a limited response to conventional therapies. Moreover, chemotherapies result in severe side effects with significant impairment of the immune system. Therefore, the identification of novel treatment strategies is mandatory.Recently, we developed a human MLLr leukemia model by inducing chromosomal rearrangements in CD34+ cells using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. This MLLr model authentically mimics patient leukemic cells and can be used as a platform for novel treatment strategies. RNA sequencing of our model revealed MYC as one of the most important key drivers to promote oncogenesis. However, in clinical trials the BRD4 inhibitor JQ-1 leading to indirect blocking of the MYC pathway shows only modest activity. We and others previously reported that epigenetic drugs targeting MAT2A or PRMT5 promote cell death in MLLr cells. Therefore, we use these drugs in combination with JQ-1 leading to augmented anti-leukemic effects. Moreover, we found activation of T, NK and iNKT cells, release of immunomodulatory cytokines and downregulation of the PD-1/PD-L1 axis upon inhibitor treatment leading to improved cytotoxicity.In summary, the inhibition of MYC and MAT2A or PRMT5 drives robust synergistic anti-leukemic activity in MLLr leukemia. Moreover, the immune system is concomitantly activated upon combinatorial inhibitor treatment, hereby further augmenting the therapeutic efficiency.

Biological Properties of Dental Pulp Stem Cells Isolated from Inflamed and Healthy Pulp and Cultured in an Inflammatory Microenvironment

IntroductionThe aim of this study was to assess whether the biological characteristics of dental pulp stem cells (DPSCs), such as viability, adhesion to dentin, mineralization, and release of immunomodulatory cytokines, are affected by the inflammatory status of the donor tissue and/or the sustained inflammatory environment. MethodsDPSCs were isolated from pulps from 3 caries-free teeth (healthy or hDPSCs), and from 3 teeth with irreversible pulpitis or deep caries (unhealthy DPSCs or uDPSCs). The cells were cultured in odontogenic and osteogenic media with or without lipopolysaccharides. Viability was analyzed by MTT assay at days 1, 3, 5, and 7; adhesion to dentin was evaluated through an environmental scanning electron microscope after 48 hours and through MTT assay; mineralization was analyzed with alizarin red staining after 21 days; and the release of proinflammatory (interleukin 6) and immunosuppressive cytokines (interleukin 10) was measured with the enzyme-linked immunosorbent assay after 24 hours and 7 days. ResultsThe inflammatory status of the pulp significantly reduced the viability and mineralization capacity of the DPSCs, although it did not affect the adhesion capacity to dentin or the secretion of the proinflammatory interleukin. The inflammatory microenvironment (lipopolysaccharide) only had a significant impact on the secretion of interleukin 6, which was augmented after 7 days. ConclusionsThe inflammatory status of the dental pulp should be taken into account when the use of DPSCs is intended either for research and/or for application in reparative or regenerative therapies.

Retrospective analysis of the effect of angiotensin receptor blockers and angiotensin converting enzyme inhibitors on the efficacy of anti-PD-1/PD-L1 immunotherapy in patients with advanced cancer.

2605 Background: Components of the renin angiotensin system (RAS) are expressed in tumors with activation leading to tumor and stromal cell release of immunomodulatory cytokines promoting an immunosuppressive microenvironment and increased infiltration of tumor associated macrophages. Angiotensin converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARB) are anti-hypertensives that inhibit the RAS. It is unknown if ACEi and ARB treatment modulates the efficacy of anti-PD-1/PD-L1 immunotherapy. Methods: A retrospective chart review was conducted to identify patients with unresectable malignancies initiating treatment with a PD-1 or PD-L1 inhibitor based regimen between 1/1/13 and 6/1/19 at Valley Hospital in New Jersey. IRB approval was obtained. Efficacy of treatment was measured by medical oncologist documentation of clinical benefit (CB = stable disease / treatment response) versus disease progression. Chi-square tests were performed to assess relationship between ACEi or ARB exposure and immunotherapy benefit. To assess for a broader anti-hypertensive effect, the relationship in patients on a beta-blocker but not ACEi/ARB was assessed. Results: 197 patients were identified of which 190 had documentation of clinical outcome. The mean age was 72.8 years with 54% female and 46% male. 56 patients were exposed to ACEi (N = 28) or ARB (N = 28) and 141 patients to neither. 37 patients were exposed to beta blocker but not ACEi or ARB. 60 patients had nonsquamous NSCLC (16 on ACEi or ARB), 26 squamous NSCLC (7 on ACEi or ARB) and 24 melanoma (10 on ACEi or ARB). 71% of ACEi/ARB exposed patients had CB by clinician assessment as opposed to 47% in those not on ACEi or ARB (p = 0.003). In ACEi exposed patients, 71% had CB versus 51% in unexposed patients (p = 0.048). In ARB exposed patients, 70% had CB versus 51% in unexposed patients (p = 0.069). By contrast, 51% of beta-blocker treated patients had CB versus 55% in untreated patients (p = 0.72). 36% of ACEi/ARB exposed patients remained on treatment at 12 months versus 22% of ACEi/ARB unexposed patients. Subset analyses were performed on the 3 largest patient populations: squamous NSCLC, nonsquamous NSCLC and melanoma. ACEi/ARB exposed melanoma patients had CB rate of 70% versus 43% in those unexposed. ACEi/ARB exposed squamous NSCLC patients had CB rate of 86% versus 47% in unexposed patients. By contrast, ACEi/ARB exposed nonsquamous NSCLC patients had a 56% CB rate versus 48% in unexposed patients. Conclusions: Based on retrospective analysis, advanced cancer patients treated with anti-PD-1/PD-L1 regimens had a statistically significant increase in clinical benefit if administered in the context of renin angiotensin system blockade. Prospective disease specific trials can further validate these findings and define the degree of clinical benefit.

P2.17-12 Eliminating Radiation Resistance of Non-Small Cell Lung Cancer By DHA Trough Abrogating Immunity Escaping via Inhibiting PD-L1 Expression

Lung cancer is a highly immune-suppressing malignancy with numerous methods to evade antitumor immune responses, including deficiencies in antigen processing and presentation, release of immunomodulatory cytokines, and inhibition of T cell activation. Our previous research also demonstrate that radiation can induce a local inflammatory response and simultaneously induce programmed death ligand 1 (PD-L1) expression that attenuate the sensitivity of radiation response in NSCLC. Current data have implicated that the molecular mechanisms by which DHA functions as radiosensitizer are varied, such as inducing apoptosis, extracellular signal-regulated protein kinases 1/2 (Erk1/2) activity, nuclear factor-kappa-B (NF-ƘB), and so on. However, there is no research about the immunity system alterations after the treatment of DHA plus radiation.In the present study, we demonstrate that combined DHA and radiotherapy synergistically enhances the anti-tumor effect by inhibiting the expression of PD-L1, eradicating the local accumulation of tumor-infiltrating regulatory T cells (iTregs) and myeloid-derived suppressor cells (MDSCs) and stimulating CD8+ T cell infiltration in the tumor microenvironment. Furthermore, DHA may also through inhibiting TGF-β, p-AKT and p-STAT3 pathways, epithelial-mesenchymal transition (EMT) process, facilitating apoptosis, and regulating TRIM21 to induce the synergistic anti-tumor effect. Radiation resistance cell lines (A549/X) was induced from A549 by conventionally fractionated radiation. The relationship between PD-L1 expression and the radiation resistance and the immune cell was investigated by immunohistochemistry (IHC) and western blot in vitro and vivo. Apoptosis was evaluated by flow cytometry. The signaling pathway proteins, epithelial-mesenchymal transition (EMT)-related protein were analyzed by western blot. PD-L1 was up-regulated after radiation in vivo and vitro. Concomitant with radiation-mediated tumor regression, combined DHA and radiotherapy synergistically enhances the anti-tumor effect by inhibiting the expression of PD-L1. DHA plus radiotherapy also reduced the local accumulation of tumor-infiltrating regulatory T cells (iTregs) and myeloid-derived suppressor cells (MDSCs) and stimulated CD8+ T cell infiltration in the tumor microenvironment. Furthermore, DHA may also through inhibiting TGF-β, p-AKT and p-STAT3 pathways, epithelial-mesenchymal transition (EMT) process, facilitating apoptosis, and regulating TRIM21 to induce the synergistic anti-tumor effect. Our study demonstrated a synergistic anti-tumor effect of DHA in combination with radiation trough abrogating immunity escaping via inhibiting PD-L1 expression, which establish a basis for the rational design of combination therapy of DHA plus radiotherapy in NSCLC.

Involvement of NFAT Transcription Factors in NK Cell Reactivity

NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), as highlighted by its important role in T and B cell development and function. With regard to NK cells, available data indicate that NFAT is dispensable for development. However, several lines of evidence including the observation that the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, influence NK reactivity implicate a role of this family of transcription factors in NK cell reactivity and function. Here we employed different genetic mouse models on the C57BL/6 background to directly study the functional role of NFAT in NK cells.We found that except for NFAT3 mRNA and protein of all family members (NFAT 1, 2, 4 and 5) was expressed in resting NK cells of wild type (WT) mice with NFAT1, 2 and 4 being most abundantly detectable. When we employed NK cells with knockout (KO) of NFAT 1, 2, and 4 in comparative in vitro analyses, we surprisingly found that lack of NFAT resulted in enhanced NK cell activation, degranulation and release of immunomodulatory cytokines like IFN-γ after co-culture with YAC-1 target cells as well as increased production of granzyme B and perforin after cytokine activation. The inhibitory effect of NFAT on NK cell effector function was further confirmed in vivo by employing WT and germ line NFAT KO animals in the syngeneic B16 melanoma model, which revealed a significantly reduced metastatic burden in NFAT KO mice. Depletion of NK cells in this model system in turn resulted in increased metastasis, however, with WT animals displaying significantly higher metastatic burden compared to NFAT KO mice. As this pointed to the fact that NFAT influences metastasis via both NK-dependent and independent mechanisms, we further generated mice with a NK cell-specific (conditional Ncr1-Cre dependent) NFAT2 KO. When these animals were employed again in analyses of B16 lung metastasis, comparative analyses with WT animals confirmed the inhibitory effect of NFAT on NK tumor immunosurveillance.Taken together, these results provide the first direct evidence for the functional involvement of NFAT in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function. DisclosuresNo relevant conflicts of interest to declare.

The immune response to primary EBV infection: a role for natural killer cells

The role of antigen-specific CD3(+)CD8(+) cytotoxic T cells in the control of primary Epstein-Barr Virus (EBV) infection is well established. However, time is required for the antigen-specific immune response to develop and expand. In contrast, innate immune responses, such as natural killer (NK) cells, are considered vital early in the infection process. We analysed the scale, phenotype and function of the NK cell response during symptomatic primary EBV infection, infectious mononucleosis (IM) and showed that NK cell numbers were significantly elevated both at diagnosis of IM and in the first month following diagnosis. There were also significant changes in cell phenotype and function, an increase in the proportion of CD56(bright) cells at diagnosis, and freshly isolated cells showing an enhanced ability to kill EBV-infected cell lines. Moreover, in our cohort of IM patients higher NK cell counts were associated with significantly lower viral load in peripheral blood. Our results suggest NK cells have an important role in the control of primary EBV infection by eliminating infected B cells and augmenting the antigen-specific T cell response via release of immunomodulatory cytokines. The magnitude of the NK cell response may ultimately determine whether primary EBV infection has a clinical outcome.

IL-10 production in cutaneous basal and squamous cell carcinomas. A mechanism for evading the local T cell immune response.

Cytokines play a vital role in the host immune response by regulating the development and function of immunocompetent cells. To explore the possibility that tumors may alter the host response via release of immunomodulatory cytokines, we studied two different skin cancers, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) as models. By RT-PCR, we found that the type 2 cytokines, IL-4 and IL-10, were strongly expressed in BCC compared with matched PBMC. Furthermore, IL-10 was more strongly expressed in SCC compared with benign growths. To identify the cell types responsible for production of these cytokines, tumor and tumor-infiltrating lymphocyte (TIL) cell lines were derived from BCC and SCC biopsy specimens. IL-2 and IFN-gamma mRNAs were expressed in TIL, while IL-10 mRNA were strongly expressed in BCC lines. In addition, IL-10 was detected in culture supernatants from BCC and SCC cell lines by ELISA and in tissue sections by immunohistology. TIL lines derived from these tumors demonstrated proliferative activity to autologous tumor cells in the presence of APCs, dependent on the addition of low concentrations of rIL-2 or neutralizing anti-IL-10 mAb in the culture medium. Furthermore, treatment of BCC with intralesional IFN-alpha induced tumor regression with concomitant up-regulation of IL-2 and down-regulation of IL-10 mRNA expression in lesions. These data suggest that tumor production of the cytokine, IL-10, may provide a mechanism for evading the local T cell-mediated immune response.

Regulation of Neutrophil Function During Exercise

In recent years there has been considerable interest in how exercise and training may affect the immune system. There is now substantial cross-sectional and epidemiological evidence that exercise causes significant changes in the distribution and function of a number of cellular and humoral immune parameters. Neutrophils represent one of the key nonspecific host defence cell populations responsible for the phagocytosis of many microbial, bacterial and viral pathogens. The neutrophil is also known to be involved in the synthesis and release of immunomodulatory cytokines that influence both T cell and B cell activities. Therefore, it plays an important role in both the efferent (phagocytosis and degranulation) and afferent (release of immunomodulatory molecules) limbs of the immune response. Neutrophils and macrophages respond both to phagocytosable particles (e.g. bacteria, viruses and cell debris) and to a number of soluble factors. There is an increase in the number of circulating neutrophils with exercise as a result of demargination of cells from endothelial tissues (mediated by catecholamines) and bone marrow (mediated by cortisol), or as part of the phagocytic and inflammatory response to exercise-induced tissue damage. Following exercise-induced mobilisation into the circulation and migration into tissues, neutrophils undergo adherence, phagocytosis (engulfment) of bacteria or tissue fragments, degranulation of cytoplasmic granules and, ultimately, activation of the respiratory burst. The capacity of the respiratory burst largely determines the cytotoxic potential of the neutrophil. The respiratory burst involves a sudden increase in nonmitochondrial oxidative metabolism, resulting in the production of the superoxide anion (O2-) and other reactive oxygen species by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex located at the plasma membrane. Although the biochemistry of the respiratory burst has been well studied, the mechanisms by which exercise and training may influence its activity are not well characterised or understood. Studies on the acute effects of exercise show that exercise generally elicits an initial activation of neutrophils-evidenced by release of cytoplasmic enzymes (degranulation) with secondary changes in key effector functions such as the phagocytic and respiratory burst activity. The nature of the functional changes is still unclear, as some studies show a transient suppression of the respiratory burst and/or phagocytic capacity immediately after exercise, while others report that moderate intensity exercise elicits an enhanced response. The variability in findings may be attributable to differences in the age, gender and initial fitness levels of the people studied, the intensity and duration of the exercise protocols used, and the different methodological procedures employed.(ABSTRACT TRUNCATED AT 400 WORDS)