Absence Of Immune Cells Research Articles

Indoleamine 2,3-dioxygenase mediates immune-independent human tumor cell resistance to olaparib, gamma radiation, and cisplatin.

Indoleamine 2,3-dioxygenase-1 (IDO) is an immunosuppressive molecule expressed by most human tumors. IDO levels correlate with poor prognosis in cancer patients and IDO inhibitors are under investigation to enhance endogenous anticancer immunosurveillance. Little is known of immune-independent functions of IDO relevant to cancer therapy. We show, for the first time, that IDO mediates human tumor cell resistance to a PARP inhibitor (olaparib), gamma radiation, cisplatin, and combined treatment with olaparib and radiation, in the absence of immune cells. Antisense-mediated reduction of IDO, alone and (in a synthetic lethal approach) in combination with antisense to the DNA repair protein BRCA2 sensitizes human lung cancer cells to olaparib and cisplatin. Antisense reduction of IDO decreased NAD+ in human tumor cells. NAD+ is essential for PARP activity and these data suggest that IDO mediates treatment resistance independent of immunity and at least partially due to a previously unrecognized role for IDO in DNA repair. Furthermore, IDO levels correlated with accumulation of tumor cells in G1 and depletion of cells in G2/M of the cell cycle, suggesting that IDO effects on cell cycle may also modulate sensitivity to radiation and chemotherapeutic agents. IDO is a potentially valuable therapeutic target in cancer treatment, independent of immune function and in combination with other therapies.

Upregulation of endogenous ICAM‐1 reduces ovarian cancer cell growth in the absence of immune cells

Ovarian cancer is a difficult-to-treat cancer with a 5-year survival rate of only ∼45%, due to late diagnosis and therapy resistance. In need of new therapeutic approaches, induction of intercellular adhesion molecule (ICAM)-1 expression might be of interest, since the expression of ICAM-1 is lower in ovarian cancer cells compared with healthy ovarian cells and correlated with decreased tumorigenicity. Whereas ICAM-1 expression on tumor cells is of importance for attracting immune cells, ICAM-1 might also induce tumorigenicity and chemoresistance. In ovarian cancer, such a role of ICAM-1 is unclear. Here, we investigated whether ICAM-1 has a cell-biological role by bidirectional modulation of ICAM-1 expression using ICAM-targeting artificial transcription factors. For a panel of ovarian cancer cells, tumor growth and cisplatin sensitivity were evaluated. Induction of ICAM-1 expression (ranging from 3- to 228-fold on mRNA level and 1.7- to 108-fold on protein level) resulted in indications of decreased ovarian cancer cell growth and reduced cisplatin sensitivity. Repression ranged from 48 to 94% on mRNA level and 47 to 91% on protein level. This study shows that, next to its established immunogenic role, ICAM-1 affects cell biological behavior of ovarian cancer cells and, importantly, that reexpression by artificial transcription factors represents a powerful approach for functional validation of genes epigenetically silenced in cancer, such as ICAM-1.

Lipopolysaccharide induces a fibrotic‐like phenotype in endothelial cells

Endothelial dysfunction is crucial in endotoxaemia-derived sepsis syndrome pathogenesis. It is well accepted that lipopolysaccharide (LPS) induces endothelial dysfunction through immune system activation. However, LPS can also directly generate actions in endothelial cells (ECs) in the absence of participation by immune cells. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge. However, the mechanism that confers endothelial resistance to LPS is not known. LPS-resistant ECs exhibit a fibroblast-like morphology, suggesting that these ECs enter a fibrotic programme in response to LPS. Thus, our aim was to investigate whether LPS is able to induce endothelial fibrosis in the absence of immune cells and explore the underlying mechanism. Using primary cultures of ECs and culturing intact blood vessels, we demonstrated that LPS is a crucial factor to induce endothelial fibrosis. We demonstrated that LPS was able and sufficient to promote endothelial fibrosis, in the absence of immune cells through an activin receptor–like kinase 5 (ALK5) activity–dependent mechanism. LPS-challenged ECs showed an up-regulation of both fibroblast-specific protein expression and extracellular matrix proteins secretion, as well as a down-regulation of endothelial markers. These results demonstrate that LPS is a crucial factor in inducing endothelial fibrosis in the absence of immune cells through an ALK5-dependent mechanism. It is noteworthy that LPS-induced endothelial fibrosis perpetuates endothelial dysfunction as a maladaptive process rather than a survival mechanism for protection against LPS. These findings are useful in improving current treatment against endotoxaemia-derived sepsis syndrome and other inflammatory diseases.

The liposome‐incorporating cell wall skeleton of Mycobacterium bovis bacillus Calmette‐Guéin can directly enhance the susceptibility of cancer cells to lymphokine‐activated killer cells through up‐regulation of natural‐killer group 2, member D ligands

• To conduct a preclinical evaluation of the ability of natural killer cells to cytolyze bladder cancer cells that were modified to show enhanced expression of natural-killer group 2, member D (NKG2D) ligands by R8-liposome-bacillus Calmette-Guéin (BCG)-cell wall skeleton (CWS) treatment. • The T24 cells and RT-112 cells were co-cultured with R8-liposome-BCG-CWS and BCG for 2, 4, or 6 h, and then the surface expression of NKG2D ligands was analyzed using TaqMan real-time quantitative RT-PCR. • Peripheral blood mononuclear cells were obtained with a conventional preparation kit, and then lymphokine-activated killer (LAK) cells were generated from these purified peripheral blood mononuclear cells via interleukin-2 stimulation. • The anti-tumour effect of LAK cells against untreated and R8-liposome-BCG-CWS co-cultured with cells of the human bladder cancer cell lines T24 and RT-112 was analyzed using the cytotoxic WST-8 assay method at 4 h of culture at various effector/target (E : T) ratios. • Major histocompatibility complex class I-related chain B (MICB) expression was increased ≈1.5-fold on T24 cells and RT-112 cells with BCG. • UL-16-binding protein (ULBP) 1 expression was also increased ≈1.5-fold on T24 cells and RT-112 cells with BCG. R8-liposome-BCG-CWS increased the surface expression of MICB 2.2-fold on T24 cells but did not increase it significantly on RT-112 cells. • ULBP1 expression was increased ≈2.2-fold on RT-112 cells, although no differences were observed between the expression of ULBP2 and 3 with R8-liposome-BCG-CWS. • T24 cells that were co-cultured with R8-liposome-BCG-CWS showed an ≈1.3-fold increase in sensitivity to cytolysis by LAK cells at an E : T ratio of 4 and RT-112 cells showed an ≈1.4-fold increase at an E : T ratio of 2. • In the present study, the induction of surface NKG2D ligands by R8-liposome-BCG-CWS rendered cancer cells more susceptible to cytolysis by LAK cells. • T24 cells and RT-112 cells, even when cultured singly in the absence of immune cells, can directly respond to R8-liposome-BCG-CWS. • The results obtained in the present study may therefore indicate a novel adoptive immunotherapy against bladder cancers.

Mesenchymal stem cells improve murine acute coxsackievirus B3-induced myocarditis

AimsCoxsackievirus B3 (CVB3)-induced myocarditis, initially considered a sole immune-mediated disease, also results from a direct CVB3-mediated injury of the cardiomyocytes. Mesenchymal stem cells (MSCs) have, besides immunomodulatory, also anti-apoptotic features. In view of clinical translation, we first analysed whether MSCs can be infected by CVB3. Next, we explored whether and how MSCs could reduce the direct CVB3-mediated cardiomyocyte injury and viral progeny release, in vitro, in the absence of immune cells. Finally, we investigated whether MSC application could improve murine acute CVB3-induced myocarditis.Methods and resultsPhase contrast pictures and MTS viability assay demonstrated that MSCs did not suffer from CVB3 infection 4–12–24–48 h after CVB3 infection. Coxsackievirus B3 RNA copy number decreased in this time frame, suggesting that no CVB3 replication took place. Co-culture of MSCs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis, oxidative stress, intracellular viral particle production, and viral progeny release in a nitric oxide (NO)-dependent manner. Moreover, MSCs required priming via interferon-γ (IFN-γ) to exert their protective effects. In vivo, MSC application improved the contractility and relaxation parameters in CVB3-induced myocarditis, which was paralleled with a reduction in cardiac apoptosis, cardiomyocyte damage, left ventricular tumour necrosis factor-α mRNA expression, and cardiac mononuclear cell activation. Mesenchymal stem cells reduced the CVB3-induced CD4− and CD8− T cell activation in an NO-dependent way and required IFN-γ priming.ConclusionWe conclude that MSCs improve murine acute CVB3-induced myocarditis via their anti-apoptotic and immunomodulatory properties, which occur in an NO-dependent manner and require priming via IFN-γ.

Effects of non-steroidal anti-inflammatory drugs on Aβ deposition in Aβ 1–42 transgenic C. elegans

Although epidemiological studies have shown that long-term treatment with non-steroidal anti-inflammatory drugs (NSAIDs) may protect against Alzheimer's disease (AD), the mechanism(s) by which NSAIDs reduce the risk of AD remain to be determined. As C. elegans possess neither inflammatory cells nor the arachidonate cascade, we could evaluate the effects of NSAIDs on amyloid β (Aβ) deposition in the absence of immune cells using Aβ-transgenic C. elegans. For this purpose, we established a strain of Aβ-transgenic C. elegans in which thioflavin S-reactive deposits are reproducibly detectable by confocal microscopy. Among the NSAIDs examined, ibuprofen and naproxen reduced the number of thioflavin S-reactive deposits. Furthermore, ibuprofen and naproxen neither affect the thioflavin S binding to Aβ nor Aβ expression in transgenic C. elegans. These data suggest that ibuprofen and naproxen, the most frequently used NSAIDs for the treatment of AD, have an inhibitory effect on Aβ deposition that is independent of the arachidonate cascade and cellular immune systems.

The Small Antitumoral Immune Response Modifier Imiquimod Interacts with Adenosine Receptor Signaling in a TLR7- and TLR8-Independent Fashion

Imiquimod, a small-molecule immune response modifier of the imidazoquinoline family, has shown profound antitumoral and antiviral efficacy both in vitro and in clinical applications in vivo. It has been demonstrated that this activity is mediated through the Toll-like receptor (TLR)7- and TLR8-signaling cascade resulting in the secretion of proinflammatory cytokines and, consecutively, induction of a tumor-directed cellular immune response. In addition, imiquimod exerts a direct proapoptotic activity in tumor cells. We demonstrate here that imiquimod induces activation of the transcription factor NF-kappaB and the downstream production of proinflammatory cytokines in the absence of TLR7 and TLR8. In Chinese hamster ovary cells stably transfected with the human adenosine receptor subtypes, we then show in radioligand-binding competition experiments that imiquimod binds to adenosine receptors at concentrations relevant in clinical settings, with highest affinities to the A(1) and A(2A) subtypes. The effect on the receptor-mediated activation of adenylyl cyclase was also studied, and these experiments revealed that imiquimod acts as an adenosine receptor antagonist. In addition, imiquimod had an inhibitory effect on adenylyl cyclase activity downstream from the receptor. Finally, using transformed human keratinocytes, we provide experimental evidence that imiquimod and A(2A) adenosine receptor-specific compounds similarly induce proinflammatory cytokines in the absence of immune cells. Thus, imiquimod appears to suppress an important feedback mechanism of inflammation by antagonism of adenosine receptor-dependent increase of cAMP and a concomitant receptor-independent inhibition of cAMP production. These novel mechanisms presumably act synergistic with the positive induction of proinflammatory cytokines and can, at least in part, explain the profound inflammation observed in some patients in vivo.

Detection of ACTH, MC‐1R and MC‐4R immunoreactivity in human terminal and vellus hair follicles

Pro‐opiomelanocortin (POMC) peptides such as ACTH and α‐MSH play an important role in the pigmentation of human skin and hair. Both peptides bind to a family of transmembrane receptors known as the melanocortin receptors (MC‐Rs). To investigate the in situ expression of α‐MSH, ACTH and MC‐Rs, we performed immunohistochemical studies on human terminal and vellus hair follicles. Immunoreactivity for ACTH, MC‐1R and MC‐4R was detected in both human terminal and vellus hair follicles, with a pronounced increase of staining intensity in the suprabulbar and bulbar region of anagen hair follicles. The MC‐1R immunoreactivity in situ was in accordance with in vitro studies showing expression of MC‐1R at the RNA level in human hair follicle keratinocytes. There was no detectable difference in the staining pattern of strong, pigmented terminal hair follicles as compared with fine, silky and non‐pigmented vellus hair follicles. These results indicate that POMC‐derived peptides may play a regulatory role in the human hair follicle independent of the pigmentation process. The expression pattern of ACTH and its receptors MC‐1R and MC‐4R in the human hair follicle correlates with the immune privilege of the human hair follicle which is characterized by the absence of immune cells in the bulbar region of anagen hair follicles. Thus, ACTH may possibly be involved in the maintenance of the peribulbar immune privilege in both, terminal and vellus hair follicles.

HIV-1 Tat enhances Kaposi sarcoma–associated herpesvirus (KSHV) infectivity

AbstractThe high frequency of Kaposi sarcoma (KS) in immunodeficiency states, particularly in patients with AIDS, has been attributed to increased replication of KS-associated herpesvirus (KSHV), a necessary cofactor for KS development. However, experimental KSHV infection of endothelial lineage cells that compose KS lesions has been difficult even in the absence of immune cells. Here we show that HIV-1 Tat protein can directly promote KSHV transmission. Full-length HIV-1 Tat and a 13–amino-acid peptide corresponding to the basic region of Tat specifically enhances the entry of KSHV into endothelial and other cells, presenting evidence for an active role of HIV-1 in the development of KSHV-associated diseases. These results can explain why AIDS-KS is more frequent and clinically more aggressive than KS in other immunodeficiency states.

Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells.

Demyelination results in severe disability in many neurodegenerative diseases and nervous system infections, and it is typically mediated by inflammatory responses. Mycobacterium leprae, the causative organism of leprosy, induced rapid demyelination by a contact-dependent mechanism in the absence of immune cells in an in vitro nerve tissue culture model and in Rag1-knockout (Rag1-/-) mice, which lack mature B and T lymphocytes. Myelinated Schwann cells were resistant to M. leprae invasion but undergo demyelination upon bacterial attachment, whereas nonmyelinated Schwann cells harbor intracellular M. leprae in large numbers. During M. leprae-induced demyelination, Schwann cells proliferate significantly both in vitro and in vivo and generate a more nonmyelinated phenotype, thereby securing the intracellular niche for M. leprae.

Effect of gliadin and other food peptides on expression of MHC class II molecules by HT-29 cells.

Expression of major histocompatibility (MHC) class II molecules by enterocytes is known to be enhanced in coeliac disease and other disorders characterised by intestinal inflammation--an effect thought to be mediated via intestinal lymphocytes. To investigate if food peptides can exert direct effects on class II expression, the influence of gliadins, casein, and beta lactoglobulin on an intestinal epithelial cell line (HT-29) was examined in the absence of immune cells. Class II expression was determined by flow cytometry and immunofluorescence microscopy using antibodies against the beta chain of all products of the gene subregions DR, DQ, and DP. MHC expression was low in HT-29 cells but could be stimulated by interferon gamma. Tryptin digested gliadin had no effect on class II expression. In the presence of interferon gamma, however, it was able to amplify MHC class II expression to mean (SEM) 150 (4)%. Casein exerted a similar effect (160 (14)%), but undigested gliadin, tryptin digested casein, and beta lactoglobulin had no influence. The observations suggest that within the concert of cytokine mediated interactions between enterocytes and lymphocytes, some dietary peptides could upregulate the presentation of food antigens, leading to a more efficient stimulation of lymphocytes, which in the case of coeliac disease might result in damage to the enterocytes.