Tn glycosylation of the MUC6 protein modulates its immunogenicity and promotes the induction of Th17-biased T cell responses

The Tn antigen (α-GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. This antigen, together with mucins, the major carriers of O-glycosylated tumor antigens in adenocarcinomas, are being evaluated as anti-cancer immunotherapeutic targets. In particular, the MUC6 protein, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. To develop anti-cancer vaccines based on the Tn antigen, we produced MUC6 proteins with different Tn density by using mixtures of recombinant ppGalNAc-T1, -T2, and -T7. The obtained glycoproteins were characterized and analyzed for their immunological properties, as compared with the non-glycosylated MUC6. We show that these various MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, Tn glycosylation of the MUC6 protein strongly affected their immunogenicity by partially abrogating Th1 cell responses, and promoting IL-17 responses. Moreover, the non-glycosylated MUC6 was more efficiently presented than MUC6:Tn glycoproteins to specific T CD4(+) hybridomas, suggesting that Tn glycosylation may affect MUC6 processing or MHC binding of the processed peptides. In conclusion, our results indicate that Tn glycosylation of the MUC6 protein strongly affects its B and T cell immunogenicity, and might favor immune escape of tumor cells.     

Glycoantigens induce human peripheral Tr1 cell differentiation with gut-homing specialization

The carbohydrate antigen (glycoantigen) PSA from an intestinal commensal bacteria is able to down-regulate inflammatory bowel disease in model mice, suggesting that stimulation with PSA results in regulatory T cell (Treg) generation. However, mechanisms of how peripheral human T cells respond and home in response to commensal antigens are still not understood. Here, we demonstrate that a single exposure to PSA induces differentiation of human peripheral CD4(+) T cells into type-Tr1 Tregs. This is in contrast to mouse models where PSA induced the production of Foxp3(+) iTregs. The human PSA-induced Tr1 cells are profoundly anergic and exhibit nonspecific bystander suppression mediated by IL-10 secretion. Most surprisingly, glycoantigen exposure provoked expression of gut homing receptors on their surface. These findings reveal a mechanism for immune homeostasis in the gut whereby exposure to commensal glycoantigens provides the requisite information to responding T cells for proper tissue localization (gut) and function (anti-inflammatory/regulatory).     

Down-regulation of cytokine-induced interleukin-8 requires inhibition of p38 mitogen-activated protein kinase (MAPK) via MAPK phosphatase 1-dependent and -independent mechanisms

Down-regulation of overabundant interleukin (IL)-8 present in cystic fibrosis (CF) airways could ease excessive neutrophil burden and its deleterious consequences for the lung. IL-8 production in airway epithelial cells, stimulated with e.g. inflammatory cytokines IL-1β and tumor necrosis factor (TNF)-α, is regulated by several signaling pathways including nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK). We previously demonstrated that the anti-inflammatory drugs dexamethasone and ibuprofen suppress NF-κB; however, only dexamethasone down-regulates cytokine-induced IL-8, highlighting the importance of non-NF-κB mechanisms. Here, we tested the hypothesis that down-regulation of cytokine-induced IL-8 requires modulation of the MAPK phosphatase (MKP)-1/p38 MAPK/mRNA stability pathway. The effects of dexamethasone (5 nm) and ibuprofen (480 μm) on this pathway and IL-8 were studied in CF (CFTE29o-, CFBE41o-) and non-CF (1HAEo-) airway epithelial cells. We observed that dexamethasone, but not ibuprofen, destabilizes IL-8 mRNA and up-regulates MKP-1 mRNA. Further, siRNA silencing of MKP-1, via p38 MAPK, leads to IL-8 overproduction and diminishes the anti-IL-8 potential of dexamethasone. However, MKP-1 overexpression does not significantly alter IL-8 production. By contrast, direct inhibition of p38 MAPK (inhibitor SB203580) efficiently suppresses IL-8 with potency comparable with dexamethasone. Similar to dexamethasone, SB203580 decreases IL-8 mRNA stability. Dexamethasone does not affect p38 MAPK activation, which excludes its effects upstream of p38 MAPK. In conclusion, normal levels of MKP-1 are necessary for a full anti-IL-8 potential of pharmacological agents; however, efficient pharmacological down-regulation of cytokine-induced IL-8 also requires direct effects on p38 MAPK and mRNA stability independently of MKP-1.     

Naloxone and ouabain in ultralow concentrations restore Na+/K+-ATPase and cytoskeleton in lipopolysaccharide-treated astrocytes

Astrocytes respond to inflammatory stimuli and may be important modulators of the inflammatory response in the nervous system. This study aimed first to assess how astrocytes in primary culture behave in response to inflammatory stimuli concerning intracellular Ca(2+) responses, expression of Toll-like receptor 4 (TLR4), Na(+)/K(+)-ATPase, actin filament organization, and expression of cytokines. In a cell culture model with lipopolysaccharide (LPS), astrocyte response was assessed first in the acute phase and then after incubation with LPS for 1-48 h. The concentration curve for LPS-stimulated Ca(2+) responses was bell-shaped, and the astrocytes expressed TLR4, which detects LPS and evokes intracellular Ca(2+) transients. After a long incubation with LPS, TLR4 was up-regulated, LPS-evoked Ca(2+) transients were expressed as oscillations, Na(+)/K(+)-ATPase was down-regulated, and the actin filaments were disorganized. Interleukin-1β (IL-1β) release was increased after 24 h in LPS. A second aim was to try to restore the LPS-induced changes in astrocytes with substances that may have dose-dependent anti-inflammatory properties. Naloxone and ouabain were tested separately in ultralow or high concentrations. Both substances evoked intracellular Ca(2+) transients for all of the concentrations from 10(-15) up to 10(-4) M. Neither substance blocked the TLR4-evoked Ca(2+) responses. Naloxone and ouabain prevented the LPS-induced down-regulation of Na(+)/K(+)-ATPase and restored the actin filaments. Ouabain, in addition, reduced the IL-1β release from reactive astrocytes. Notably, ultralow concentrations (10(-12) M) of naloxone and ouabain showed these qualities. Ouabain seems to be more potent in these effects of the two tested substances.     

Effects of carotenoids, immune activation and immune suppression on the intensity of chronic coccidiosis in greenfinches

Allocation trade-offs of carotenoids between their use in the immune system and production of integumentary colouration have been suggested as a proximate mechanism maintaining honesty of signal traits. We tested how dietary carotenoid supplementation, immune activation and immune suppression affect intensity of coccidian infection in captive greenfinches Carduelis chloris, a passerine with carotenoid-based plumage. Immune activation with phytohaemagglutinin (PHA) decreased body mass among birds not supplemented with lutein, while among the carotenoid-fed birds, PHA had no effect on mass dynamics. Immune suppression with dexamethasone (DEX) induced loss of body mass and reduced the swelling response to PHA. DEX and PHA increased the concentration of circulating heterophils. Lutein supplementation increased plasma carotenoid levels but had no effect on the swelling response induced by PHA. PHA and DEX treatments did not affect plasma carotenoids. Immune stimulation by PHA suppressed the infection, but only among carotenoid-supplemented birds. Priming of the immune system can thus aid in suppressing chronic infection but only when sufficient amount of carotenoids is available. Our experiment shows the importance of carotenoids in immune response, but also the complicated nature of this impact, which could be the reason for inconsistent results in studies investigating the immunomodulatory effects of carotenoids. The findings about involvement of carotenoids in modulation of an immune response against coccidiosis suggest that carotenoid-based ornaments may honestly signal individuals’ ability to manage chronic infections.     

Role of a small G protein Ras in cellular immune response of the beet armyworm, Spodoptera exigua

Insect cellular immune responses accompany cytoskeletal rearrangement of hemocytes to exhibit filopodial and pseudopodial extension of their cytoplasm. Small G proteins are postulated to be implicated in the hemocyte cellular processes to perform phagocytosis, nodulation, and encapsulation behaviors. A small G protein ras gene (Se-Ras) was cloned from cDNAs prepared from hemocytes of the beet armyworm, Spodoptera exigua. The open reading frame of Se-Ras encoded 179 amino acids with a predicted molecular weight of 20.0 kDa, in which 114 residues at amino terminus were predicted to be a GTP binding domain. It showed high sequence similarities (86.1-92.8%) with known ras genes in other insects. Se-Ras was constitutively expressed in all developmental stages from egg to adult without any significant change in expression levels in response to bacterial challenge. A specific double strand RNA (dsRNA) could knockdown its expression in the hemocytes after 48 h post-injection. While the RNA interference (RNAi) did not show any change in total or differential hemocyte counts, it impaired hemocyte behaviors. The RNAi of Se-Ras significantly suppressed hemocyte spreading, cytoskeleton extension, and nodulation behaviors in response to bacterial challenge. Release of prophenoloxidase from oenocytoids was significantly inhibited by the RNAi, which resulted in significant suppression in PO activation in response to an inducer, PGE₂. These results suggest that Se-Ras is implicated in mediating cellular processes of S. exigua hemocytes. This is the first report of Ras role in insect cellular immune response.     

Mathematical model of a three-stage innate immune response to a pneumococcal lung infection

Pneumococcal pneumonia is a leading cause of death and a major source of human morbidity. The initial immune response plays a central role in determining the course and outcome of pneumococcal disease. We combine bacterial titer measurements from mice infected with Streptococcus pneumoniae with mathematical modeling to investigate the coordination of immune responses and the effects of initial inoculum on outcome. To evaluate the contributions of individual components, we systematically build a mathematical model from three subsystems that describe the succession of defensive cells in the lung: resident alveolar macrophages, neutrophils and monocyte-derived macrophages. The alveolar macrophage response, which can be modeled by a single differential equation, can by itself rapidly clear small initial numbers of pneumococci. Extending the model to include the neutrophil response required additional equations for recruitment cytokines and host cell status and damage. With these dynamics, two outcomes can be predicted: bacterial clearance or sustained bacterial growth. Finally, a model including monocyte-derived macrophage recruitment by neutrophils suggests that sustained bacterial growth is possible even in their presence. Our model quantifies the contributions of cytotoxicity and immune-mediated damage in pneumococcal pathogenesis.     

A mathematical model of combined bacillus Calmette-Guerin (BCG) and interleukin (IL)-2 immunotherapy of superficial bladder cancer

We report a mathematical model that describes the growth of superficial bladder cancer and the effect thereupon of immunotherapy based on the administration of Bacillus Calmette-Guerin (BCG) combined or not with interleukin-2 (IL-2). Intravesical instillations of BCG performed after surgical removal of tumors represents an established treatment with approximately 50% success rate. So far, attempts to improve this efficiency have not led to essential changes. However, convincing clinical results have been reported on the combination of IL-2 to BCG, even though this is still not applied in current practice. The present model provides insights into the dynamical outcomes arising in the bladder from the interactions of immune cells with tumor cells in the course of BCG therapy associated or not with IL-2. Specifically, from the simulations performed using seven ordinary and non-linear differential equations we obtained indications on the conditions that would result in successful bladder cancer treatment. We show that immune cells -effector lymphocytes and antigen-presenting cells-expand and reach a sustainable plateau under BCG treatment, which may account for its beneficial effect, resulting from inflammatory “side-effects” which eliminate residual or eventual newly arising tumor cells, providing thus protection from further cancer development. We find, however, that IL-2 does not actually potentiate the effect of BCG as regards tumor cell eradication. Hence, associating both under the conditions simulated should not result in more efficient treatment of bladder cancer patients.     

Some implications of Scale Relativity theory in avascular stages of growth of solid tumors in the presence of an immune system response

We present a traveling-wave analysis of a reduced mathematical model describing the growth of a solid tumor in the presence of an immune system response in the framework of Scale Relativity theory. Attention is focused upon the attack of tumor cells by tumor-infiltrating cytotoxic lymphocytes (TICLs), in a small multicellular tumor, without necrosis and at some stage prior to (tumor-induced) angiogenesis. For a particular choice of parameters, the underlying system of partial differential equations is able to simulate the well-documented phenomenon of cancer dormancy and propagation of a perturbation in the tumor cell concentration by cnoidal modes, by depicting spatially heterogeneous tumor cell distributions that are characterized by a relatively small total number of tumor cells. This behavior is consistent with several immunomorphological investigations. Moreover, the alteration of certain parameters of the model is enough to induce soliton like modes and soliton packets into the system, which in turn result in tumor invasion in the form of a standard traveling wave. In the same framework of Scale Relativity theory, a very important feature of malignant tumors also results, that even in avascular stages they might propagate and invade healthy tissues, by means of a diffusion on a Newtonian fluid.     

Plasticity and limitations of extended egg retention in oviparous Zootoca vivipara (Reptilia: Lacertidae)

The transition between oviparity and viviparity in reptiles is generally accepted to be a gradual process, the result of selection for increasingly prolonged egg retention within the oviduct. We examined egg retention plasticity in an oviparous strain of the lacertid lizard Zootoca vivipara, a species having both oviparous and viviparous populations. We forced a group of female Z. vivipara to retain their clutch in utero by keeping them in dry substrata, and assessed the effect on embryonic development and hatching success, along with offspring phenotype and locomotor performance. Forced egg retention for one additional week affected the developmental stage of embryos at oviposition, as well as hatchling robustness and locomotor performance. However, embryos from forced clutch retention treatment reached one stage unit more than control embryos at oviposition time. Embryos from control eggs were more developed than embryos from experimental eggs after approximately the same period of external incubation, showing that embryonic development is retarded during the period of extended egg retention, despite the high temperature inside the mother's body. Significant differences in external incubation time were only found in one of the two years of study. Hatching success was much lower in the experimental group with forced egg retention (21.1%) than in the control group (95.4%). Therefore, we conclude that there are limitations that hinder the advance of intrauterine embryonic development beyond the normal time of oviposition, and that extended egg retention does not represent clear advantages in this population of Z. vivipara. Nevertheless, the fact that some eggs are successful after forced egg retention could be advantageous for the females that are able to retain their clutch under unfavourable climatic conditions. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 75–82.