Showing papers by "Ivan Zanoni published in 2014"

The nature of activatory and tolerogenic dendritic cell-derived signal 2.

Abstract: One of the most interesting issues in immunology is how the innate and adaptive branches of the immune system cooperate in vertebrate organisms to respond and destroy invading microorganisms without destroying self-tissues. More than 20 years ago, Charles Janeway proposed the innate immune recognition theory (1). He hypothesized the existence of innate receptors (Pattern recognition receptors, PRRs) that, by recognizing molecular structures associated to pathogens (PAMPs) and being expressed by antigen presenting cells (APCs) and epithelial cells, could alert the immune system to the presence of a pathogen, making it possible to mount an immediate inflammatory response. Moreover, by transducing the alert signal in professional APCs and inducing the expression of costimulatory molecules, these receptors could control the activation of lymphocytes bearing clonal antigen-specific receptors, thereby promoting appropriate adaptive immune responses. Since adaptive immunity can be activated also following sterile inflammatory conditions, it was subsequently proposed by Polly Matzinger that the innate immune system could be also activated by endogenous danger signals, generically called danger associated molecular patterns (DAMPs) (2). The first prediction has been amply confirmed by the discovery of Toll-like receptors (3–5) and cytoplasmic PRRs such as RIG-like receptors (6). Other PRR families such as the NOD-like receptors and C-type lectins exert immunogenic or tolerogenic signals (7–9) and may recognize not strictly pathogens but also endogenous danger signals (10, 11). Dendritic cells (DCs) have been identified as the cells of the innate immune system that, by sensing PAMPs or DAMPs transduce signals to the nucleus. This leads to a transcriptional reprograming of DCs with the consequent expression of three signals, namely signal 1 (MHC + peptide), signal 2 (surface costimulatory molecules), and signal 3 (cytokines) necessary for the priming of antigen-specific naive T cell responses (signal 1 and 2) and T cell polarization (signal 3). The reason why DCs are superior with respect to other professional APCs in naive T cell activation has not been unequivocally defined. It has not been established whether DCs can provide a special “signal 2” or simply very high levels, compared with other APCs, of commonly expressed signals 1 and 2, so that a naive T cell could reach the threshold of activation. A second aspect of DC biology needs also to be taken into account. Concerning the question of how self-tissues are not destroyed following the initiation of adaptive immune responses, different mechanisms of central and peripheral auto-reactive T cell tolerization have been proposed (12). In particular, it has been defined that high affinity T cells are deleted in the thymus, while low affinity auto-reactive T cells or T cells specific for tissue-sequestered antigens that do not have access to the thymus are controlled in the periphery. In a simplified vision of how peripheral T cell tolerance could be induced and maintained, it was thought that, in resting conditions, immature DCs, expressing low levels of signal 1 and low or no levels of signal 2, were able to induce T cell unresponsiveness. Nevertheless, it is now clear that a fundamental contribution to the peripheral tolerance is due to the conversion of naive T cells into peripheral regulatory T cells (pTreg cells) and it is also clear that DCs need to receive a specific conditioning to become able to induce pTreg cell differentiation. Even more intriguing is that also DCs activated through PRRs, with particular Toll-like receptor (TLR) agonists, are capable of generating pTreg cell conversion if these agonists induce the production of the appropriate cytokines. Thus, what is emerging is that immature DCs are not able to induce tolerance by default but need to receive specific signals in order to acquire the ability of transferring to T cells a tolerogenic, rather than an activatory, signal 2. Given these premises, this special issue covers the following topics: The responses induced specifically in DCs by PAMPs and DAMPs and the consequences of these responses. The DAMP and PAMP receptors expressed by different DC subsets and the consequences in the activation of adaptive immune responses. How DCs induce and maintain peripheral T cell tolerance and the stimuli that confer tolerogenicity.

Modulation of CD14 and TLR4·MD-2 activities by a synthetic lipid A mimetic.

Abstract: Monosaccharide lipid A mimetics based on a glucosamine core linked to two fatty acid chains and bearing one or two phosphate groups have been synthesized. Compounds 1 and 2, each with one phosphate group, were practically inactive in inhibiting LPS-induced TLR4 signaling and cytokine production in HEK-blue cells and murine macrophages, but compound 3, with two phosphate groups, was found to be active in efficiently inhibiting TLR4 signal in both cell types. The direct interaction between compound 3 and the MD-2 coreceptor was investigated by NMR spectroscopy and molecular modeling/docking analysis. This compound also interacts directly with the CD14 receptor, stimulating its internalization by endocytosis. Experiments on macrophages show that the effect on CD14 reinforces the activity on MD-2·TLR4 because compound 3's activity is higher when CD14 is important for TLR4 signaling (i.e., at low LPS concentration). The dual targeting of MD-2 and CD14, accompanied by good solubility in water and lack of toxicity, suggests the use of monosaccharide 3 as a lead compound for the development of drugs directed against TLR4-related syndromes.

Murein lytic enzyme TgaA of Bifidobacterium bifidum MIMBb75 modulates dendritic cell maturation through its cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) amidase domain.

Abstract: Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system.

Wiskott-Aldrich syndrome protein deficiency in natural killer and dendritic cells affects antitumor immunity

Abstract: Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency caused by reduced or absent expression of the WAS protein (WASP). WAS patients are affected by microthrombocytopenia, recurrent infections, eczema, autoimmune diseases, and malignancies. Although immune deficiency has been proposed to play a role in tumor pathogenesis, there is little evidence on the correlation between immune cell defects and tumor susceptibility. Taking advantage of a tumor-prone model, we show that the lack of WASP induces early tumor onset because of defective immune surveillance. Consistently, the B16 melanoma model shows that tumor growth and the number of lung metastases are increased in the absence of WASP. We then investigated the in vivo contribution of Was(-/-) NK cells and DCs in controlling B16 melanoma development. We found fewer B16 metastases developed in the lungs of Was(-/-) mice that had received WT NK cells as compared with mice bearing Was(-/-) NK cells. Furthermore, we demonstrated that Was(-/-) DCs were less efficient in inducing NK-cell activation in vitro and in vivo. In summary, for the first time, we demonstrate in in vivo models that WASP deficiency affects resistance to tumor and causes impairment in the antitumor capacity of NK cells and DCs.

rBet v 1 immunotherapy of sensitized mice with Streptococcus thermophilus as vehicle and adjuvant

Abstract: Lactobacilli are able to induce upregulation of co-stimulatory molecules in DCs with Th1 cytokines production and increase in Treg activity. This could explain the observed effectiveness of the prolonged administration of lactobacilli in the prevention of allergic disorders in infants and envisage the possible use of bacteria expressing the allergen for the specific immunotherapy of allergic diseases. Hence, we evaluated Streptococcus thermophilus (ST) expressing rBet v 1 as allergen delivery tool and adjuvant factor for immunotherapy in Betv1-sensitized mice. rBet v 1 gene was introduced and expressed in ST (ST[rBet v 1]). BALB/c mice were sensitized with rBet v 1 and then treated with either ST alone, ST[rBet v 1], or the combination of ST and rBet v 1, for 20 days. After 2 aerosol challenges, Treg frequency, in vitro allergen-induced cytokines, rBet v 1-specific IgE and IgG2a, and bronchial histology were made in harvested spleen, sera, and lung. Results were compared with those obtained from not-treat...