The long pentraxin PTX3: A prototypical sensor of tissue injury and a regulator of homeostasis

Italian Ministry of Health. Grant Number: RF2011‐02348358 AIRC—Associazione Italiana per la Ricerca sul Cancro. Grant Number: AIRC 5x1000 cod. project 9962 Fondazione CARIPLO. Grant Number: 2015/056

Immune inhibitory receptors : regulated expression and suppression

The immune system protects against disease by identifying and eliminating pathogens, while leaving healthy host cells unaffected. Regulatory mechanisms are required to prevent excess or inappropriate immune cell activation and to ultimately terminate the immune response, thereby restoring homeostasis. One important regulatory mechanism is the expression of immune inhibitory receptors, which suppress activation signals initiated by immune receptors. Immune inhibitory receptors are characterized by the presence of Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs) in the intracellular tail of the receptor, which are crucial for the inhibitory function. Further research on inhibitory receptors is essential to fully understand their distinct roles in infection or inflammation and during the various stages of the immune response. This thesis centers on the characterization and function of the novel immune inhibitory receptor termed signal inhibitory receptor on leukocytes-1 (SIRL-1), and on the expression and function of the inhibitory receptor leukocyte-associated Ig-like receptor-1 (LAIR-1). Utilizing an in silico search for novel inhibitory receptors we identified SIRL-1. SIRL-1 contains two ITIMs and is exclusively expressed on human phagocytes. As a classical ITIM-bearing receptor, SIRL-1 is capable of inhibiting Fc?RI-mediated signaling and recruiting SH2 domain-containing phosphatases. The expression of SIRL-1 is regulated; pattern recognition receptor-induced phagocyte activation leads to a down-regulation of SIRL-1 expression. Furthermore, cross-linking of SIRL-1 decreases Reactive Oxygen Species (ROS) production in primary human phagocytes. ROS production by phagocytes is crucial for bacterial killing, and SIRL-1 cross-linking on primary neutrophils results in reduced killing of internalized bacteria. Notably, phagocytosis and cytokine production by phagocytes are not affected by SIRL-1 cross-linking, pointing to a selective regulation of ROS production. We propose that SIRL-1 on phagocytes sets an activation threshold to prevent inappropriate production of oxygen radicals. Upon infection, SIRL-1 is down-regulated, allowing microbial killing and clearance of the pathogen. Collagens are high affinity ligands for the inhibitory receptor LAIR-1, which is expressed on the majority of human peripheral blood mononuclear cells. The activating collagen receptor glycoprotein VI (GPVI) is structurally related to LAIR-1 and LAIR-1 cross-linking abrogates collagen-induced GPVI signaling when both receptors are ectopically expressed on the same cell. Interestingly, we identified a subset of morphologically distinct megakaryocytes which co-express GPVI and LAIR-1. We report for the first time that a primary cell co-expresses these collagen receptors with opposite signaling properties. Since megakaryocytes mature in the collagen-rich environment of the bone marrow, these findings may point to a role for LAIR-1 in the control of megakaryocyte migration. The most prominent splice variants of LAIR-1 are LAIR-1a and LAIR-1b, with LAIR-1b lacking 17 amino acids in the stalk region of the extracellular domain. We show that B and T cells express the LAIR-1b isoform, while granulocytes solely express LAIR-1a. We found that LAIR-1a has enhanced binding to collagen compared to LAIR-1b. Through its adhesive properties, LAIR-1a could affect neutrophil transmigration and perhaps megakaryocyte retention in the bone marrow niche. These hypotheses are subject of further investigation. Taken together, this thesis contributes to the understanding of the role of inhibitory receptors in immune regulatio

Effector/memory T cells of the weanling mouse exhibit Type 2 cytokine polarization in vitro and in vivo in the advanced stages of acute energy deficit.

Our objective was to determine whether the polarizing cytokine profile of the effector/memory T-cell compartment reflects the profound decline of cell-mediated inflammatory competence that characterizes acute prepubescent malnutrition. Weanling C57BL/6J mice were permitted free access to a complete purified diet, free access to an isocaloric low-protein diet or restricted intake of the complete diet for 14 days. First, interleukin (IL)-4 and interferon (IFN)-gamma concentrations generated in vitro by splenic and nodal effector/memory T cells were assessed following exposure to plate-bound anti-CD3. Second, net systemic production of IFN-gamma and IL-4 by the effector/memory T-cell compartment was assessed by the in vivo cytokine capture assay following anti-CD3 stimulation. In vitro stimulation generated less IFN-gamma (P=.002) but more IL-4 (P=.05) by T cells from the restricted-intake group relative to the age-matched control group. Similarly, in vivo stimulation generated low serum levels of antibody-captured IFN-gamma in the restricted-intake group vis-à-vis the age-matched control group (P=.01), while the IL-4 response was sustained (P=.39). By contrast, the 14-day low-protein model exhibited no change in T-cell cytokine signature either in vitro or in vivo. However, following extended consumption of the low-protein diet (26 days), carcass energy losses exceeded those of the 14-day protocol and serum levels of in vivo antibody-captured IFN-gamma were low after anti-CD3 challenge relative to the age-matched control group (P=.02), while levels of captured IL-4 remained unaffected (P=.07). Acute weanling malnutrition elicits a Type 2 polarizing cytokine character on the part of the effector/memory T-cell compartment, but only in the most advanced stages of energy decrement

Adoptively Transferred Dendritic Cells Restore Primary Cell-Mediated Inflammatory Competence to Acutely Malnourished Weanling Mice

Immune depression associated with prepubescent malnutrition underlies a staggering burden of infection-related morbidity. This investigation centered on dendritic cells as potentially decisive in this phenomenon. C57BL/6J mice, initially 19 days old, had free access for 14 days to a complete diet or to a low-protein formulation that induced wasting deficits of protein and energy. Mice were sensitized by i.p. injection of sheep red blood cells on day 9, at which time one-half of the animals in each dietary group received a simultaneous injection of 106 syngeneic dendritic cells (JAWS II). All mice were challenged with the immunizing antigen in the right hind footpad on day 13, and the 24-hour delayed hypersensitivity response was assessed as percentage increase in footpad thickness. The low-protein diet reduced the inflammatory immune response, but JAWS cells, which exhibited immature phenotypic and functional characteristics, increased the response of both the malnourished group and the controls. By contrast, i.p. injection of 106 syngeneic T cells did not influence the inflammatory immune response of mice subjected to the low-protein protocol. Antigen-presenting cell numbers limited primary inflammatory cell-mediated competence in this model of wasting malnutrition, an outcome that challenges the prevailing multifactorial model of malnutrition-associated immune depression. Thus, a new dendritic cell-centered perspective emerges regarding the cellular mechanism underlying immune depression in acute pediatric protein and energy deficit

Signal inhibitory receptor on leukocytes-1 (SIRL-1) negatively regulates the oxidative burst in human phagocytes.

ROS production is an important effector mechanism mediating intracellular killing of microbes by phagocytes. Inappropriate or untimely ROS production can lead to tissue damage, thus tight regulation is essential. We recently characterized signal inhibitory receptor on leukocytes-1 (SIRL-1) as an inhibitory receptor expressed by human phagocytes. Here, we demonstrate that ligation of SIRL-1 dampens Fc receptor-induced ROS production in primary human phagocytes. In accordance, SIRL-1 engagement on these cells impairs the microbicidal activity of neutrophils, without affecting phagocytosis. The inhibition of ROS production may result from reduced ERK activation, since co-ligation of Fc receptors and SIRL-1 on phagocytes inhibited phosphorylation of ERK. Importantly, we demonstrate that microbial and inflammatory stimuli cause rapid downregulation of SIRL-1 expression on the surface of primary neutrophils and monocytes. In accordance, SIRL-1 expression levels on neutrophils in bronchoalveolar lavage fluid from patients with neutrophilic airway inflammation are greatly reduced. We propose that SIRL-1 on phagocytes sets an activation threshold to prevent inappropriate production of oxygen radicals. Upon infection, SIRL-1 expression is downregulated, allowing microbial killing and clearance of the pathogen