Exposure to heat shock and other stressful conditions activates in cells of all organisms a speci... more Exposure to heat shock and other stressful conditions activates in cells of all organisms a specific genetic program. This enhances the synthesis of proteins with a protective role against cellular damage, called heat shock proteins (hsps). Furthermore, in the mammalian nervous system, a considerable amount of hsps is also synthesized under normal conditions suggesting that they play an important role in the metabolism of unstressed cells. In this study we analysed the constitutive expression of proteins belonging to the hsp70 and hsp90 family in the rat cerebellum using immunocytochemistry with specific monoclonal antibodies. Our results showed that an intense immunostaining was evident, but was restricted in certain distinct cerebellar areas only, while no differences in the distribution of the two hsps were found. The strongest response was detected in the Purkinje neurons but deep cerebellar nuclei were also positive. In no case glial cells were found to be reactive for hsps despite their strong response for specific markers like glial fibrillary acid protein (astrocytes) and cyclic nucleotide phosphodiesterase (oligodendrocytes). These data indicate that both the hsp70 and hsp90 family have fundamental physiological functions in cerebellar neurons while they seem to play only a minor role in the metabolism of glial cells.
Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here ... more Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here we report that the CCRL2/chemerin/CMKLR1 axis represents a selective pathway for the homing of natural killer (NK) cells to the lung. C–C motif chemokine receptor-like 2 (CCRL2) is a nonsignaling seven-transmembrane domain receptor able to control lung tumor growth. CCRL2 constitutive or conditional endothelial cell targeted ablation, or deletion of its ligand chemerin, were found to promote tumor progression in a Kras/p53Flox lung cancer cell model. This phenotype was dependent on the reduced recruitment of CD27– CD11b+ mature NK cells. Other chemotactic receptors identified in lung-infiltrating NK cells by single-cell RNA sequencing (scRNA-seq), such as Cxcr3, Cx3cr1, and S1pr5, were found to be dispensable in the regulation of NK-cell infiltration of the lung and lung tumor growth. scRNA-seq identified CCRL2 as the hallmark of general alveolar lung capillary endothelial cells. CCRL2 expression was epigenetically regulated in lung endothelium and it was upregulated by the demethylating agent 5-aza-2′-deoxycytidine (5-Aza). In vivo administration of low doses of 5-Aza induced CCRL2 upregulation, increased recruitment of NK cells, and reduced lung tumor growth. These results identify CCRL2 as an NK-cell lung homing molecule that has the potential to be exploited to promote NK cell–mediated lung immune surveillance.
To gain more information about the relationship between the structure of IFN-gamma and its activi... more To gain more information about the relationship between the structure of IFN-gamma and its activity, a peptide corresponding to a hydrophilic peak between amino acids 4 and 16 was used to immunize mice and generate mAb. mAb IGMB-15 reacts to both native and rIFN-gamma and neutralizes the antiproliferative activity of IFN-gamma without affecting its antiviral activity or its ability to up-regulate HLA-DR Ag expression. Moreover, we observed that mAb IGMB-15 was unable to inhibit the binding of radiolabeled IFN-gamma to its cellular receptor. These findings show that the NH2-terminal region may somehow be involved in the biologic activity of IFN-gamma. Besides, the capability of mAb IGMB-15 to inhibit the antiproliferative but not the antiviral activity of IFN-gamma in the same cell (HEp-2) suggests the presence of different elements involved in signal transduction, which may account for the multiple activities of the lymphokine.
COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. Howe... more COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of ...
Inhibitors of phosphodiesterase-4 (PDE4) are small-molecule drugs that, by increasing the intrace... more Inhibitors of phosphodiesterase-4 (PDE4) are small-molecule drugs that, by increasing the intracellular levels of cAMP in immune cells, elicit a broad spectrum of anti-inflammatory effects. As such, PDE4 inhibitors are actively studied as therapeutic options in a variety of human diseases characterized by an underlying inflammatory pathogenesis. Dendritic cells (DCs) are checkpoints of the inflammatory and immune responses, being responsible for both activation and dampening depending on their activation status. This review shows evidence that PDE4 inhibitors modulate inflammatory DC activation by decreasing the secretion of inflammatory and Th1/Th17-polarizing cytokines, although preserving the expression of costimulatory molecules and the CD4+ T cell-activating potential. In addition, DCs activated in the presence of PDE4 inhibitors induce a preferential Th2 skewing of effector T cells, retain the secretion of Th2-attracting chemokines and increase the production of T cell regulat...
Background Tanimilast is a novel and selective inhaled inhibitor of phosphodiesterase-4 in advanc... more Background Tanimilast is a novel and selective inhaled inhibitor of phosphodiesterase-4 in advanced clinical development for chronic obstructive pulmonary disease (COPD). Tanimilast is known to exert prominent anti-inflammatory activity when tested in preclinical experimental models as well as in human clinical studies. Recently, we have demonstrated that it also finely tunes, rather than suppressing, the cytokine network secreted by activated dendritic cells (DCs). This study was designed to characterize the effects of tanimilast on T-cell polarizing properties of DCs and to investigate additional functional and phenotypical features induced by tanimilast. Methods DCs at day 6 of culture were stimulated with LPS in the presence or absence of tanimilast or the control drug budesonide. After 24 h, DCs were analyzed for the expression of surface markers of maturation and activation by flow cytometry and cocultured with T cells to investigate cell proliferation and activation/polarizat...
Histone deacetylase inhibitors (HDIs) are promising drugs for the treatment of inflammatory disea... more Histone deacetylase inhibitors (HDIs) are promising drugs for the treatment of inflammatory diseases. However, their therapeutical exploitation is slowed down by severe adverse manifestations that can hardly be foreseen, mainly due to incomplete knowledge of how HDIs impact the delicate balance of inflammatory mediators. In this work, we characterized the effects of the HDI trichostatin A (TSA) on the expression of TNFAIP3, which is a crucial inhibitor of the classical NF-kB pathway and an LPS-induced negative feedback regulator. The accumulation of TNFAIP3 mRNA after LPS stimulation showed biphasic behavior, with one wave within the first hour of stimulation and a second wave several hours later, which were both reduced by TSA. By using inhibition and knockdown approaches, we identified two temporally and mechanistically distinct modes of action. The first wave of TNAIP3 accumulation was directly blunted by the histone deacetylase (HDAC) blockade. By contrast, the second wave was d...
C27. PROTEINASES AND THEIR INHIBITORS IN INFLAMMATION AND INJURY, 2010
University Hospital of Brescia, Brescia, Italy, Meakins Christie Laboratories, McGill University,... more University Hospital of Brescia, Brescia, Italy, Meakins Christie Laboratories, McGill University, Montreal, Canada, McGill University, 1 2 ... Corresponding author's email: pini@med.unibs.it ... Rationale The pathogenesis of emphysema in patients with AATD ...
Antibodies to interleukin-2 (IL-2) have been reported in the sera of patients under therapies inv... more Antibodies to interleukin-2 (IL-2) have been reported in the sera of patients under therapies involving this cytokine and in the sera of patients infected with human immunodeficiency virus (HIV). Our study proves that auto-antibodies to IL-2 are present also, at a lower titre, in healthy individuals. These antibodies were affinity purified and studied for their capability to interfere with the in vitro biological activity of IL-2. Data obtained show that human anti-IL-2 antibodies can interfere with lymphocyte proliferation both in the lymphokine activated killer (LAK) cell assay and in the mixed lymphocyte reaction (MLR). However, the kinetics of inhibition by anti-IL-2 antibodies differs from LAK cell assay to MLR as the former are always inhibited in a time-independent manner, and the latter only by adding antibodies at the outset of culture. The neutralizing activity observed in vitro suggests that such antibodies play a part in the elaborate cytokine network by which the immune system regulates the amplitude and duration of its response.
Basic fibroblast growth factor (bFGF) induces a protein kinase C (PKC)-dependent mitogenic respon... more Basic fibroblast growth factor (bFGF) induces a protein kinase C (PKC)-dependent mitogenic response in transformed fetal bovine aortic endothelial GM 7373 cells. A long-lasting interaction of bFGF with the cell is required to induce cell proliferation. bFGF-treated cells are in fact committed to proliferate only after they have entered the phase S of the cell cycle, 12-14 h after the beginning of bFGF treatment. Before that time, the mitogenic response to bFGF is abolished by 1) removal of extracellular bFGF by suramin, 2) addition of neutralizing anti-bFGF antibodies to the culture medium, 3) inhibition of PKC activity by the protein kinase inhibitor H-7, and 4) down-regulation of PKC by cotreatment with phorbol ester. Thus the requirement for a prolonged interaction of bFGF with the cell reflects the requirement for a prolonged activation of PKC. Similar conclusions can be drawn for the PKC activators 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol. The two mo...
Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host res... more Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001—is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide—a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, de...
Phosphodiesterase 4 (PDE4) inhibitors are immunomodulatory drugs approved to treat diseases assoc... more Phosphodiesterase 4 (PDE4) inhibitors are immunomodulatory drugs approved to treat diseases associated with chronic inflammatory conditions, such as COPD, psoriasis and atopic dermatitis. Tanimilast (international non-proprietary name of CHF6001) is a novel, potent and selective inhaled PDE4 inhibitor in advanced clinical development for the treatment of COPD. To begin testing its potential in limiting hyperinflammation and immune dysregulation associated to SARS-CoV-2 infection, we took advantage of an in vitro model of dendritic cell (DC) activation by SARS-CoV-2 genomic ssRNA (SCV2-RNA). In this context, Tanimilast decreased the release of pro-inflammatory cytokines (TNF-α and IL-6), chemokines (CCL3, CXCL9, and CXCL10) and of Th1-polarizing cytokines (IL-12, type I IFNs). In contrast to β-methasone, a reference steroid anti-inflammatory drug, Tanimilast did not impair the acquisition of the maturation markers CD83, CD86 and MHC-II, nor that of the lymph node homing receptor CCR7...
Exposure to heat shock and other stressful conditions activates in cells of all organisms a speci... more Exposure to heat shock and other stressful conditions activates in cells of all organisms a specific genetic program. This enhances the synthesis of proteins with a protective role against cellular damage, called heat shock proteins (hsps). Furthermore, in the mammalian nervous system, a considerable amount of hsps is also synthesized under normal conditions suggesting that they play an important role in the metabolism of unstressed cells. In this study we analysed the constitutive expression of proteins belonging to the hsp70 and hsp90 family in the rat cerebellum using immunocytochemistry with specific monoclonal antibodies. Our results showed that an intense immunostaining was evident, but was restricted in certain distinct cerebellar areas only, while no differences in the distribution of the two hsps were found. The strongest response was detected in the Purkinje neurons but deep cerebellar nuclei were also positive. In no case glial cells were found to be reactive for hsps despite their strong response for specific markers like glial fibrillary acid protein (astrocytes) and cyclic nucleotide phosphodiesterase (oligodendrocytes). These data indicate that both the hsp70 and hsp90 family have fundamental physiological functions in cerebellar neurons while they seem to play only a minor role in the metabolism of glial cells.
Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here ... more Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here we report that the CCRL2/chemerin/CMKLR1 axis represents a selective pathway for the homing of natural killer (NK) cells to the lung. C–C motif chemokine receptor-like 2 (CCRL2) is a nonsignaling seven-transmembrane domain receptor able to control lung tumor growth. CCRL2 constitutive or conditional endothelial cell targeted ablation, or deletion of its ligand chemerin, were found to promote tumor progression in a Kras/p53Flox lung cancer cell model. This phenotype was dependent on the reduced recruitment of CD27– CD11b+ mature NK cells. Other chemotactic receptors identified in lung-infiltrating NK cells by single-cell RNA sequencing (scRNA-seq), such as Cxcr3, Cx3cr1, and S1pr5, were found to be dispensable in the regulation of NK-cell infiltration of the lung and lung tumor growth. scRNA-seq identified CCRL2 as the hallmark of general alveolar lung capillary endothelial cells. CCRL2 expression was epigenetically regulated in lung endothelium and it was upregulated by the demethylating agent 5-aza-2′-deoxycytidine (5-Aza). In vivo administration of low doses of 5-Aza induced CCRL2 upregulation, increased recruitment of NK cells, and reduced lung tumor growth. These results identify CCRL2 as an NK-cell lung homing molecule that has the potential to be exploited to promote NK cell–mediated lung immune surveillance.
To gain more information about the relationship between the structure of IFN-gamma and its activi... more To gain more information about the relationship between the structure of IFN-gamma and its activity, a peptide corresponding to a hydrophilic peak between amino acids 4 and 16 was used to immunize mice and generate mAb. mAb IGMB-15 reacts to both native and rIFN-gamma and neutralizes the antiproliferative activity of IFN-gamma without affecting its antiviral activity or its ability to up-regulate HLA-DR Ag expression. Moreover, we observed that mAb IGMB-15 was unable to inhibit the binding of radiolabeled IFN-gamma to its cellular receptor. These findings show that the NH2-terminal region may somehow be involved in the biologic activity of IFN-gamma. Besides, the capability of mAb IGMB-15 to inhibit the antiproliferative but not the antiviral activity of IFN-gamma in the same cell (HEp-2) suggests the presence of different elements involved in signal transduction, which may account for the multiple activities of the lymphokine.
COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. Howe... more COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of ...
Inhibitors of phosphodiesterase-4 (PDE4) are small-molecule drugs that, by increasing the intrace... more Inhibitors of phosphodiesterase-4 (PDE4) are small-molecule drugs that, by increasing the intracellular levels of cAMP in immune cells, elicit a broad spectrum of anti-inflammatory effects. As such, PDE4 inhibitors are actively studied as therapeutic options in a variety of human diseases characterized by an underlying inflammatory pathogenesis. Dendritic cells (DCs) are checkpoints of the inflammatory and immune responses, being responsible for both activation and dampening depending on their activation status. This review shows evidence that PDE4 inhibitors modulate inflammatory DC activation by decreasing the secretion of inflammatory and Th1/Th17-polarizing cytokines, although preserving the expression of costimulatory molecules and the CD4+ T cell-activating potential. In addition, DCs activated in the presence of PDE4 inhibitors induce a preferential Th2 skewing of effector T cells, retain the secretion of Th2-attracting chemokines and increase the production of T cell regulat...
Background Tanimilast is a novel and selective inhaled inhibitor of phosphodiesterase-4 in advanc... more Background Tanimilast is a novel and selective inhaled inhibitor of phosphodiesterase-4 in advanced clinical development for chronic obstructive pulmonary disease (COPD). Tanimilast is known to exert prominent anti-inflammatory activity when tested in preclinical experimental models as well as in human clinical studies. Recently, we have demonstrated that it also finely tunes, rather than suppressing, the cytokine network secreted by activated dendritic cells (DCs). This study was designed to characterize the effects of tanimilast on T-cell polarizing properties of DCs and to investigate additional functional and phenotypical features induced by tanimilast. Methods DCs at day 6 of culture were stimulated with LPS in the presence or absence of tanimilast or the control drug budesonide. After 24 h, DCs were analyzed for the expression of surface markers of maturation and activation by flow cytometry and cocultured with T cells to investigate cell proliferation and activation/polarizat...
Histone deacetylase inhibitors (HDIs) are promising drugs for the treatment of inflammatory disea... more Histone deacetylase inhibitors (HDIs) are promising drugs for the treatment of inflammatory diseases. However, their therapeutical exploitation is slowed down by severe adverse manifestations that can hardly be foreseen, mainly due to incomplete knowledge of how HDIs impact the delicate balance of inflammatory mediators. In this work, we characterized the effects of the HDI trichostatin A (TSA) on the expression of TNFAIP3, which is a crucial inhibitor of the classical NF-kB pathway and an LPS-induced negative feedback regulator. The accumulation of TNFAIP3 mRNA after LPS stimulation showed biphasic behavior, with one wave within the first hour of stimulation and a second wave several hours later, which were both reduced by TSA. By using inhibition and knockdown approaches, we identified two temporally and mechanistically distinct modes of action. The first wave of TNAIP3 accumulation was directly blunted by the histone deacetylase (HDAC) blockade. By contrast, the second wave was d...
C27. PROTEINASES AND THEIR INHIBITORS IN INFLAMMATION AND INJURY, 2010
University Hospital of Brescia, Brescia, Italy, Meakins Christie Laboratories, McGill University,... more University Hospital of Brescia, Brescia, Italy, Meakins Christie Laboratories, McGill University, Montreal, Canada, McGill University, 1 2 ... Corresponding author's email: pini@med.unibs.it ... Rationale The pathogenesis of emphysema in patients with AATD ...
Antibodies to interleukin-2 (IL-2) have been reported in the sera of patients under therapies inv... more Antibodies to interleukin-2 (IL-2) have been reported in the sera of patients under therapies involving this cytokine and in the sera of patients infected with human immunodeficiency virus (HIV). Our study proves that auto-antibodies to IL-2 are present also, at a lower titre, in healthy individuals. These antibodies were affinity purified and studied for their capability to interfere with the in vitro biological activity of IL-2. Data obtained show that human anti-IL-2 antibodies can interfere with lymphocyte proliferation both in the lymphokine activated killer (LAK) cell assay and in the mixed lymphocyte reaction (MLR). However, the kinetics of inhibition by anti-IL-2 antibodies differs from LAK cell assay to MLR as the former are always inhibited in a time-independent manner, and the latter only by adding antibodies at the outset of culture. The neutralizing activity observed in vitro suggests that such antibodies play a part in the elaborate cytokine network by which the immune system regulates the amplitude and duration of its response.
Basic fibroblast growth factor (bFGF) induces a protein kinase C (PKC)-dependent mitogenic respon... more Basic fibroblast growth factor (bFGF) induces a protein kinase C (PKC)-dependent mitogenic response in transformed fetal bovine aortic endothelial GM 7373 cells. A long-lasting interaction of bFGF with the cell is required to induce cell proliferation. bFGF-treated cells are in fact committed to proliferate only after they have entered the phase S of the cell cycle, 12-14 h after the beginning of bFGF treatment. Before that time, the mitogenic response to bFGF is abolished by 1) removal of extracellular bFGF by suramin, 2) addition of neutralizing anti-bFGF antibodies to the culture medium, 3) inhibition of PKC activity by the protein kinase inhibitor H-7, and 4) down-regulation of PKC by cotreatment with phorbol ester. Thus the requirement for a prolonged interaction of bFGF with the cell reflects the requirement for a prolonged activation of PKC. Similar conclusions can be drawn for the PKC activators 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol. The two mo...
Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host res... more Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001—is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide—a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, de...
Phosphodiesterase 4 (PDE4) inhibitors are immunomodulatory drugs approved to treat diseases assoc... more Phosphodiesterase 4 (PDE4) inhibitors are immunomodulatory drugs approved to treat diseases associated with chronic inflammatory conditions, such as COPD, psoriasis and atopic dermatitis. Tanimilast (international non-proprietary name of CHF6001) is a novel, potent and selective inhaled PDE4 inhibitor in advanced clinical development for the treatment of COPD. To begin testing its potential in limiting hyperinflammation and immune dysregulation associated to SARS-CoV-2 infection, we took advantage of an in vitro model of dendritic cell (DC) activation by SARS-CoV-2 genomic ssRNA (SCV2-RNA). In this context, Tanimilast decreased the release of pro-inflammatory cytokines (TNF-α and IL-6), chemokines (CCL3, CXCL9, and CXCL10) and of Th1-polarizing cytokines (IL-12, type I IFNs). In contrast to β-methasone, a reference steroid anti-inflammatory drug, Tanimilast did not impair the acquisition of the maturation markers CD83, CD86 and MHC-II, nor that of the lymph node homing receptor CCR7...
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Papers by Laura Tiberio