Low P-Selectin Glycoprotein Ligand-1 Expression in Neutrophils Associates with Disease Activity and Deregulated NET Formation in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the generation of anti-DNA autoantibodies due to exposure of immune cells to excessive amounts of extracellular DNA. Lack of P-selectin in mice induces the development of a lupus-like syndrome and patients with cutaneous lupus have reduced P-selectin expression in skin vessels. Using flow cytometry we analyzed in healthy donors and patients the expression of P-selectin Glycoprotein Ligand-1 (PSGL-1) in circulating neutrophils and the implication of PSGL-1/P-selectin interaction in neutrophil extracellular traps (NETs) generation. We found a statistical significance that neutrophils from active SLE patients have a reduced expression of PSGL-1 and low levels of PSGL-1 in neutrophils from SLE patients associated with the presence of anti-dsDNA antibodies, clinical lung involvement, Raynaud's phenomenon, and positive lupus anticoagulant. PSGL-1 is present along the DNA in the NET. In healthy donors, neutrophil interaction with immobilized P-selectin triggers Syk activation, increases the NETs percentage and reduces the amount of DNA extruded in the NETs. In active SLE patients, neutrophil interaction with P-selectin does not activate Syk or reduce the amount of DNA extruded in the NETs, that might contribute to increase the extracellular level of DNA and hence, to disease pathogenesis.

Targeted nanotherapy with everolimus reduces inflammation and fibrosis in scleroderma-related interstitial lung disease developed by PSGL-1 deficient mice.

BACKGROUND AND PURPOSE: Interstitial lung disease (ILD) is the main cause of mortality in systemic sclerosis (SSc), and current therapies available are of low efficacy or high toxicity. Thus, the identification of innovative less toxic and high efficacy therapeutic approaches to ILD treatment is an urgent need. The interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin initiates leukocyte extravasation and deletion of the corresponding gene (Selplg) induces a SSc-like syndrome with high incidence of ILD in aged mice. EXPERIMENTAL APPROACH: Aged PSGL-1 KO (Selplg-/- ) mice were used to assess the therapeutic effects of nanotherapy with everolimus, included in liposomes decorated with high MW hyaluronic acid (LipHA+Ev) and administered intratracheally to specifically target CD44-expressing lung cells. KEY RESULTS: PSGL-1 KO mice had increased numbers of CD45+ and CD45- cells, including alveolar and interstitial macrophages, eosinophils, granulocytes and NK cells, and myofibroblasts in bronchoalveolar lavage (BAL). CD45+ and CD45- cells expressing pro-inflammatory and pro-fibrotic cytokines were also increased. Lungs from PSGL-1 KO mice showed increased immune cell infiltration and apoptosis and exacerbated interstitial and peribronchial fibrosis. Targeted nanotherapy with LipHA+Ev decreased the myofibroblasts in BAL, cells producing proinflammatory and profibrotic cytokines, and the degree of lung inflammation at histology. LipHA+Ev treatment also decreased the severity of peribronchial and interstitial lung fibrosis, from moderate to mild levels. CONCLUSIONS AND IMPLICATIONS: In PSGL-1 KO mice, targeted nanotherapy with LipHA+Ev was an effective treatment for SSc-ILD, reducing the number of inflammatory and fibrotic cells in BAL and reducing inflammation and fibrosis in lungs.

IGF-1 facilitates extinction of conditioned fear.

Insulin-like growth factor-1 (IGF-1) plays a key role in synaptic plasticity, spatial learning, and anxiety-like behavioral processes. While IGF-1 regulates neuronal firing and synaptic transmission in many areas of the central nervous system, its signaling and consequences on excitability, synaptic plasticity, and animal behavior dependent on the prefrontal cortex remain unexplored. Here, we show that IGF-1 induces a long-lasting depression of the medium and slow post-spike afterhyperpolarization (mAHP and sAHP), increasing the excitability of layer 5 pyramidal neurons of the rat infralimbic cortex. Besides, IGF-1 mediates a presynaptic long-term depression of both inhibitory and excitatory synaptic transmission in these neurons. The net effect of this IGF-1-mediated synaptic plasticity is a long-term potentiation of the postsynaptic potentials. Moreover, we demonstrate that IGF-1 favors the fear extinction memory. These results show novel functional consequences of IGF-1 signaling, revealing IGF-1 as a key element in the control of the fear extinction memory.

miR-320c Regulates SERPINA1 Expression and Is Induced in Patients With Pulmonary Disease.

INTRODUCTION: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting in lung and liver disease with a great clinical variability. MicroRNAs have been identified as disease modifiers; therefore miRNA deregulation could play an important role in disease heterogeneity. Members of miR-320 family are involved in regulating of multiple processes including inflammation, and have potential specific binding sites in the 3'UTR region of SERPINA1 gene. In this study we explore the involvement of miR-320c, a member of this family, in this disease. METHODS: Firstly in vitro studies were carried out to demonstrate regulation of SERPINA1 gene by miR-320. Furthermore, the expression of miR-320c was analyzed in the blood of 98 individuals with different AAT serum levels by using quantitative PCR and expression was correlated to clinical parameters of the patients. Finally, HL60 cells were used to analyze induction of miR-320c in inflammatory conditions. RESULTS: Overexpression of miR-320 members in human HepG2 cells led to inhibition of SERPINA1 expression. Analysis of miR-320c expression in patient's samples revealed significantly increased expression of miR-320c in individuals with pulmonary disease. Additionally, HL60 cells treated with the pro-inflammatory factor lipopolysaccharide (LPS) showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation. CONCLUSION: Our findings demonstrate that miR-320c inhibits SERPINA1 expression in a hepatic cell line and its levels in blood are associated with lung disease in a cohort of patients with different AAT serum levels. These results suggest that miR-320c can play a role in AAT regulation and could be a biomarker of inflammatory processes in pulmonary diseases.

Relevance of PSGL-1 Expression in B Cell Development and Activation.

PSGL-1 is expressed in all plasma cells, but only in a small percentage of circulating B cells. Patients with systemic sclerosis (SSc) show reduced expression of PSGL-1 in B cells and increased prevalence of pulmonary arterial hypertension. PSGL-1 deficiency leads to a SSc-like syndrome and SSc-associated pulmonary hypertension in female mice. In this work, the expression of PSGL-1 was assessed during murine B cell development in the bone marrow and in several peripheral and spleen B cell subsets. The impact of PSGL-1 absence on B cell biology was also evaluated. Interestingly, the percentage of PSGL-1 expressing cells and PSGL-1 expression levels decreased in the transition from common lymphoid progenitors to immature B cells. PSGL-1-/- mice showed reduced frequencies of peripheral B cells and reduced B cell lineage-committed precursors in the bone marrow. In the spleen of WT mice, the highest percentages of PSGL-1+ populations were shown by Breg (90%), B1a (34.7%), and B1b (19.1%), while only 2.5-8% of B2 cells expressed PSGL-1; however, within B2 cells, the class-switched subsets showed the highest percentages of PSGL-1+ cells. Interestingly, PSGL-1-/- mice had increased IgG+ and IgD+ subsets and decreased IgA+ population. Of note, the percentage of PSGL-1+ cells was increased in all the B cell subclasses studied in peritoneal fluid. Furthermore, PSGL-1 engagement during in vitro activation with anti-IgM and anti-CD40 antibodies of human peripheral B cells, blocked IL-10 expression by activated human B cells. Remarkably, PSGL-1 expression in circulating plasma cells was reduced in pulmonary arterial hypertension patients. In summary, although the expression of PSGL-1 in mature B cells is low, the lack of PSGL-1 compromises normal B cell development and it may also play a role in the maturation and activation of peripheral naïve B cells.

miR-320c Regulates SERPINA1 Expression and Is Induced in Patients With Pulmonary Disease.

INTRODUCTION: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting in lung and liver disease with a great clinical variability. MicroRNAs have been identified as disease modifiers; therefore miRNA deregulation could play an important role in disease heterogeneity. Members of miR-320 family are involved in regulating of multiple processes including inflammation, and have potential specific binding sites in the 3'UTR region of SERPINA1 gene. In this study we explore the involvement of miR-320c, a member of this family, in this disease. METHODS: Firstly in vitro studies were carried out to demonstrate regulation of SERPINA1 gene by miR-320. Furthermore, the expression of miR-320c was analyzed in the blood of 98 individuals with different AAT serum levels by using quantitative PCR and expression was correlated to clinical parameters of the patients. Finally, HL60 cells were used to analyze induction of miR-320c in inflammatory conditions. RESULTS: Overexpression of miR-320 members in human HepG2 cells led to inhibition of SERPINA1 expression. Analysis of miR-320c expression in patient's samples revealed significantly increased expression of miR-320c in individuals with pulmonary disease. Additionally, HL60 cells treated with the pro-inflammatory factor lipopolysaccharide (LPS) showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation. CONCLUSION: Our findings demonstrate that miR-320c inhibits SERPINA1 expression in a hepatic cell line and its levels in blood are associated with lung disease in a cohort of patients with different AAT serum levels. These results suggest that miR-320c can play a role in AAT regulation and could be a biomarker of inflammatory processes in pulmonary diseases.

Spontaneous Pulmonary Hypertension Associated With Systemic Sclerosis in P-Selectin Glycoprotein Ligand 1-Deficient Mice.

OBJECTIVE: Pulmonary arterial hypertension (PAH), one of the major complications of systemic sclerosis (SSc), is a rare disease with unknown etiopathogenesis and noncurative treatments. As mice deficient in P-selectin glycoprotein ligand 1 (PSGL-1) develop a spontaneous SSc-like syndrome, we undertook this study to analyze whether they develop PAH and to examine the molecular mechanisms involved. METHODS: Doppler echocardiography was used to estimate pulmonary pressure, immunohistochemistry was used to assess vascular remodeling, and myography of dissected pulmonary artery rings was used to analyze vascular reactivity. Angiotensin II (Ang II) levels were quantified by enzyme-linked immunosorbent assay, and Western blotting was used to measure Ang II type 1 receptor (AT1 R), AT2 R, endothelial cell nitric oxide synthase (eNOS), and phosphorylated eNOS expression in lung lysates. Flow cytometry allowed us to determine cytokine production by immune cells and NO production by endothelial cells. In all cases, there were 4-8 mice per experimental group. RESULTS: PSGL-1-/- mice showed lung vessel wall remodeling and a reduced mean ± SD expression of pulmonary AT2 R (expression ratio [relative to ß-actin] in female mice age >18 months: wild-type mice 0.799 ± 0.508 versus knockout mice 0.346 ± 0.229). With aging, female PSGL-1-/- mice had impaired up-regulation of estrogen receptor α (ERα) and developed lung vascular endothelial dysfunction coinciding with an increase in mean ± SEM pulmonary Ang II levels (wild-type 48.70 ± 5.13 pg/gm lung tissue versus knockout 78.02 ± 28.09 pg/gm lung tissue) and a decrease in eNOS phosphorylation, leading to reduced endothelial NO production. These events led to a reduction in the pulmonary artery acceleration time:ejection time ratio in 33% of aged female PSGL-1-/- mice, indicating pulmonary hypertension. Importantly, we found expanded populations of interferon-γ-producing PSGL-1-/- T cells and B cells and a reduced presence of regulatory T cells. CONCLUSION: The absence of PSGL-1 induces a reduction in Treg cells, NO production, and ERα expression and causes an increase in Ang II in the lungs of female mice, favoring the development of PAH.

Deregulated PSGL-1 Expression in B Cells and Dendritic Cells May Be Implicated in Human Systemic Sclerosis Development.

Systemic sclerosis (SSc) is an autoimmune disorder with high morbidity and mortality, is difficult to diagnose early, and has no curative treatment. PSGL-1 is a leukocyte receptor whose deficiency in mice promotes an SSc-like disease. ADAM8, a metalloprotease that cleaves PSGL-1, is implicated in inflammatory processes. Our goal was to evaluate whether PSGL-1 and ADAM8 contribute to the pathogenesis of human SSc. We found that patients with SSc presented increased PSGL-1 expression on monocytes, dendritic cells, and T cells and decreased expression of PSGL-1 on B cells. PSGL-1 on monocytes from SSc patients failed to induce Syk phosphorylation or IL-10 production after interaction with P-selectin. Up to 60% of the IL-10-producing B cells expressed PSGL-1, pointing to a regulatory role for PSGL-1 in B cells, and PSGL-1+ B cells from SSc patients had decreased IL-10 production. ADAM8 expression was increased on antigen-presenting cells and T lymphocytes of SSc patients. Patients treated with calcium antagonists had lower levels of ADAM8 on APCs and T lymphocytes. Multivariate analysis indicated that the high percentage of ADAM8-expressing plasmacytoid dendritic cells discriminated patients from healthy donors. High PSGL-1 expression on dendritic cells was associated with the presence of interstitial lung disease.