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.

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.

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.

Ratio of Circulating Estrogen Receptors Beta and Alpha (ERß/ERα) Indicates Endoscopic Activity in Patients with Crohn's Disease.

BACKGROUND: Data supporting a role of female hormones and/or their receptors in inflammatory bowel disease (IBD) are increasing, but most of them are derived from animal models. Estrogen receptors alpha (ERα) and beta (ERß) participate in immune and inflammatory response, among a variety of biological processes. Their effects are antagonistic, and the net action of estrogens may depend on their relative proportions. AIM: To determine the possible association between the balance of circulating ERß and ERα (ERß/ERα) and IBD risk and activity. METHODS: Serum samples from 145 patients with IBD (79 Crohn's disease [CD] and 66 ulcerative colitis [UC]) and 39 controls were retrospectively studied. Circulating ERα and ERß were measured by ELISA. Disease activities were assessed by clinical and endoscopic indices specific for CD and UC. RESULTS: Low values of ERß/ERα ratio were directly associated with clinical (p = 0.019) and endoscopic (p = 0.002) disease activity. Further analyses by type of IBD confirmed a strong association between low ERß/ERα ratio and CD clinical (p = 0.011) and endoscopic activity (p = 0.002). The receiver operating curve (ROC) analysis showed that an ERß/ERα ratio under 0.85 was a good marker of CD endoscopic activity (area under the curve [AUC]: 0.84; p = 0.002; sensitivity: 70%; specificity: 91%). ERß/ERα ratio was not useful to predict UC activity. CONCLUSIONS: An ERß/ERα ratio under 0.85 indicated CD endoscopic activity. The determination of serum ERß/ERα might be a useful noninvasive screening tool for CD endoscopic activity.

P-Selectin preserves immune tolerance in mice and is reduced in human cutaneous lupus.

Mice deficient in P-Selectin presented altered immunity/tolerance balance. We have observed that the absence of P-Selectin promotes splenomegaly with reduced naïve T cell population, elevated activated/effector T cell subset, increased germinal center B and Tfh populations and high production of autoreactive antibodies. Moreover, 1.5-3-month-old P-selectin KO mice showed reduced IL-10-producing leukocytes in blood and a slightly reduced Treg population in the skin. With aging and, coinciding with disease severity, there is an increase in the IL17+ circulating and dermal T cell subpopulations and reduction of dermal Treg. As a consequence, P-Selectin deficient mice developed a progressive autoimmune syndrome showing skin alterations characteristic of lupus prone mice and elevated circulating autoantibodies, including anti-dsDNA. Similar to human SLE, disease pathogenesis was characterized by deposition of immune complexes in the dermoepidermal junction and renal glomeruli, and a complex pattern of autoantibodies. More important, skin biopsies of cutaneous lupus erythematosus patients did not show increased expression of P-Selectin, as described for other inflammatory diseases, and the number of vessels expressing P-Selectin was reduced.

PSGL-1 on Leukocytes is a Critical Component of the Host Immune Response against Invasive Pneumococcal Disease.

Bacterial uptake by phagocytic cells is a vital event in the clearance of invading pathogens such as Streptococcus pneumoniae. A major role of the P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes against invasive pneumococcal disease is described in this study. Phagocytosis experiments using different serotypes demonstrated that PSGL-1 is involved in the recognition, uptake and killing of S. pneumoniae. Co-localization of several clinical isolates of S. pneumoniae with PSGL-1 was demonstrated, observing a rapid and active phagocytosis in the presence of PSGL-1. Furthermore, the pneumococcal capsular polysaccharide and the main autolysin of the bacterium--the amidase LytA--were identified as bacterial ligands for PSGL-1. Experimental models of pneumococcal disease including invasive pneumonia and systemic infection showed that bacterial levels were markedly increased in the blood of PSGL-1-/- mice. During pneumonia, PSGL-1 controls the severity of pneumococcal dissemination from the lung to the bloodstream. In systemic infection, a major role of PSGL-1 in host defense is to clear the bacteria in the systemic circulation controlling bacterial replication. These results confirmed the importance of this receptor in the recognition and clearance of S. pneumoniae during invasive pneumococcal disease. Histological and cellular analysis demonstrated that PSGL-1-/- mice have increased levels of T cells migrating to the lung than the corresponding wild-type mice. In contrast, during systemic infection, PSGL-1-/- mice had increased numbers of neutrophils and macrophages in blood, but were less effective controlling the infection process due to the lack of this functional receptor. Overall, this study demonstrates that PSGL-1 is a novel receptor for S. pneumoniae that contributes to protection against invasive pneumococcal disease.