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.

Prevention of excitotoxicity-induced processing of BDNF receptor TrkB-FL leads to stroke neuroprotection.

Neuroprotective strategies aimed to pharmacologically treat stroke, a prominent cause of death, disability, and dementia, have remained elusive. A promising approach is restriction of excitotoxic neuronal death in the infarct penumbra through enhancement of survival pathways initiated by brain-derived neurotrophic factor (BDNF). However, boosting of neurotrophic signaling after ischemia is challenged by downregulation of BDNF high-affinity receptor, full-length tropomyosin-related kinase B (TrkB-FL), due to calpain-degradation, and, secondarily, regulated intramembrane proteolysis. Here, we have designed a blood-brain barrier (BBB) permeable peptide containing TrkB-FL sequences (TFL457 ) which prevents receptor disappearance from the neuronal surface, early induced after excitotoxicity. In this way, TFL457 interferes TrkB-FL cleavage by both proteolytic systems and increases neuronal viability via a PLCγ-dependent mechanism. By preserving downstream CREB and MEF2 promoter activities, TFL457 initiates a feedback mechanism favoring increased levels in excitotoxic neurons of critical prosurvival mRNAs and proteins. This neuroprotective peptide could be highly relevant for stroke therapy since, in a mouse ischemia model, it counteracts TrkB-FL downregulation in the infarcted brain, efficiently decreases infarct size, and improves neurological outcome.

Distinctive pattern of cytokine production and adhesion molecule expression in peripheral blood memory CD4+ T cells from patients with active Crohn's disease.

An expansion of both circulating and intestinal lamina propria CD4+ CD45RO+ T cells has been described in patients with Crohn's disease. We studied both the cytokine profile and the expression of adhesion molecules on this T-cell subset. Peripheral blood CD4+ CD45RO+ T cells from patients with Crohn's disease (n=45) were assessed by flow cytometry and RT-PCR methods. The cytokine profile was also measured in intestinal lamina propria from seven patients. They were classified according to the CDAI and the results were compared with those of patients with ulcerative colitis (n=21) and noninflammatory intestinal conditions (n=15), and healthy controls (n=39). The mean percentage of circulating CD4+ CD45RO+ T cells producing intracellular TNF was higher in active than in inactive Crohn's disease patients (p < 0.001), active (p = 0.49) and inactive ulcerative colitis (p = 0.019), and healthy controls (p =0. 017). TNF expression correlated with CDAI (p < 0.001). An increased expression of intracellular IL-2, IL-6, and IL-10 in active Crohn's disease patients was also found. CD62L was downregulated in active Crohn's disease patients while no differences were observed in CD49d and CD11a expression. Lamina propria CD4+ CD45RO+ T cells from active Crohn's disease lesions showed an increased intracellular staining of TNF, IFN-gamma, and IL-10. Both peripheral and intestinal mucosa CD4+ CD45RO+ T cells from active Crohn's disease patients show an increased production of TNF. In addition, the circulating CD4+ CD45RO+ T-cell subset expresses a pattern of adhesion molecules that promotes homing to extranodal lymphoid tissues. This T-cell subset may play a relevant role in the immunopathogenesis of Crohn's disease.

Active Crohn's disease patients show a distinctive expansion of circulating memory CD4+CD45RO+CD28null T cells.

In a previous study we found an expansion of circulating memory (CD45RO(+)) CD4(+) T cells in patients with Crohn's disease (CD). The aim of this work was to investigate the phenotypic and functional characteristics of this T-cell subset in CD. We analyzed in peripheral blood CD4(+)CD45RO(+) T cells from CD patients the expression of surface markers associated to immune activation, costimulation, and apoptosis. In sorted CD4(+)CD45RO(+) T cells apoptosis was quantified by fluorescent annexin V binding. Healthy subjects and patients with ulcerative colitis and acute bacterial enterocolitis served as control groups. An increased percentage of memory CD4(+)CD45RO(+) T cells lacking the expression of costimulatory receptor CD28 was detected in patients with active CD when compared to the other groups evaluated. This expanded CD4(+)CD45RO(+)CD28(null) T-cell subset expressed mostly the effector-cell marker CD57(+). Both CD28 downregulation and CD57 expression correlated to CDAI and surrogate markers of disease activity. These phenotypic changes observed on CD4(+)CD45RO(+) T cells from active CD returned to values similar to healthy controls after clinical remission. Moreover, this memory CD28(null) T-cell subset might express more intracytoplasmic TNF and IFN-gamma than their CD28(+) counterpart. Significantly lower frequencies of memory CD4(+)CD45RO(+) T cells expressing CD95 apoptosis receptor were found in patients with active CD. Moreover, sorted CD4(+)CD45RO(+)and CD4(+)CD45RO(+) CD28(null) T cells from patients with active CD exhibited a lower apoptotic rate than that found in healthy controls and inactive CD patients. According to our data, circulating T lymphocytes from active CD patients show distinctive phenotypic and functional changes, characterized by an expansion of memory CD4(+)CD45RO(+)CD28(null) T cells expressing effector-associated cell surface molecules and displaying enhanced resistance to apoptosis.

Apoptotic rate: a new indicator for the quantification of the incidence of apoptosis in cell cultures.

BACKGROUND: Late apoptotic cells divide into apoptotic bodies and are missed by current detection methods. This results in an artificially low apoptotic index (AI). METHODS: This study proposes a flow cytometry-based ratiometric method that uses an internal reference standard of microbeads combined with fluorescein-annexin V binding and 7-aminoactinomycin D to enumerate viable, necrotic, and early and late apoptotic cells within specific subsets of a heterogeneous culture. RESULTS: In the absence of cell growth, the number of apoptotic cells that undergo fragmentation into apoptotic bodies in culture can also be determined accurately by this method. This information can then be used to obtain the apoptotic rate (AR), a new indicator of apoptosis that calculates the proportion of cells that have undergone apoptosis with respect to the total number of seeded cells. The main limitation of the method is that the AR is only suitable for the study of apoptosis in noncycling cells. CONCLUSIONS: This study reveals the superiority of the proposed method over the widely used Nicoletti method and current annexin-V binding methods. The AI did not reflect the true incidence of lymphocyte apoptosis, neither in response to lectins or phorbol esters, nor to serum deprivation. AR was more sensitive than AI, detecting apoptosis at lower concentrations of cell death inducers in all the subsets studied.