Beneficial effect of temporary methotrexate interruption on B and T cell responses upon SARS-CoV-2 vaccination in patients with rheumatoid arthritis or psoriatic arthritis.

B and T cell responses were evaluated in patients with rheumatoid arthritis (RA) or psoriatic arthritis (PsA) after 1 or 2 weeks of methotrexate (MTX) withdrawal following each COVID-19 vaccine dose and compared with those who maintained MTX. Adult RA and PsA patients treated with MTX were recruited and randomly assigned to 3 groups: MTX-maintenance (n = 72), MTX-withdrawal for 1 week (n = 71) or MTX-withdrawal for 2 weeks (n = 73). Specific antibodies to several SARS-CoV-2 antigens and interferon (IFN)-γ and interleukin (IL)-21 responses were assessed. MTX withdrawal in patients without previous COVID-19 was associated with higher levels of anti-RBD IgG and neutralising antibodies, especially in the 2-week withdrawal group and with higher IFN-γ secretion upon stimulation with pools of SARS-CoV-2 S peptides. No increment of RA/PsA relapses was detected across groups. Our data indicate that two-week MTX interruption following COVID-19 vaccination in patients with RA or PsA improves humoral and cellular immune responses.

Resistance to Experimental Visceral Leishmaniasis in Mice Infected With Leishmania infantum Requires Batf3.

Unveiling the protective immune response to visceral leishmaniasis is critical for a rational design of vaccines aimed at reducing the impact caused by this fatal, if left untreated, vector-borne disease. In this study we sought to determine the role of the basic leucine zipper transcription factor ATF-like 3 (Batf3) in the evolution of infection with Leishmania infantum, the causative agent of human visceral leishmaniasis in the Mediterranean Basin and Latin America. For that, Batf3-deficient mice in C57BL/6 background were infected with an L. infantum strain expressing the luciferase gene. Bioluminescent imaging, as well as in vitro parasite titration, demonstrated that Batf3-deficient mice were unable to control hepatic parasitosis as opposed to wild-type C57BL/6 mice. The impaired microbicide capacities of L. infantum-infected macrophages from Batf3-deficient mice mainly correlated with a reduction of parasite-specific IFN-γ production. Our results reinforce the implication of Batf3 in the generation of type 1 immunity against infectious diseases.

CCR7 as a novel therapeutic target in t-cell PROLYMPHOCYTIC leukemia.

T-cell prolymphocytic leukemia (T-PLL) is a poor prognostic disease with very limited options of efficient therapies. Most patients are refractory to chemotherapies and despite high response rates after alemtuzumab, virtually all patients relapse. Therefore, there is an unmet medical need for novel therapies in T-PLL. As the chemokine receptor CCR7 is a molecule expressed in a wide range of malignancies and relevant in many tumor processes, the present study addressed the biologic role of this receptor in T-PLL. Furthermore, we elucidated the mechanisms of action mediated by an anti-CCR7 monoclonal antibody (mAb) and evaluated whether its anti-tumor activity would warrant development towards clinical applications in T-PLL. Our results demonstrate that CCR7 is a prognostic biomarker for overall survival in T-PLL patients and a functional receptor involved in the migration, invasion, and survival of leukemic cells. Targeting CCR7 with a mAb inhibited ligand-mediated signaling pathways and induced tumor cell killing in primary samples. In addition, directing antibodies against CCR7 was highly effective in T-cell leukemia xenograft models. Together, these findings make CCR7 an attractive molecule for novel mAb-based therapeutic applications in T-PLL, a disease where recent drug screen efforts and studies addressing new compounds have focused on chemotherapy or small molecules. SUPPLEMENTARY INFORMATION: Supplementary information accompanies this paper at 10.1186/s40364-020-00234-z.

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.

Endothelial MT1-MMP targeting limits intussusceptive angiogenesis and colitis via TSP1/nitric oxide axis.

Pathological angiogenesis contributes to cancer progression and chronic inflammatory diseases. In inflammatory bowel disease, the microvasculature expands by intussusceptive angiogenesis (IA), a poorly characterized mechanism involving increased blood flow and splitting of pre-existing capillaries. In this report, mice lacking the protease MT1-MMP in endothelial cells (MT1iΔEC ) presented limited IA in the capillary plexus of the colon mucosa assessed by 3D imaging during 1% DSS-induced colitis. This resulted in better tissue perfusion, preserved intestinal morphology, and milder disease activity index. Combined in vivo intravital microscopy and lentiviral rescue experiments with in vitro cell culture demonstrated that MT1-MMP activity in endothelial cells is required for vasodilation and IA, as well as for nitric oxide production via binding of the C-terminal fragment of MT1-MMP substrate thrombospondin-1 (TSP1) to CD47/αvß3 integrin. Moreover, TSP1 levels were significantly higher in serum from IBD patients and in vivo administration of an anti-MT1-MMP inhibitory antibody or a nonamer peptide spanning the αvß3 integrin binding site in TSP1 reduced IA during mouse colitis. Our results identify MT1-MMP as a new actor in inflammatory IA and a promising therapeutic target for inflammatory bowel 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.

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.

The metalloprotease ADAM8 is associated with and regulates the function of the adhesion receptor PSGL-1 through ERM proteins.

The P-selectin glycoprotein ligand-1 (PSGL-1) is involved in the initial contact of leukocytes with activated endothelium, and its adhesive function is regulated through its proteolytic processing. We have found that the metalloprotease ADAM8 is both associated with PSGL-1 through the ezrin­radixin­moesin actin-binding proteins and able to cause the proteolytic cleavage of this adhesion receptor. Accordingly, ADAM8 knockdown increases PSGL-1 expression, and functional assays show that ADAM8 is able to reduce leukocyte rolling on P-selectin and hence on activated endothelial cells. We conclude that ADAM8 modulates the expression and function of PSGL-1.

Functional role of P-selectin glycoprotein ligand 1/P-selectin interaction in the generation of tolerogenic dendritic cells.

Dendritic cells (DCs) have a key role in both the generation of the immune response and the induction of tolerance to self-Ags. In this work, the possible role of P-selectin glycoprotein ligand 1 (PSGL-1) on the tolerogenic activity of human DCs was explored. We found that the engagement of PSGL-1 by P-selectin on DCs induced the expression of c-Fos, IDO, IL-10, and TGF-beta genes. Remarkably, stimulation of DCs through PSGL-1 with P-selectin enhanced their capability to generate CD4(+)CD25(+)Foxp3(+) regulatory T cells, which expressed high levels of TGF-beta1 mRNA, synthesized IL-10, and suppressed the proliferation of autologous CD4(+)CD25(-) T cells. Accordingly, we found that DCs from PSGL-1(-/-) mice expressed higher levels of MHC class II molecules, and exhibited an enhanced immunogenicity compared with wild-type mice. In addition, the percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells in the thymus of PSGL-1-deficient animals was significantly reduced. Our data reveal an unexpected role of PSGL-1 on the tolerogenic function of DCs, and the regulation of the immune response.

Myosin IIA is involved in the endocytosis of CXCR4 induced by SDF-1alpha.

Endocytosis of chemokine receptors regulates signal transduction initiated by chemokines, but the molecular mechanisms underlying this process are not fully defined. In this work, we assessed the involvement of the motor protein nonmuscle myosin heavy chain IIA (MIIA) in the endocytosis of CXCR4 induced by SDF-1alpha (also known as CXCL12) in T lymphocytes. Overexpression of the C-terminal half of MIIA inhibited the ligand-induced endocytosis of CXCR4, but not that of transferrin receptor. Targeting MIIA either by silencing its expression with small interfering RNA (siRNA) or by blebbistatin treatment also inhibited endocytosis of CXCR4. Inhibition of endocytosis of CXCR4 by targeting endogenous MIIA resulted in an increased migration of T cells induced by SDF-1alpha, and in the inhibition of the HIV-1-Env antifusogenic activity of this chemokine. Coimmunoprecipitation and protein-protein binding studies demonstrated that MIIA interacts with both the cytoplasmic tail of CXCR4 and beta-arrestin. Moreover, SDF-1alpha promotes a rapid MIIA-beta-arrestin dissociation. Our data reveal a novel role for MIIA in CXCR4 endocytosis, which involves its dynamic association with beta-arrestin and highlights the role of endogenous MIIA as a regulator of CXCR4 internalization and, therefore, the onset of SDF-1alpha signaling.