Search

Hematopoiesis is a highly regulated process and it’s widely described in bone marrow, the classical niche for hematopoietic stem cells (HSCs). The lungs are a novel niche of HSCs, as recently described by Lefrançais in 2017, however, little is known about the nature of these cells. Isthmin 1 (ISM1) is a secreted molecule important in developmental hematopoiesis in zebrafish; moreover, it is known that murine lungs express high levels of ISM1 and its secretion is altered after LPS challenge. In this work we performed multiparametric flow cytometry identifying a subpopulation of lung-resident HSCs expressing ISM1. Then we wanted to assess if HSCs ISM1+ cells are affected under inflammatory insults. Acute infection in mice with Pseudomonas aeruginosa showed that lung-derived HSCs ISM1+ are increased, indicating that the lung-HSCs are perturbed. Due to our model produced systemic inflammation we sought to investigate whether circulating ISM1+ cells were affected, we found that the percentage and absolute numbers of ISM1+ cells in blood were slightly diminished. Since ISM1 is a secreted molecule we evaluated its plasmatic levels by ELISA. We discovered a reduction in ISM1 plasmatic levels during murine sepsis with Pseudomonas aeruginosa. Then, we sought to analyze ISM1 levels in human inflammation; we retrieved samples from pediatric individuals with sepsis and adult patients with 2th and 3th grade burn injuries, and in both cases, we detected reduced levels of plasmatic ISM1. Our data suggest that ISM1 is likely a regulator of hematopoiesis in the lung but also may have a function in the regulation of systemic inflammation in mice and humans. We suggest that ISM1 is a novel biomarker of systemic inflammation.

Salmonella infects and survives within B cells, but the mechanism used by the bacterium to promote its survival in these cells is unknown. In macrophages, flagellin secreted by Salmonella activates the Nod-like receptor (NLR) family CARD domain containing protein 4 (NLRC4) inflammasome, leading to the production of IL-1β and pyroptosis of infected cells. In this study, we demonstrated that the NLRC4 inflammasome is functional in B cells; however, in Salmonella-infected B cells, IL-1β secretion is prevented through the downregulation of NLRC4 expression. A functional Salmonella pathogenicity island 1 type III secretion system appears to be required for this process. Furthermore, infection induces Yap phosphorylation and promotes the interaction of Yap with Hck, thus preventing the transcriptional activation of NLRC4. The ability of Salmonella to inhibit IL-1β production also prevents B cell death; thus, B cells represent an ideal niche in which Salmonella resides, thereby promoting its persistence and dissemination.

Introduction Rheumatoid Arthritis (RA) is a systemic and chronic inflammatory disease, which affects 1–1.5% of population worldwide. Synovial membrane, cartilage and bone are the main sites affected by inflammation. The inflammatory process in RA leads to increased production of inflammatory cytokines IL-8, TNF-a, IL-6, IL-1b, IL-15, IL-18 e IL-17A in synovial fluid and serum. In addition to Th17, others cells such as gd T cells, iNKTs, mast cells and neutrophils also produce IL-17A. In mouse model of RA, IL-17A producing neutrophils seems to be important for the onset of the disease. Thus, we hypothesize that the population of IL-17A producing neutrophils is increased in patients with RA. Objective To analyze the frequency of IL-17A producing neutrophils in peripheral blood of patients with RA. Methods Peripheral blood from 68 patients with rheumatoid arthritis and 15 healthy donors has been collected, erythrocytes were lysed and peripheral blood leukocytes were stained for surface marker CD177 and for intracellular detection of IL-17A. Cells were analyzed by flow cytometry. It was also evaluated the frequency TH17 cells by flow cytometry using standard protocol. Results IL-17A producing neutrophils are increased in peripheral blood of patients with RA (0–19.5% Mean 1.66%) compared with healthy donors where no neutrophils IL-17A+ were observed (0–0.1% mean 0.05%). Surprisingly, Th17 cells were not detected in peripheral blood of patients with RA. Conclusion Neutrophils might be the most important source of IL-17A during the inflammatory response observed in patients with RA since we do not detect TH17 cells in these patients.

To study the processing and presentation of endogenously synthesized Ag to class II MHC-restricted T cells, hen egg lysozyme (HEL), either tagged with a peptide that confers retention in the endoplasmic reticulum (HEL.KDEL), or in the secretory form (HELs), was stably expressed in LK-35.2 B hybridoma cells. Presentation of HEL peptides bound to class II molecules was assessed by activation of specific T cell hybridomas recognizing seven different epitopes derived from exogenous HEL. The presentation of endogenously synthesized HEL was not caused by reuptake of secreted of shed Ag. All the HEL epitopes examined were efficiently presented after processing of endogenous HEL by HELs-transfected LK-35.2 cells. Processing of HEL tagged with KDEL, however, gave rise to presentation of only six of the seven HEL epitopes. The epitope included in the HEL sequence 112-124 was not presented by HEL.KDEL-transfected B cells. In addition, two of the four T cell hybridomas recognizing HEL 116-129 together with I-Ak molecules were not activated by HEL.KDEL, and three other epitopes were presented with lower efficiency as compared with HELs. Thus, endogenously synthesized HEL in secretory form gives rise to a set of class II-binding epitopes indistinguishable from exogenous HEL, whereas endoplasmic reticulum-retained HEL generates a similar but not identical set of epitopes. The endosomal protease inhibitor leupeptin prevented presentation of the epitope 108-116, but not 46-61, both by HELs and HEL.KDEL transfected cells, indicating a requirement for endosomal processing in both cases. In addition, the presentation of peptides derived from endogenously synthesized, either secretory or endoplasmic reticulum-retained HEL, could be inhibited by lysosomotropic amines, further indicating that the intracellular route of class II molecules presenting peptides derived from endogenous Ag intersects the acidic endosomal compartment.

Osteoarthritis (OA) is a chronic-degenerative arthropathy with high incidence and prevalence in which cartilage is destroyed due to extracellular matrix erosion (collagen and proteoglycan’s loss) and death of chondrocytes mainly by chondroptosis (program cell death) that could be clearance by neighboring chondrocytes. In order to confirm chondrocyte’s phagocytic capacity we performed a phagocytosis assay with Salmonella thyphimurium-GFP (moi 30) and we observed that after 30 min chondrocytes were able to phagocytose bacteria. We also found that 60% of chondrocytes, either from normal or OA tissue, were able to phagocyte 2 or more apoptotic chondrocytes. In addition, we found that less than the 5% of the chondrocytes express CD36 and the percentage of CD36 positive cells increases after the contact with apopotic cell. These data confirm that chondrocytes have the capacity to clearance apoptotic cells and that CD36 is involve in this process. Suggesting that chondrocytes could be able to eliminate apoptotic chondrocytes in the tissue in order to maintain homeostasis cartilage.

The reconstitution of the T CD8 lymphocyte population in patients subject to hematopoietic cell transplantation (HCT) depends largely of homeostatic peripheral expansion (HPE) of the T CD8 lymphocytes that were not eliminated during the pre-transplant conditioning regimen or the ones that are contained in the transplanted cells. In order to have an efficient HPE, MHC-I molecules, IL-7 and IL-15 are required. Evidences obtained in our lab indicate that the low and/or heterogeneous MHC-I levels of expression correlate with a inefficient reconstitution of T CD8 lymphocytes in patients that received allogeneic HCT. The deficiency in MHC-I expression could be due to a decrease in the expression of the NLRP12 protein, which has been proposed as a positive regulator of classical and non-classical MHC-I molecules expression. Therefore the objective of the following project is to determine the involvement of NALP12 in the MHC-I expression in monocytes from patients subject to HCT. We studied a cohort of 11 patients, 6 of which received autologous transplant and 5 with allogeneic transplant. Most of the patients present normal levels of T CD8 lymphocytes and a homogeneous expression of MHC-I. The patients that received autologous transplantation show a correlation of r=0.56. Our results suggest that NALP12 could act as an enhancer for the expression of the MHC-I molecules and that this expression could be affected in patients subject to allogeneic HPC transplantation.

Type 1 Diabetes (T1D) is a chronic, autoimmune disease, characterized by specific destruction of the insulin producing pancreatic β cells. This destruction is mediated by auto-reactive T cells. Lymphocytes NKT play an important role in type 1 diabetes (T1D) pathogenesis. Data from the NOD mouse shows a numerically and functionally deficient NKT cell, and those produce subnormal levels of IL-4 and IFNγ when stimulated. Similar defects have been suggested in humans, although they are not conclusive, some studies predict a role for NKT cells in T1D development. Class I Restricted T Cell Associated Molecule (CRTAM) is expressed at the cell surface of activated human invariant NKT cells (iNKT), CD8 and CD4 T cells. Recently we analyzed a cohort of 42 T1D patients, 103 relatives (mother, father and sibling), and 40 healthy controls. We found a significant low percentage and absolute number for iNKT in T1D patients, and identified two cell populations among the iNKT cells (iNKThi and iNKTlo). High levels of Va24/Vb11 (TCR) were observed for the iNKThi whereas the iNKTlo expressed low levels of TCR and it was CRTAM positive.