The prognosis of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) remains unsatisfactory and, despite major advances in genomic studies, the biological mechanisms ...underlying chemoresistance are still poorly understood. We conducted for the first time a large-scale differential multi-omics investigation on DLBCL patient's samples in order to identify new biomarkers that could early identify patients at risk of R/R disease and to identify new targets that could determine chemorefractoriness. We compared a well-characterized cohort of R/R versus chemosensitive DLBCL patients by combining label-free quantitative proteomics and targeted RNA sequencing performed on the same tissues samples. The cross-section of both data levels allowed extracting a sub-list of 22 transcripts/proteins pairs whose expression levels significantly differed between the two groups of patients. In particular, we identified significant targets related to tumor metabolism (Hexokinase 3), microenvironment (IDO1, CXCL13), cancer cells proliferation, migration and invasion (S100 proteins) or BCR signaling pathway (CD79B). Overall, this study revealed several extremely promising biomarker candidates related to DLBCL chemorefractoriness and highlighted some new potential therapeutic drug targets. The complete datasets have been made publically available and should constitute a valuable resource for the future research.
Disseminated intravascular coagulation (DIC) is a severe complication of septic shock. Polymorphonuclear neutrophils (PMNs) may play a key role in septic shock-induced DIC via the release of ...neutrophils extracellular traps (NETs). NETs capture invading pathogens, but also act as a pro-coagulant surface at the interface between immunity and thrombosis. During septic shock-induced DIC, neutrophil activation may result in excessive NET formation. Herein, we originally report the presence of circulating NETs in human blood during septic shock-induced DIC.
To investigate NET formation during shock-induced DIC neutrophils were isolated from patients in septic shock associated with (n = 3) or without (n = 3) DIC. Neutrophils from healthy donors (n = 3) were stimulated in vitro with ionomycin as NET formation positive controls. PMNs smears were stained with mouse anti-human FITC anti-myeloperoxidase antibody and the blue-fluorescent DAPI nucleic acid stain. NETs were identified as elongated extracellular DNA fibers associated to myeloperoxidase detected by immunofluorescence.
NETs were unambiguously observed in PMNs from septic shock patients with DIC but not from patients without DIC. NETs features in DIC+ patients were undistinguishable from those observed in ionomycin-induced PMNs from healthy donors. Fluorescence images of NETs were associated to extracellular cytoplasmic expansions.
Our data report for the first time the direct visualization of circulating NETs in patients with septic shock-induced DIC. The in vivo relevance of previously reported indirect markers of NETosis (neutrophil side fluorescence) is confirmed.
•Netosis features can be detected in blood PMNs using simple fluorescence microscopy.•PMNs purified from patients with DIC-induced septic shock display NETosis features.•Morphological analysis of PMNs can distinguish septic shock patients with DIC.
Neutrophils serve as the frontline defenders in the host's response to infections. However, the available methods for assessing the activated status of neutrophils are still limited. The immature ...cells that appear during sepsis are large with complex cytoplasmic components and rich nucleic acids, making them diagnosable by cell population data analysis using the automated cell counter. The changes are expressed as increased forward scattered light, side fluorescence light, and side fluorescence distribution width. Additionally, changes in side fluorescence light may indicate the neutrophil extracellular trap formation and can be useful for the diagnosis of sepsis-associated disseminated intravascular coagulation.
Neutrophils extracellular traps (NETs) have recently emerged as a new potential link between inflammation, immunity, and thrombosis and could play a key role in septic shock-induced disseminated ...intravascular coagulation (DIC) pathogenesis. The objective of our study was to investigate a potential link between NETosis and septic shock-induced DIC.
Twenty patients with septic shock (10 without and 10 with DIC according to JAAM 2006 score) were prospectively included in our study. Vascular cell activation was assessed by microparticle (MP) measurement. NETosis was investigated at days 1, 3, and 7 using two different approaches: probing and measurement of neutrophil DNA decompaction by neutrophil-side fluorescence light (NEUT-SFL) as recorded by an automated blood cell cytometer and the assessment of nucleosomes and NETs (DNA-bound myeloperoxidase, DNA-MPO).
Endothelial-derived CD105-MPs, leucocyte-derived CD11a-MPs/leucocyte, and neutrophil-derived CD66b-MPs/neutrophil count ratios significantly increased in DIC compared with non-DIC patients, indicating on-going cell activation (P <0.05). NEUT-SFL, indicating DNA decompaction, was significantly higher in DIC patients. Circulating nucleosomes and DNA-MPO were increased in DIC patients (P <0.05). There were significant correlations between: nucleosomes and NETs (r = 0.397, P = 0.004), NEUT-SFL and nucleosomes (r = 0.243, P = 0.032), NEUT-SFL and DNA-MPO (r = 0.266, P = 0.024).
NEUT-SFL, NETs, and elevated nucleosome concentrations were all correlated to DIC (P <0.05). We have shown that NETosis is significantly correlated to septic shock-induced DIC.
B-cell receptor (BCR) signaling is crucial for the pathophysiology of most mature B-cell lymphomas/leukemias and has emerged as a therapeutic target whose effectiveness remains limited by the ...occurrence of mutations. Therefore, deciphering the cellular program activated downstream this pathway has become of paramount importance for the development of innovative therapies. Using an original ex vivo model of BCR-induced proliferation of chronic lymphocytic leukemia cells, we generated 108 temporal transcriptional and proteomic profiles from 1 h up to 4 days after BCR activation. This dataset revealed a structured temporal response composed of 13,065 transcripts and 4027 proteins, comprising a leukemic proliferative signature consisting of 430 genes and 374 proteins. Mathematical modeling of this complex cellular response further highlighted a transcriptional network driven by 14 early genes linked to proteins involved in cell proliferation. This group includes expected genes (EGR1/2, NF-kB) and genes involved in NF-kB signaling modulation (TANK, ROHF) and immune evasion (KMO, IL4I1) that have not yet been associated with leukemic cells proliferation. Our study unveils the BCR-activated proliferative genetic program in primary leukemic cells. This approach combining temporal measurements with modeling allows identifying new putative targets for innovative therapy of lymphoid malignancies and also cancers dependent on ligand-receptor interactions.
A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. ...Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However, functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells, pointing out the need of mandatory BCR co-factors in this process. Here, we investigated benefits of several BCR co-stimulatory molecules (IL-2, IL-4, IL-15, IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand, IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition, we established a proliferative advantage for ZAP70 positive CLL cells, associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover, the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies.
Toll-like receptors play crucial roles in the sepsis-induced systemic inflammatory response. Septic shock mortality correlates with overexpression of neutrophilic TLR2 and TLR9, while the role of ...TLR4 overexpression remains a debate. In addition, TLRs are involved in the pathogenesis of viral infections such as COVID-19, where the single-stranded RNA of SARS-CoV-2 is recognized by TLR7 and TLR8, and the spike protein activates TLR4.
In this study, we conducted a comprehensive analysis of TLRs 1-10 expressions in white blood cells from 71 patients with bacterial and viral infections. Patients were divided into 4 groups based on disease type and severity (sepsis, septic shock, moderate, and severe COVID-19) and compared to 7 healthy volunteers.
We observed a significant reduction in the expression of TLR4 and its co-receptor CD14 in septic shock neutrophils compared to the control group (p < 0.001). Severe COVID-19 patients exhibited a significant increase in TLR3 and TLR7 levels in neutrophils compared to controls (p < 0.05). Septic shock patients also showed a similar increase in TLR7 in neutrophils along with elevated intermediate monocytes (CD14+CD16+) compared to the control group (p < 0.005 and p < 0.001, respectively). However, TLR expression remained unchanged in lymphocytes.
This study provides further insights into the mechanisms of TLR activation in various infectious conditions. Additional analysis is needed to assess their correlation with patient outcome and to evaluate the impact of TLR-pathway modulation during septic shock and severe COVID-19.
To investigate the contribution of neutrophil activation as innate immune cells during septic shock-induced disseminated intravascular coagulation.
Prospective study.
One University Hospital ICU.
...Hundred patients with septic shock. Thirty-five patients had disseminated intravascular coagulation according to Japanese Association for Acute Medicine 2006 score.
None.
Neutrophil chromatin decondensation was assessed by measuring neutrophil fluorescence (NEUT-side-fluorescence light) labeled by a fluorochrome-based polymethine reagent using a routine automated flow cytometer Sysmex XN20 (Sysmex, Kobe, Japan) and neutrophil-derived CD66b microparticles by prothrombinase assay. Measurements in disseminated intravascular coagulation and no disseminated intravascular coagulation patients showed that a mean value of NEUT-side-fluorescence light above 57.3 arbitrary units had a sensitivity of 90.91% and a specificity of 80.60% for disseminated intravascular coagulation diagnosis. NEUT-side-fluorescence light was correlated to the CD66b microparticles/neutrophil count, a surrogate of neutrophil activation associated with septic shock-induced disseminated intravascular coagulation.
NEUT-side-fluorescence light, routinely available, could prove an accurate biomarker of neutrophil activation.
The intestine‐specific caudal‐related homeobox gene‐2 (CDX2) homeobox gene, while being a tumor suppressor in the gut, is ectopically expressed in a large proportion of acute leukemia and is ...associated with poor prognosis. Here, we report that turning on human CDX2 expression in the hematopoietic lineage of mice induces acute monoblastic leukemia, characterized by the decrease in erythroid and lymphoid cells at the benefit of immature monocytic and granulocytic cells. One of the highly stimulated genes in leukemic bone marrow cells was BMP and activin membrane‐bound inhibitor (Bambi), an inhibitor of transforming growth factor‐β (TGF‐β) signaling. The CDX2 protein was shown to bind to and activate the transcription of the human BAMBI promoter. Moreover, in a leukemic cell line established from CDX2‐expressing mice, reducing the levels of CDX2 or Bambi stimulated the TGF‐β‐dependent expression of Cd11b, a marker of monocyte maturation. Taken together, this work demonstrates the strong oncogenic potential of the homeobox gene CDX2 in the hematopoietic lineage, in contrast with its physiological tumor suppressor activity exerted in the gut. It also reveals, through BAMBI and TGF‐β signaling, the involvement of CDX2 in the perturbation of the interactions between leukemia cells and their microenvironment.
Abnormal expression of the homeodomain transcription factor caudal‐related homeobox gene‐2 (CDX2) is associated with poor prognosis of leukemia patients. Here, we report that turning on human CDX2 expression in the hematopoietic lineage of mice induces acute monoblastic leukemia, through inhibition of the lymphocytic lineage and accumulation of immature monoblasts mediated by the stimulation of the BMP and activin membrane‐bound inhibitor that blocked transforming growth factor‐β‐dependent differentiation of monocytes.
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