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Pancreatic ductal adenocarcinoma (PDAC) is characterized by a KRAS-driven inflammatory program and a desmoplastic stroma, which contribute to the profoundly chemoresistant phenotype. The tumor stroma contains an abundance of cancer-associated fibroblasts (CAF), which engage in extensive paracrine cross-talk with tumor cells to perpetuate protumorigenic inflammation. IL1α, a pleiotropic, tumor cell-derived cytokine, plays a critical role in shaping the stromal landscape. To provide insights into the molecular mechanisms regulating IL1A expression in PDAC, we performed transcriptional profiling of The Cancer Genome Atlas datasets and pharmacologic screening in PDAC cells and identified p38α MAPK as a key positive regulator of IL1A expression. Both genetic and pharmacologic inhibition of p38 MAPK significantly diminished IL1α production in vitro. Chromatin- and coimmunoprecipitation analyses revealed that p38 MAPK coordinates the transcription factors Sp1 and the p65 subunit of NFκB to drive IL1A overexpression. Single-cell RNA sequencing of a highly desmoplastic murine PDAC model, Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT), confirmed that p38 MAPK inhibition significantly decreases tumor cell-derived Il1a and attenuates the inflammatory CAF phenotype in a paracrine IL1α-dependent manner. Furthermore, p38 MAPK inhibition favorably modulated intratumoral immunosuppressive myeloid populations and augmented chemotherapeutic efficacy to substantially reduce tumor burden and improve overall survival in PKT mice. These findings illustrate a cellular mechanism of tumor cell-intrinsic p38-p65/Sp1-IL1α signaling that is responsible for sustaining stromal inflammation and CAF activation, offering an attractive therapeutic approach to enhance chemosensitivity in PDAC., Significance: Inhibition of p38 MAPK suppresses tumor cell-derived IL1α and attenuates the inflammatory stroma and immunosuppressive tumor microenvironment to overcome chemotherapeutic resistance in pancreatic cancer., (©2024 American Association for Cancer Research.)

Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent antiinflammatory and antitumor activity in several preclinical models and has proven both safe and efficacious in clinical trials for advanced gastrointestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared with posttransplant cyclophosphamide (PTCy). Strikingly, we found Minnelide improved survival, weight loss, and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score, and colonic length. Notably, in addition to decreased donor T cell infiltration early after aHSCT, several regulatory cell populations, including Tregs, ILC2s, and myeloid-derived stem cells in the colon were increased, which together may account for Minnelide's GVHD suppression after aHSCT. Importantly, Minnelide's GVHD prevention was accompanied by preservation of graft-versus-tumor activity. As Minnelide possesses anti-acute myeloid leukemia (anti-AML) activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for patients with AML undergoing aHSCT.

Pancreatic ductal adenocarcinoma (PDAC) is a significant contributor to cancer-related morbidity and mortality, and it is known for its resistance to conventional treatment regimens, including chemotherapy and immune checkpoint blockade (ICB)-based therapies. We have previously shown that Urolithin A (Uro A), a gut microbial metabolite derived from pomegranates, can target and inhibit KRAS -dependent PI3K/AKT/mTOR signaling pathways to overcome therapeutic resistance and improve survival in PDAC. However, the effect of Uro A on the tumor immune microenvironment and its ability to enhance ICB efficacy has not been explored. This study demonstrates that Uro A treatment reduces stromal fibrosis and reinvigorates the adaptive T-cell immune response to overcome resistance to PD-1 blockade in a genetically engineered mouse model (GEMM) of PDAC. Flow cytometric-based analysis of Uro A-treated mouse tumors revealed a significant attenuation of immunosuppressive tumor-associated M2-like macrophages with a concurrent increase in the infiltration of CD4 + and CD8 + T cells with memory-like phenotype along with reduced expression of the exhaustion-associated protein, PD-1. Importantly, the combination of Uro A treatment with anti-PD-1 immunotherapy promoted enhancement of the antitumor response with increased infiltration of CD4 + Th1 cells, ultimately resulting in a remarkable improvement in overall survival in GEMM of PDAC. Overall, our findings provide preclinical evidence for the potential of Uro A as a novel therapeutic agent to increase sensitivity to immunotherapy in PDAC and warrant further mechanistic exploration in preclinical and clinical studies., Significance: Immunotherapeutic agents are ineffective against pancreatic cancer, mainly due to the immunosuppressive tumor microenvironment and stromal desmoplasia. Our current study demonstrates the therapeutic utility of a novel gut microbial metabolite, Uro A, to remodel the stromal-immune microenvironment and improve overall survival with anti-PD-1 therapy in pancreatic cancer., (© 2023 The Authors; Published by the American Association for Cancer Research.)

We have shown that KRAS-TP53 genomic coalteration is associated with immune-excluded microenvironments, chemoresistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. By treating KRAS-TP53 cooperativity as a model for high-risk biology, we now identify cell-autonomous Cxcl1 as a key mediator of spatial T-cell restriction via interactions with CXCR2+ neutrophilic myeloid-derived suppressor cells in human PDAC using imaging mass cytometry. Silencing of cell-intrinsic Cxcl1 in LSL-KrasG12D/+;Trp53R172H/+;Pdx-1Cre/+(KPC) cells reprograms the trafficking and functional dynamics of neutrophils to overcome T-cell exclusion and controls tumor growth in a T cell-dependent manner. Mechanistically, neutrophil-derived TNF is a central regulator of this immunologic rewiring, instigating feed-forward Cxcl1 overproduction from tumor cells and cancer-associated fibroblasts (CAF), T-cell dysfunction, and inflammatory CAF polarization via transmembrane TNF-TNFR2 interactions. TNFR2 inhibition disrupts this circuitry and improves sensitivity to chemotherapy in vivo. Our results uncover cancer cell-neutrophil cross-talk in which context-dependent TNF signaling amplifies stromal inflammation and immune tolerance to promote therapeutic resistance in PDAC., Significance: By decoding connections between high-risk tumor genotypes, cell-autonomous inflammatory programs, and myeloid-enriched/T cell-excluded contexts, we identify a novel role for neutrophil-derived TNF in sustaining immunosuppression and stromal inflammation in pancreatic tumor microenvironments. This work offers a conceptual framework by which targeting context-dependent TNF signaling may overcome hallmarks of chemoresistance in pancreatic cancer. This article is highlighted in the In This Issue feature, p. 1275., (©2023 American Association for Cancer Research.)

Background & Aims: We have shown that reciprocally activated rat sarcoma (RAS)/mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and Janus kinase/signal transducer and activator of transcription 3 (STAT3) pathways mediate therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC), while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance and alters stromal architecture. We now determine whether MEKi+STAT3i reprograms the cancer-associated fibroblast (CAF) and immune microenvironment to overcome resistance to immune checkpoint inhibition in PDAC., Methods: CAF and immune cell transcriptomes in MEKi (trametinib)+STAT3i (ruxolitinib)-treated vs vehicle-treated Ptf1a Cre/+ ;LSL-Kras G12D/+; Tgfbr2 flox/flox (PKT) tumors were examined via single-cell RNA sequencing (scRNAseq). Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats associated protein 9 silencing of CAF-restricted Map2k1/Mek1 or Stat3, or both, enabled interrogation of CAF-dependent effects on immunologic remodeling in orthotopic models. Tumor growth, survival, and immune profiling via mass cytometry by time-of-flight were examined in PKT mice treated with vehicle, anti-programmed cell death protein 1 (PD-1) monotherapy, and MEKi+STAT3i combined with anti-PD1., Results: MEKi+STAT3i attenuates Il6/Cxcl1-expressing proinflammatory and Lrrc15-expressing myofibroblastic CAF phenotypes while enriching for Ly6a/Cd34-expressing CAFs exhibiting mesenchymal stem cell-like features via scRNAseq in PKT mice. This CAF plasticity is associated with M2-to-M1 reprogramming of tumor-associated macrophages, and enhanced trafficking of cluster of differentiation 8 + T cells, which exhibit distinct effector transcriptional programs. These MEKi+STAT3i-induced effects appear CAF-dependent, because CAF-restricted Mek1/Stat3 silencing mitigates inflammatory-CAF polarization and myeloid infiltration in vivo. Addition of MEKi+STAT3i to PD-1 blockade not only dramatically improves antitumor responses and survival in PKT mice but also augments recruitment of activated/memory T cells while improving their degranulating and cytotoxic capacity compared with anti-PD-1 monotherapy. Importantly, treatment of a patient who has chemotherapy-refractory metastatic PDAC with MEKi (trametinib), STAT3i (ruxolitinib), and PD-1 inhibitor (nivolumab) yielded clinical benefit., Conclusions: Combined MEKi+STAT3i mitigates stromal inflammation and enriches for CAF phenotypes with mesenchymal stem cell-like properties to overcome immunotherapy resistance in PDAC., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Partial/complete pathologic response following neoadjuvant chemotherapy (NAC) in pancreatic cancer (PDAC) patients undergoing pancreatectomy is associated with improved survival. We sought to determine whether neutrophil-to-lymphocyte ratio (NLR) dynamics predict pathologic response following chemotherapy in PDAC, and if manipulating NLR impacts chemosensitivity in preclinical models and uncovers potential mechanistic underpinnings underlying these effects., Methods: Pathologic response in PDAC patients (n=94) undergoing NAC and pancreatectomy (7/2015-12/2019) was dichotomized as partial/complete or poor/absent. Bootstrap-validated multivariable models assessed associations between pre-chemotherapy NLR (%neutrophils÷%lymphocytes) or NLR dynamics during chemotherapy (ΔNLR = pre-surgery-pre-chemotherapy NLR) and pathologic response, disease-free survival (DFS), and overall survival (OS). To preclinically model effects of NLR attenuation on chemosensitivity, Ptf1a Cre/+ ; Kras LSL-G12D/+ ;Tgfbr2 flox/flox (PKT) mice and C57BL/6 mice orthotopically injected with Kras LSL-G12D/+ ;Trp53 LSL-R172H/+ ;Pdx1 Cre (KPC) cells were randomized to vehicle, gemcitabine/paclitaxel alone, and NLR-attenuating anti-Ly6G with/without gemcitabine/paclitaxel treatment., Results: In 94 PDAC patients undergoing NAC (median:4 months), pre-chemotherapy NLR (p<0.001) and ΔNLR attenuation during NAC (p=0.002) were independently associated with partial/complete pathologic response. An NLR score = pre-chemotherapy NLR+ΔNLR correlated with DFS (p=0.006) and OS (p=0.002). Upon preclinical modeling, combining NLR-attenuating anti-Ly6G treatment with gemcitabine/paclitaxel-compared with gemcitabine/paclitaxel or anti-Ly6G alone-not only significantly reduced tumor burden and metastatic outgrowth, but also augmented tumor-infiltrating CD107a + -degranulating CD8 + T-cells (p<0.01) while dampening inflammatory cancer-associated fibroblast (CAF) polarization (p=0.006) and chemoresistant IL-6/STAT-3 signaling in vivo. Neutrophil-derived IL-1β emerged as a novel mediator of stromal inflammation, inducing inflammatory CAF polarization and CAF-tumor cell IL-6/STAT-3 signaling in ex vivo co-cultures., Conclusions: Therapeutic strategies to mitigate neutrophil-CAF-tumor cell IL-1β/IL-6/STAT-3 signaling during NAC may improve pathologic responses and/or survival in PDAC., Funding: Supported by KL2 career development grant by Miami CTSI under NIH Award UL1TR002736, Stanley Glaser Foundation, American College of Surgeons Franklin Martin Career Development Award, and Association for Academic Surgery Joel J. Roslyn Faculty Award (to J. Datta); NIH R01 CA161976 (to N.B. Merchant); and NCI/NIH Award P30CA240139 (to J. Datta and N.B. Merchant)., Competing Interests: Id, AB, ND, PS, SM, VG, SS, JE, PH, DK, NM, JD No competing interests declared, (© 2022, de Castro Silva et al.)

Obesity causes chronic inflammation and changes in gut microbiome. However, how this contributes to poor survival and therapy resistance in patients with pancreatic cancer remain undetermined. Our current study shows that high fat diet-fed obese pancreatic tumor bearing mice do not respond to standard of care therapy with gemcitabine and paclitaxel when compared to corresponding control diet-fed mice. C57BL6 mice were put on control and high fat diet for 1 month following with pancreatic tumors were implanted in both groups. Microbiome of lean (control) and obese (high fat diet fed) mice was analyzed. Fecal matter transplant from control mice to obese mice sensitized tumors to chemotherapy and demonstrated extensive cell death. Analysis of gut microbiome showed an enrichment of queuosine (Q) producing bacteria in obese mice and an enrichment of S-adenosyl methionine (SAM) producing bacteria in control diet-fed mice. Further, supplementation of obese animals with SAM sensitized pancreatic tumors to chemotherapy. Treatment of pancreatic cancer cells with Q increased PRDX1 involved in oxidative stress protection. In parallel, tumors in obese mice showed increase in CD133 + treatment refractory tumor populations compared to control animals. These observations indicated that microbial metabolite Q accumulation in high fat diet-fed mice protected tumors from chemotherapy induced oxidative stress by upregulating PRDX1. This protection could be reversed by treatment with SAM. We conclude that relative concentration of SAM and queuosine in fecal samples of pancreatic cancer patients can be developed as a potential biomarker and therapeutic target in chemotherapy refractory pancreatic cancer.

In pancreatic cancer, the robust fibroinflammatory stroma contributes to immune suppression and renders tumors hypoxic, altering intratumoral metabolic pathways and leading to poor survival. One metabolic enzyme activated during hypoxia is lactate dehydrogenase A (LDHA). As a result of its promiscuous activity under hypoxia, LDHA produces L-2 hydroxyglutarate (L-2HG), an epigenetic modifier, that regulates the tumor transcriptome. However, the role of L-2HG in remodeling the pancreatic tumor microenvironment is not known. Here we used mass spectrometry to detect L-2HG in serum samples from patients with pancreatic cancer, comprising tumor cells as well as stromal cells. Both hypoxic pancreatic tumors as well as serum from patients with pancreatic cancer accumulated L-2HG as a result of promiscuous activity of LDHA. This abnormally accumulated L-2HG led to H3 hypermethylation and altered gene expression, which regulated a critical balance between stemness and differentiation in pancreatic tumors. Secreted L-2HG inhibited T-cell proliferation and migration, suppressing antitumor immunity. In a syngeneic orthotopic model of pancreatic cancer, inhibition of LDH with GSK2837808A decreased L-2HG, induced tumor regression, and sensitized tumors to anti-PD1 therapy. In conclusion, hypoxia-mediated promiscuous activity of LDH produces L-2HG in pancreatic tumor cells, regulating the stemness-differentiation balance and contributing to immune evasion. Targeting LDH can be developed as a potential therapy to sensitize pancreatic tumors to checkpoint inhibitor therapy. SIGNIFICANCE: This study shows that promiscuous LDH activity produces L-2HG in pancreatic tumor and stromal cells, modulating tumor stemness and immune cell function and infiltration in the tumor microenvironment., (©2021 American Association for Cancer Research.)

Pancreatic adenocarcinoma is a devastating disease with an abysmal survival rate of 9%. A robust fibro-inflammatory and desmoplastic stroma, characteristic of pancreatic cancer, contribute to the challenges in developing viable therapeutic strategies in this disease. Apart from constricting blood vessels and preventing efficient drug delivery to the tumor, the stroma also contributes to the aggressive biology of cancer along with its immune-evasive microenvironment. In this study, we show that in pancreatic tumors, the developing stroma increases tumor initiation frequency in pancreatic cancer cells in vivo by enriching for CD133 + aggressive "stem-like" cells. Additionally, the stromal fibroblasts secrete IL6 as the major cytokine, increases glycolytic flux in the pancreatic tumor cells, and increases lactate efflux in the microenvironment via activation of the STAT signaling pathway. We also show that the secreted lactate favors activation of M2 macrophages in the tumor microenvironment, which excludes CD8 + T cells in the tumor. Our data additionally confirms that the treatment of pancreatic tumors with anti-IL6 antibody results in tumor regression as well as decreased CD133 + population within the tumor. Furthermore, inhibiting the lactate efflux in the microenvironment reduces M2 macrophages, and makes pancreatic tumors more responsive to anti-PD1 therapy. This suggests that stromal IL6 driven metabolic reprogramming plays a significant role in the development of an immune-evasive microenvironment. In conclusion, our study shows that targeting the metabolic pathways affected by stromal IL6 can make pancreatic tumors amenable to checkpoint inhibitor therapy.

Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic background of the tumor and better understanding of the tumor microenvironment, immune checkpoint inhibitor therapy (targeting CTLA4, PD1, PDL1) has not been very successful against PDAC. The robust desmoplastic stroma, along with an extensive extracellular matrix (ECM) that is rich in hyaluronan, plays an integral role in this immune evasion. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on the one hand and influence the hyaluronan synthesis in the ECM on the other. The rate-limiting enzyme of the pathway, glutamine-fructose amidotransferase 1 (GFAT1), uses glutamine and fructose 6-phosphate to eventually synthesize uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). In the current manuscript, we targeted this glutamine-utilizing enzyme by a small molecule glutamine analog (6-diazo-5-oxo-l-norleucine [DON]). Our results showed that DON decreased the self-renewal potential and metastatic ability of tumor cells. Further, treatment with DON decreased hyaluronan and collagen in the tumor microenvironment, leading to an extensive remodeling of the ECM and an increased infiltration of CD8+ T cells. Additionally, treatment with DON sensitized pancreatic tumors to anti-PD1 therapy, resulting in tumor regression and prolonged survival.

Presence of quiescent, therapy evasive population often described as cancer stem cells (CSC) or tumor initiating cells (TIC) is often attributed to extreme metastasis and tumor recurrence. This population is typically enriched in a tumor as a result of microenvironment or chemotherapy induced stress. The TIC population adapts to this stress by turning on cell cycle arrest programs that is a "fail-safe" mechanism to prevent expansion of malignant cells to prevent further injury. Upon removal of the "stress" conditions, these cells restart their cell cycle and regain their proliferative nature thereby resulting in tumor relapse. Growth Arrest Specific 5 (GAS5) is a long-non-coding RNA that plays a vital role in this process. In pancreatic cancer, CD133+ population is a typical representation of the TIC population that is responsible for tumor relapse. In this study, we show for the first time that emergence of CD133+ population coincides with upregulation of GAS5, that reprograms the cell cycle to slow proliferation by inhibiting GR mediated cell cycle control. The CD133+ population further routed metabolites like glucose to shunt pathways like pentose phosphate pathway, that were predominantly biosynthetic in spite of being quiescent in nature but did not use it immediately for nucleic acid synthesis. Upon inhibiting GAS5, these cells were released from their growth arrest and restarted the nucleic acid synthesis and proliferation. Our study thus showed that GAS5 acts as a molecular switch for regulating quiescence and growth arrest in CD133+ population, that is responsible for aggressive biology of pancreatic tumors.

Platelet activation and platelet-derived cytokines contribute to the vascular inflammation and increased thrombotic activity known to occur in patients with sickle cell anemia (SCA). CD40 ligand (CD40L), a platelet-associated pro-inflammatory molecule that promotes endothelial cell activation, is elevated in the circulation of SCA patients. We sought to evaluate the association of CD40L and inflammation with sickle-related clinical complications and laboratory variables in SCA patients. Soluble CD40L, thrombospondin (TSP)-1 and tumor necrosis factor (TNF)-α were determined in the platelet-poor plasma of healthy individuals and steady-state SCA patients by ELISA. Lifetime clinical complications were verified by detailed review of patients' medical records. We found that plasma CD40L was associated with acute chest syndrome (ACS), and that SCA patients with a lifetime history of ACS (ACS+) presented significantly higher plasma CD40L and TSP-1 than patients who had never experienced ACS (ACS-). In the ACS+ group, both platelet-derived proteins (CD40L and TSP-1) correlated with mean corpuscular volume, mean corpuscular hemoglobin and reticulocyte hemoglobin, while in the ACS- group, CD40L correlated with low red blood cell counts, hemoglobin, hematocrit and lactate dehydrogenase, and TSP-1 correlated with reticulocyte percentage and white blood cell count. As expected, CD40L and TSP-1 correlated with platelet counts in both groups. These data highlight the possible role of platelet activation in ACS and suggest that plasma sCD40L, together with TSP-1, may represent a potential marker of susceptibility to ACS in SCA., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified., (Copyright© Ferrata Storti Foundation.)

Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association with other therapies for SCD.

The endothelium plays an important role in sickle cell anemia (SCA) pathophysiology, interacting with red cells, leukocytes and platelets during the vaso-occlusive process and undergoing activation and dysfunction as a result of intravascular hemolysis and chronic inflammation. Blood outgrowth endothelial cells (BOECs) can be isolated from adult peripheral blood and have been used in diverse studies, since they have a high proliferative capacity and a stable phenotype during in vitro culture. This study aimed to establish BOEC cultures for use as an in vitro study model for endothelial function in sickle cell anemia. Once established, BOECs from steady-state SCA individuals (SCA BOECs) were characterized for their adhesive and inflammatory properties, in comparison to BOECs from healthy control individuals (CON BOECs). Cell adhesion assays demonstrated that control individual red cells adhered significantly more to SCA BOEC than to CON BOEC. Despite these increased adhesive properties, SCA BOECs did not demonstrate significant differences in their expression of major endothelial adhesion molecules, compared to CON BOECs. SCA BOECs were also found to be pro-inflammatory, producing a significantly higher quantity of the cytokine, IL-8, than CON BOECs. From the results obtained, we suggest that BOEC may be a good model for the in vitro study of SCA. Data indicate that endothelial cells of sickle cell anemia patients may have abnormal inflammatory and adhesive properties even outside of the chronic inflammatory and vaso-occlusive environment of patients., (© 2013.)

Chronic vascular inflammation and endothelial activation may initiate vaso-occlusion in sickle cell disease (SCD). TNFSF14 (CD258; LIGHT), a recently-identified pro-thrombotic and pro-inflammatory tumour necrosis factor (TNF)-superfamily cytokine, has a potent activating effect on endothelial cells. We evaluated whether TNFSF14 production is altered in SCD and whether platelets contribute to this production. TNFSF14 was measured in platelet-free plasma from healthy-control individuals (CON), steady-state sickle cell anaemia (SCA), SCA on hydroxycarbamide therapy (SCAHC) and haemoglobin SC (HbSC) patients. Mean plasma TNFSF14 was significantly increased in SCA, SCAHC and HbSC, compared to CON individuals. In SCA/SCAHC patients, plasma TNFSF14, showed no correlation with haematological variables, but was significantly correlated with serum lactate dehydrogenase and inflammatory markers (CD40LG , IL8 and ICAM1). Platelet-membrane TNFSF14 expression was significantly augmented on SCA platelets, and correlated with platelet activation; furthermore, measurement of platelet TNFSF14 release indicated that platelets may be a major source of circulating TNFSF14 in SCA. Interestingly, high plasma TNFSF14 was significantly associated with elevated tricuspid regurgitant velocity (≥2·5 m/s) in a population of SCA/SCAHC patients. The pro-inflammatory and atherogenic cytokine, TNFSF14, could contribute to endothelial activation and inflammation in SCA; future investigations may confirm whether this protein contributes to major clinical complications of the disease, such as pulmonary hypertension, and represents a potential therapeutic target., (© 2012 Blackwell Publishing Ltd.)

Neutrophils participate in the initiation and progression of rheumatoid arthritis (RA) although the exact mechanisms responsible for neutrophil accumulation in rheumatoid joints are not understood. This study compared the adhesive and chemotactic functions of neutrophils from RA patients in activity (DAS28 > 3.2) and not in activity (DAS28 < 2.6) and observed the effects of different treatment approaches on these functions. Neutrophils were isolated from healthy controls (CON), and patients with active or inactive RA in use of therapy not specific for RA (NSAIDs), in use of DMARDs and in use of anti-TNF-α therapy. Adhesive and chemotactic properties were evaluated using in vitro assays; adhesion molecule expression was assessed by flow cytometry and real-time PCR and circulating chemokines were determined by ELISA. No significant alterations in the adhesive and chemotactic properties of neutrophils from active RA were observed when compared to CON neutrophils, independently of treatment regimen. In contrast, neutrophils from RA patients in disease remission presented reduced adhesive properties and a lower spontaneous chemotactic capacity, in association with decreased adhesion molecule expression, although profiles of alterations differed for those patients on DMARDs and those on anti-TNF-α therapy. Circulating levels of the major neutrophilic chemokines, IL-8 and epithelial neutrophil activating peptide-78, were also significantly decreased in those patients demonstrating a clinical response. Remission of RA appears to be associated with ameliorations in aspects important for neutrophil adhesion and chemotaxis; whether these alterations contribute to decrease neutrophil migration to the synovial fluid, with consequent improvements in the clinical manifestations of RA, remains to be determined., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)