Survey of Changes in Absolute Lymphocyte Counts and Peripheral Immune Repertoire Diversity after External Beam Radiotherapy.

Radiation-induced lymphopenia (RIL) is associated with worse outcomes in patients with multiple solid tumors. Hypofractionated radiation therapy (HFRT) reduces RIL compared with conventionally fractionated radiation therapy (CFRT). However, fractionation effects on immune repertoire (IR) diversity are unknown. RNA-based T- and B-cell receptor sequencing was performed on peripheral lymphocytes collected prospectively before radiation therapy and <4 weeks after the final radiation fraction. Patients received CFRT (≤3 Gy/day × ≥10 days ± chemotherapy, n = 13) or HFRT (≥5 Gy/day × ≤5 days, n = 10), per institutional standards of care. Immune repertoire diversity parameters analyzed were number of unique CDR3 receptors (uCDR3), Shannon entropy, and sample clonality (percentage of all receptors represented by the top 10 clones). RIL was severe with concurrent chemotherapy (median %Δ ALC -58.8%, -12.5%, and -28.6% in patients treated with CFRT and chemo, CFRT alone, and HFRT, respectively). CFRT and concurrent chemotherapy was associated with more severe diversity restriction in all examined parameters than either HFRT or CFRT alone. Increased immune repertoire diversity despite decreased ALC was more common in patients treated with HFRT than CFRT and significantly less common in patients treated with concurrent chemotherapy (P < 0.001). Radiation-induced changes in immune repertoire diversity are variably reflected in the peripheral ALC. Both HFRT and CFRT caused RIL, but HFRT was associated with improved immune repertoire diversity despite RIL. The addition of chemotherapy may potentiate radiation-induced restriction in immune repertoire diversity. As immune repertoire diversity is associated with response to immunotherapy, these findings may have implications for radiation therapy/chemotherapy/immunotherapy combinations. Further studies are required to understand the relationship between radiation, circulating lymphocyte populations, immune repertoire diversity and response to treatment.

Impact of Recombinant Granulocyte Colony-Stimulating Factor During Neoadjuvant Therapy on Outcomes of Resected Pancreatic Cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by chronic inflammation and a tolerogenic immune response. The granulocyte colony-stimulating factor (G-CSF)-neutrophil axis promotes oncogenesis and progression of PDAC. Despite frequent use of recombinant G-CSF in the management and prevention of chemotherapy-induced neutropenia, its impact on oncologic outcomes of patients with resected PDAC is unclear. PATIENTS AND METHODS: This cohort study assessing the impact of G-CSF administration was conducted on 351 patients with PDAC treated with neoadjuvant therapy (NAT) and pancreatic resection at a high-volume tertiary care academic center from 2014 to 2019. Participants were identified from a prospectively maintained database and had a median follow-up of 45.8 months. RESULTS: Patients receiving G-CSF (n=138; 39.3%) were younger (64.0 vs 66.7 years; P=.008), had lower body mass index (26.5 vs 27.9; P=.021), and were more likely to receive 5-FU-based chemotherapy (42.0% vs 28.2%; P<.0001). No differences were observed in baseline or clinical tumor staging. Patients receiving G-CSF were more likely to have an elevated (>5.53) post-NAT neutrophil-to-lymphocyte ratio (45.0% vs 29.6%; P=.004). G-CSF recipients also demonstrated higher circulating levels of neutrophil extracellular traps (+709 vs -619 pg/mL; P=.006). On multivariate analysis, G-CSF treatment was associated with perineural invasion (hazard ratio [HR], 2.65; 95% CI, 1.16-6.03; P=.021) and margin-positive resection (HR, 1.67; 95% CI, 1.01-2.77; P=.046). Patients receiving G-CSF had decreased overall survival (OS) compared with nonrecipients (median OS, 29.2 vs 38.7 months; P=.001). G-CSF administration was a negative independent predictor of OS (HR, 2.02; 95% CI, 1.45-2.79; P<.0001). In the inverse probability weighted analysis of 301 matched patients, neoadjuvant G-CSF administration was associated with reduced OS. CONCLUSIONS: In patients with localized PDAC receiving NAT prior to surgical extirpation, G-CSF administration may be associated with worse oncologic outcomes and should be further evaluated.

Acute pancreatitis induces a transient hypercoagulable state in murine models.

BACKGROUND/OBJECTIVES: Although understudied, risk of venous thromboembolism (VTE) appears to be increased during acute pancreatitis (AP). We aimed to further characterize a hypercoagulable state associated with AP utilizing thromboelastography (TEG), a readily available, point of care test. METHODS: AP was induced in C57/Bl6 mice using l-arginine and caerulein. TEG was performed with citrated native samples. The maximum amplitude (MA) and coagulation index (CI), a composite marker of coagulability, were evaluated. Platelet aggregation was assessed using whole blood collagen-activated platelet impedance aggregometry. Circulating tissue factor (TF), the initiator of extrinsic coagulation, was measured with ELISA. A VTE model using IVC ligation followed by measurement of clot size and weight was evaluated. After IRB approval and consent, blood samples from patients hospitalized with a diagnosis of AP were evaluated by TEG. RESULTS: Mice with AP displayed a significant increase in MA and CI, consistent with hypercoagulability. Hypercoagulability peaked at 24 h after induction of pancreatitis, then returned to baseline by 72 h. AP resulted in significantly increased platelet aggregation and elevated circulating TF. Increased clot formation with AP was observed in an in vivo model of deep vein thrombosis. In a proof of concept, correlative study, over two thirds of patients with AP demonstrated an elevated MA and CI compared to the normal range, consistent with hypercoagulability. CONCLUSIONS: Murine acute pancreatitis results in a transient hypercoagulable state that can be assessed by TEG. Correlative evidence for hypercoagulability was also demonstrated in human pancreatitis. Further study to correlate coagulation measures to incidence of VTE in AP is warranted.

Chloroquine reduces neutrophil extracellular trap (NET) formation through inhibition of peptidyl arginine deiminase 4 (PAD4).

Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

Translucency characteristics of permanent maxillary central incisor in Indian population: An analytical cross-sectional study.

Background: Understanding the optical characteristics, especially the translucency patterns (TPs) in teeth can assist clinicians in creating biomimetic restorations. Aim: This study assessed the differences in enamel TPs and the area of translucency (AOT) in the most dominant teeth of an individual's smile according to their age and gender. Materials and Methods: This cross-sectional study was conducted on 140 consented individuals. Based on the age, there were four groups with 35 subjects in each. The digital imaging method is used to classify and differentiate different TPs. The AOT was measured with the assistance of ImageJ 1.51j8 software. Statistical Analysis Used: One-way ANOVA with a post hoc test was used to test the statistical significance difference among the various age groups, and an independent t-test was used to test the variations between genders. Results: The outcome of this study showed that the AOT was the highest for the 50 and above age group as compared to other age groups (P = 0.003). Although there were variations in the TPs between the two genders, the AOT showed no significant difference. Conclusion: The area of incisal third translucency in the upper central incisors varied between different age groups. However, gender had no influence in the area of incisal translucency. Wide variations in the TPs were seen based on age and gender.

Sequestsome-1/p62-targeted small molecules for pancreatic cancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by heightened autophagy and systemic immune dysfunction. Modest improvements in clinical outcomes have been demonstrated in completed clinical trials targeting autophagy with combination hydroxychloroquine (HCQ) and chemotherapy. Recent mechanistic insights into the role of autophagy-dependent immune evasion have prompted the need for more precise and druggable targets of autophagy inhibition. Sequestosome-1 (SQSTM-1) is a multidomain scaffold protein with well-established roles in autophagy, tumor necrosis factor alpha (TNFα)- and NF-κB-related signaling pathways. SQSTM1 overexpression is frequently observed in PDAC, correlating with clinical stage and outcome. Given the unique molecular structure of SQSTM-1 and its diverse activity, identifying means of limiting SQSTM-1-dependent autophagy to promote an effective immune response in PDAC could be a promising treatment strategy.

Intrapleural interleukin-2-expressing oncolytic virotherapy enhances acute antitumor effects and T-cell receptor diversity in malignant pleural disease.

OBJECTIVE: The mainstay of treatment for patients with malignant pleural disease is fluid drainage and systemic therapy. A tumor-specific oncolytic virus or T-cell-activating interleukin-2 immunotherapy may provide an opportunity for local control. We previously developed a vaccinia virus-expressing interleukin-2, an oncolytic virus that mediated tumor regression in preclinical peritoneal tumor models with expansion of tumor-infiltrating lymphocytes. We evaluated the antitumor efficacy and immune modulatory effects of vaccinia virus-expressing interleukin-2 in malignant pleural disease. METHODS: A murine model of malignant pleural disease was established with percutaneous intrapleural deposition of the Lewis lung carcinoma cell line and monitored with bioluminescent imaging. After intrapleural or systemic administration of vaccinia viruses (vaccinia virus yellow fluorescent protein control, vaccinia virus-expressing interleukin-2), systemic anti-programmed cell death-1 antibody, or combination therapy (vaccinia virus-expressing interleukin-2 and anti-programmed cell death-1), tumor mass, immune cell infiltration, T-cell receptor diversity, and survival were assessed. RESULTS: Intrapleural vaccinia virus resulted in significant tumor regression compared with phosphate-buffered saline control (P < .05). Inclusion of the interleukin-2 transgene further increased intratumoral CD8+ T cells (P < .01) and programmed cell death-1 expression on CD8+ tumor-infiltrating lymphocytes (P < .001). Intrapleural vaccinia virus-expressing interleukin-2 was superior to systemic vaccinia virus-expressing interleukin-2, with reduced tumor burden (P < .0001) and improved survival (P < .05). Intrapleural vaccinia virus-expressing interleukin-2 alone or combined treatment with systemic anti-programmed cell death-1 reduced tumor burden (P < .01), improved survival (P < .01), and increased intratumoral αß T-cell receptor diversity (P < .05) compared with systemic anti-programmed cell death-1 monotherapy. CONCLUSIONS: Intrapleural vaccinia virus-expressing interleukin-2 reduced tumor burden and enhanced survival in a murine malignant pleural disease model. Increased CD8+ tumor-infiltrating lymphocytes and αß T-cell receptor diversity are associated with enhanced response. Clinical trials will enable assessment of intrapleural vaccinia virus-expressing interleukin-2 therapy in patients with malignant pleural disease.

Baseline Plasma Inflammatory Profile Is Associated With Response to Neoadjuvant Chemotherapy in Patients With Pancreatic Adenocarcinoma.

Despite its increased application in pancreatic ductal adenocarcinoma (PDAC), complete response to neoadjuvant therapy (NAT) is rare. Given the critical role of host immunity in regulating cancer, we sought to correlate baseline inflammatory profiles to significant response to NAT. PDAC patients receiving NAT were classified as responders (R) or nonresponders (NR) by carbohydrate antigen 19-9 response, pathologic tumor size, and lymph node status in the resected specimen. Baseline (treatment-naive) plasma was analyzed to determine levels of 27 inflammatory mediators. Logistic regression was used to correlate individual mediators with response. Network analysis and Pearson correlation maps were derived to determine baseline inflammatory mediator profiles. Forty patients (20R and 20NR) met study criteria. The R showed significantly higher overall survival (59.4 vs. 21.25 mo, P=0.002) and disease-free survival (50.97 vs. 10.60 mo, P=0.005), compared with NR. soluble interleukin-2 receptor alpha was a significant predictor of no response to NAT (P=0.045). Analysis of inflammatory profiles using the Pearson heat map analysis followed by network analysis depicted increased inflammatory network complexity in NR compared with R (1.69 vs. 1), signifying a more robust baseline inflammatory status of NR. A panel of inflammatory mediators identified by logistic regression and Fischer score analysis was used to create a potential decision tree to predict NAT response. We demonstrate that baseline inflammatory profiles are associated with response to NAT in PDAC, and that an upregulated inflammatory status is associated with a poor response to NAT. Further analysis into the role of inflammatory mediators as predictors of chemotherapy response is warranted.

Defining best practices for tissue procurement in immuno-oncology clinical trials: consensus statement from the Society for Immunotherapy of Cancer Surgery Committee.

Immunotherapy is now a cornerstone for cancer treatment, and much attention has been placed on the identification of prognostic and predictive biomarkers. The success of biomarker development is dependent on accurate and timely collection of biospecimens and high-quality processing, storage and shipping. Tumors are also increasingly used as source material for the generation of therapeutic T cells. There have been few guidelines or consensus statements on how to optimally collect and manage biospecimens and source material being used for immunotherapy and related research. The Society for Immunotherapy of Cancer Surgery Committee has brought together surgical experts from multiple subspecialty disciplines to identify best practices and to provide consensus on how best to access and manage specific tissues for immuno-oncology treatments and clinical investigation. In addition, the committee recommends early integration of surgeons and other interventional physicians with expertise in biospecimen collection, especially in clinical trials, to optimize the quality of tissue and the validity of correlative clinical studies in cancer immunotherapy.

HMGB1 Promotes Myeloid Egress and Limits Lymphatic Clearance of Malignant Pleural Effusions.

Pleural effusions, when benign, are attributed to cardiac events and suffusion of fluid within the pleural space. When malignant, lymphatic obstruction by tumor and failure to absorb constitutively produced fluid is the predominant formulation. The prevailing view has been challenged recently, namely that the lymphatics are only passive vessels, carrying antigenic fluid to secondary lymphoid sites. Rather, lymphatic vessels can be a selective barrier, efficiently coordinating egress of immune cells and factors within tissues, limiting tumor spread and immune pathology. An alternative explanation, offered here, is that damage associated molecular pattern molecules, released in excess, maintain a local milieu associated with recruitment and retention of immune cells associated with failed lymphatic clearance and functional lymphatic obstruction. We found that levels of high mobility group box 1 (HMGB1) were equally elevated in both benign and malignant pleural effusions (MPEs) and that limited diversity of T cell receptor expressing gamma and delta chain were inversely associated with these levels in MPEs. Acellular fluid from MPEs enhanced γδ T cell proliferation in vitro, while inhibiting cytokine production from γδ T cells and monocytes as well as restricting monocyte chemotaxis. Novel therapeutic strategies, targeting HMGB1 and its neutralization in such effusions as well as direct delivery of immune cells into the pleural space to reconstitute normal physiology should be considered.

A Randomized Phase II Preoperative Study of Autophagy Inhibition with High-Dose Hydroxychloroquine and Gemcitabine/Nab-Paclitaxel in Pancreatic Cancer Patients.

PURPOSE: We hypothesized that autophagy inhibition would increase response to chemotherapy in the preoperative setting for patients with pancreatic adenocarcinoma. We performed a randomized controlled trial to assess the autophagy inhibitor hydroxychloroquine in combination with gemcitabine and nab-paclitaxel. PATIENTS AND METHODS: Participants with potentially resectable tumors were randomized to two cycles of nab-paclitaxel and gemcitabine (PG) alone or with hydroxychloroquine (PGH), followed by resection. The primary endpoint was histopathologic response in the resected specimen. Secondary clinical endpoints included serum CA 19-9 biomarker response and margin negative R0 resection. Exploratory endpoints included markers of autophagy, immune infiltrate, and serum cytokines. RESULTS: Thirty-four patients in the PGH arm and 30 in the PG arm were evaluable for the primary endpoint. The PGH arm demonstrated statistically improved Evans grade histopathologic responses (P = 0.00016), compared with control. In patients with elevated CA 19-9, a return to normal was associated with improved overall and recurrence-free survival (P < 0.0001). There were no differences in serious adverse events between arms and chemotherapy dose number was equivalent. The PGH arm had greater evidence of autophagy inhibition in their resected specimens (increased SQSTM1, P = 0.027, as well as increased immune cell tumor infiltration, P = 0.033). Overall survival (P = 0.59) and relapse-free survival (P = 0.55) did not differ between the two arms. CONCLUSIONS: The addition of hydroxychloroquine to preoperative gemcitabine and nab-paclitaxel chemotherapy in patients with resectable pancreatic adenocarcinoma resulted in greater pathologic tumor response, improved serum biomarker response, and evidence of autophagy inhibition and immune activity.

Prognostic Value of the Systemic Immune-Inflammation Index (SII) After Neoadjuvant Therapy for Patients with Resected Pancreatic Cancer.

BACKGROUND: The systemic immune-inflammation index (SII), calculated using absolute platelet, neutrophil, and lymphocyte counts, has recently emerged as a predictor of survival for patients with pancreatic ductal adenocarcinoma (PDAC) when assessed at diagnosis. Neoadjuvant therapy (NAT) is increasingly used in the treatment of PDAC. However, biomarkers of response are lacking. This study aimed to determine the prognostic significance of SII before and after NAT and its association with the pancreatic tumor biomarker carbohydrate-antigen 19-9 (CA 19-9). METHODS: This study retrospectively analyzed all PDAC patients treated with NAT before pancreatic resection at a single institution between 2007 and 2017. Pre- and post-NAT lab values were collected to calculate SII. Absolute pre-NAT, post-NAT, and change in SII after NAT were evaluated for their association with clinical outcomes. RESULTS: The study analyzed 419 patients and found no significant correlation between pre-NAT SII and clinical outcomes. Elevated post-NAT SII was an independent, negative predictor of overall survival (OS) when assessed as a continuous variable (hazard ratio [HR], 1.0001; 95% confidence interval [CI] 1.00003-1.00014; p = 0.006). Patients with a post-NAT SII greater than 900 had a shorter median OS (31.9 vs 26.1 months; p = 0.050), and a post-NAT SII greater than 900 also was an independent negative predictor of OS (HR, 1.369; 95% CI 1.019-1.838; p = 0.037). An 80% reduction in SII independently predicted a CA 19-9 response after NAT (HR, 4.22; 95% CI 1.209-14.750; p = 0.024). CONCLUSION: Post-treatment SII may be a useful prognostic marker in PDAC patients receiving NAT.

Different measures of HMGB1 location in cancer immunology.

HMGB1 is the most abundant non-histone nuclear protein. It regulates transcriptional access to open areas of chromatin and limits release of DNA with apoptotic death, serving to both inhibit apoptosis and promote DNA repair. When HMGB1 is translocated to the cytosol with many types of cellular stress, it is a powerful inducer of autophagy. It can also be released by activated immune cells and damaged or dying cells into the extracellular space, where it acts as a damage associated molecular pattern (DAMP) molecule, contributing to the pathogenesis and progression of cancer. Here, the most common methodologies to not only measure HMGB1 but also to effectively determine its subcellular localization, which dictates many of HMGB1's different functions, are reviewed.

The platelet NLRP3 inflammasome is upregulated in a murine model of pancreatic cancer and promotes platelet aggregation and tumor growth.

Platelets are activated in solid cancers, including pancreatic ductal adenocarcinoma (PDA), a highly aggressive malignancy with a devastating prognosis and limited therapeutic options. The mechanisms by which activated platelets regulate tumor progression are poorly understood. The nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3) inflammasome is a key inflammatory mechanism recently identified in platelets, which controls platelet activation and aggregation. In an orthotopic PDA mouse model involving surgical implantation of Panc02 murine cancer cells into the tail of the pancreas, we show that the NLRP3 inflammasome in circulating platelets is upregulated in pancreatic cancer. Pharmacological inhibition or genetic ablation of NLRP3 in platelets resulted in decreased platelet activation, platelet aggregation, and tumor progression. Moreover, interfering with platelet NLRP3 signaling significantly improved survival of tumor-bearing mice. Hence, the platelet NLRP3 inflammasome plays a critical role in PDA and might represent a novel therapeutic target.

Making cold malignant pleural effusions hot: driving novel immunotherapies.

Malignant pleural effusions, arising from either primary mesotheliomas or secondary malignancies, heralds advanced disease and poor prognosis. Current treatments, including therapeutic thoracentesis and tube thoracostomy, are largely palliative. The immunosuppressive environment within the pleural cavity includes myeloid derived suppressor cells, T-regulatory cells, and dysfunctional T cells. The advent of effective immunotherapy with checkpoint inhibitors and adoptive cell therapies for lung cancer and other malignancies suggests a renewed examination of local and systemic therapies for this malady. Prior strategies reporting remarkable success, including instillation of the cytokine interleukin-2, perhaps coupled with checkpoint inhibitors, should be further evaluated in the modern era.

Enhanced Neutrophil Extracellular Trap Formation in Acute Pancreatitis Contributes to Disease Severity and Is Reduced by Chloroquine.

Background: Neutrophil extracellular traps (NETs) are generated when activated neutrophils, driven by PAD4, release their DNA, histones, HMGB1, and other intracellular granule components. NETs play a role in acute pancreatitis, worsening pancreatic inflammation, and promoting pancreatic duct obstruction. The autophagy inhibitor chloroquine (CQ) inhibits NET formation; therefore, we investigated the impact of CQ mediated NET inhibition in murine models of pancreatitis and human correlative studies. Methods: L-arginine and choline deficient ethionine supplemented (CDE) diet models of acute pancreatitis were studied in wild type and PAD4-/- mice, incapable of forming NETs. Isolated neutrophils were stimulated to induce NET formation and visualized with fluorescence microscopy. CQ treatment (0.5 mg/ml PO) was initiated after induction of pancreatitis. Biomarkers of NET formation, including cell-free DNA, citrullinated histone H3 (CitH3), and MPO-DNA conjugates were measured in murine serum and correlative human patient serum samples. Results: We first confirmed the role of NETs in the pathophysiology of acute pancreatitis by demonstrating that PAD4-/- mice had decreased pancreatitis severity and improved survival compared to wild-type controls. Furthermore, patients with severe acute pancreatitis had elevated levels of cell-free DNA and MPO-DNA conjugates, consistent with NET formation. Neutrophils from mice with pancreatitis were more prone to NET formation and CQ decreased this propensity to form NETs. CQ significantly reduced serum cell-free DNA and citrullinated histone H3 in murine models of pancreatitis, increasing survival in both models. Conclusions: Inhibition of NETs with CQ decreases the severity of acute pancreatitis and improves survival. Translating these findings into clinical trials of acute pancreatitis is warranted.

TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia.

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3-/-) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1ß (IL-1ß), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3-/- mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.

Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps.

BACKGROUND: The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis. METHODS: Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated. RESULTS: The addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE. CONCLUSION: NETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer. TRIAL REGISTRATION: This study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013).

The NLRP3 inflammasome and bruton's tyrosine kinase in platelets co-regulate platelet activation, aggregation, and in vitro thrombus formation.

Cleavage of interleukin-1ß (IL-1ß) is a key inflammatory event in immune cells and platelets, which is mediated by nucleotide-binding domain leucine rich repeat containing protein (NLRP3)-dependent activation of caspase-1. In immune cells, NLRP3 and caspase-1 form inflammasome complexes with the adaptor proteins apoptosis-associated speck-like protein containing a CARD (ASC) and bruton's tyrosine kinase (BTK). In platelets, however, the regulatory triggers and the functional effects of the NLRP3 inflammasome are unknown. Here, we show in vitro that the platelet NLRP3 inflammasome contributes to platelet activation, aggregation, and thrombus formation. NLRP3 activity, as monitored by caspase-1 activation and cleavage and secretion of IL-1ß, was upregulated in activated platelets, which was dependent on platelet BTK. Pharmacological inhibition or genetic ablation of BTK in platelets led to decreased platelet activation, aggregation, and in vitro thrombus formation. We identify a functionally relevant link between BTK and NLRP3 in platelets, with potential implications in disease states associated with abnormal coagulation and inflammation.