Hisakazu Fujita

Toll-like receptor agonists stimulate human neutrophil migration via activation of mitogen-activated protein kinases

Human neutrophil migratory responses to Toll‐like receptor (TLR) agonists were studied using videomicroscopy. When challenged with lipopolysaccharide (LPS, TLR4 agonist) or N‐palmitoyl‐S‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐(R)‐cysteinyl‐seryl‐(lysyl)(3)‐lysine (P3CSK4, TLR2 agonist), neutrophils displayed enhanced motility, which was found to reflect increased random migration but not directed migration (chemotaxis). Enhanced neutrophil motility was detected within 10 min after stimulation with LPS or P3CSK4, and was sustained for more than 80 min. Stimulation of neutrophils with LPS or P3CSK4 resulted in the activation of extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK), which preceded neutrophil migration. TLR‐mediated neutrophil migration was strongly suppressed by pretreatment of cells with U0126 (MAPK/ERK kinase inhibitor) but not with U0124 (an inactive analogue of U0126) or SB203580 (a p38 MAPK inhibitor), and was almost completely abolished by pretreatment of cells with U0126 and SB203580 in combination. Randomly migrating neutrophils in response to LPS or P3CSK4 displayed directed migration when further challenged with gradient concentrations of N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) or platelet‐activating factor (PAF). These findings indicate that TLR agonists stimulate human neutrophil migration via the activation of ERK and p38 MAPK, and FMLP‐ or PAF‐induced neutrophil chemotaxis is not affected by the pre‐exposure of cells to TLR agonists.

Diagnostic value of hemostatic parameters in bone marrow transplant-associated thrombotic microangiopathy

We investigated hemostatic parameters in a prospective study of 16 patients who received bone marrow transplants (BMT). We found a significant rise in the levels of fibrinogen, plasmin-α2 antiplasmin inhibitor complex, tissue-plasminogen activator·plasminogen activator inhibitor complex (t-PA·PAI), von Willebrand factor antigen, and thrombomodulin on day 14 after transplant compared with values before transplant. Protein C and thrombin-antithrombin III levels did not change significantly. No significant changes in prothrombin time ratio, activated partial thromboplastin time, or protein S were detected. Patients who had grades II–IV graft-versus-host disease (GVHD) (n = 6) showed a significantly higher level of t-PA·PAI on day 14 compared with those with grades 0–I GVHD (n = 10) (P = 0.0062). Three patients with grades II–IV GVHD developed thrombotic microangiopathy (TMA) on days 19, 19 and 62. In these patients, we noted significantly lower levels of fibrinogen (P = 0.0383), and significantly higher levels of t-PA·PAI (P = 0.0008) and thrombomodulin (P = 0.0001) on day 14 compared with those patients who did not develop TMA. These results suggest that prothrombotic states and endothelial damage may be caused by the conditioning regimen and/or acute GVHD during BMT; thrombomodulin values on day 14 post BMT may be useful in surveillance for TMA because of endothelial cell injury.

Calpain‐mediated regulation of the distinct signaling pathways and cell migration in human neutrophils

We studied the mechanisms underlying calpain inhibition‐mediated human neutrophil migration. MAPKs, including ERK, p38, and JNK, MEK1/2, MAPK kinase 3/6 (MKK3/6), PI‐3K/Akt, c‐Raf, and p21‐activated kinase (PAK; an effector molecule of Rac) were rapidly (within 30 s) activated in neutrophils upon exposure to calpain inhibitors (PD150606 and N‐acetyl‐Leu‐Leu‐Nle‐CHO) but not PD145305 (inactive analog of PD150606). Following activation of these pathways, neutrophils displayed active migration (chemotaxis), which was sustained for more than 45 min. The studies with pharmacological inhibitors suggest that calpain inhibition‐mediated neutrophil migration is mediated by activation of MEK/ERK, p38, JNK, PI‐3K/Akt, and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor‐induced phosphorylation of distinct signaling molecules (PAK, c‐Raf, MEK1/2, ERK, MKK3/6, p38, JNK, and Akt) as well as cell migration, suggesting that the PTX‐sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. Differentiated neutrophil‐like HL‐60 cells but not undifferentiated cells displayed cell migration and activation of MAPKs and PI‐3K/Akt on calpain inhibition. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signaling pathways and cell migration in resting neutrophils, and this regulatory system develops during differentiation into mature neutrophils.

Calpain inhibition induces activation of the distinct signalling pathways and cell migration in human monocytes

We have recently reported that constitutively active calpain negatively regulates activation of the distinct signalling pathways and cell migration in human neutrophils. Here, we report that a similar regulatory system is also functioning in human monocytes, but not lymphocytes. Calpain was constitutively active in resting human monocytes, but not lymphocytes. Mitogen‐activated protein kinases, including extracellular signal‐regulated kinase (ERK), p38 and c‐Jun N‐terminal kinase (JNK), phosphatidylinositol 3‐kinase (PI3K)/Akt and p21‐activated kinase (PAK, an effector molecule of Rac) were rapidly (within 1 min) activated in monocytes, but not lymphocytes, upon exposure to calpain inhibitors (PD150606 and N‐acetyl‐Leu‐Leu‐Nle‐CHO), but not PD145305 (the inactive analogue of PD150606). Following activation of these signalling pathways, monocytes displayed active migration within 5 min after exposure to calpain inhibitors, and active migration was sustained for more than 45 min. The micropipette method revealed that calpain inhibition‐mediated monocyte migration was chemotaxis, not random migration. The studies with pharmacological inhibitors suggest that calpain inhibition‐mediated monocyte migration is mediated by activation of ERK, p38, JNK, PI3K/Akt and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor‐induced phosphorylation of distinct signalling molecules (PAK, ERK, p38, JNK and Akt) as well as cell migration, suggesting that the PTX‐sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signalling pathways and cell migration in resting monocytes, but not lymphocytes.

Stimulation of human formyl peptide receptors by calpain inhibitors: homology modeling of receptors and ligand docking simulation.

In the present study, the activity of Topoisomerase IIβ (TopoIIβ) is evaluated during peroxide induced double stranded DNA breaks (DSBs) repair in primary neurons. The results showed that the TopoIIβ levels were enhanced during recovery from peroxide mediated damage (PED) along with Ku70, PARP-1, pol beta, and WRN helicase. Furthermore, siRNA mediated knock-down of TopoIIβ in primary neurons conferred enhanced susceptibility to PED in neurons. DSBs in neurons are repaired through two pathways, one promoted by Ku70, while the other is by PARP-1 dependent manner. Participation of TopoIIβ in both pathways was assessed by analysis of the interaction of TopoIIβ with Ku70 and PARP-1 using co-immunoprecipitation experiments in extracts of neurons under peroxide treatment and recovery. The results of these studies showed a strong interaction of TopoIIβ with Ku70 as well as PARP-1 suggesting that TopoIIβ is associated both in Ku70 and PARP-dependent pathways in DSBs repair in primary neurons. The study has thus established that TopoIIβ is an essential component in DSBs repair in primary neurons in both Ku70 and PARP-1 dependent pathways. We suppose that the interaction of TopoIIβ may provide stabilization of the repair complex, which may assist in maintenance of tensional integrity in genomic DNA.Calpain inhibitors, including peptide aldehydes (N-acetyl-Leu-Leu-Nle-CHO and N-acetyl-Leu-Leu-Met-CHO) and α-mercapto-acrylic acid derivatives (PD150606 and PD151746), have been shown to stimulate phagocyte functions via activation of human formyl peptide receptor (hFPR) and/or hFPR-like 1 (hFPRL1). Using the homology modeling of the receptors and the ligand docking simulation, here we show that these calpain inhibitors could bind to the putative N-formyl-Met-Leu-Phe (fMLF) binding site on hFPR and/or hFPRL1. The studies with HEK-293 cells stably expressing hFPR or hFPRL1 showed that the concentrations of calpain inhibitors required to induce an increase in cytoplasmic free Ca(2+) ([Ca(2+)](i)) was much higher (>100 folds) than those of fMLF and Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm). HEK-293 cells expressing hFPR or hFPRL1 with the mutated fMLF binding site never exhibited the [Ca(2+)](i) response to calpain inhibitors. When the optimal concentrations of each stimulus were used, pretreatment of cells with fMLF or WKYMVm abolished an increase in [Ca(2+)](i) induced by calpain inhibitors as well as the same stimulus, whereas pretreatment of cells with calpain inhibitors significantly suppressed, but never abolished, the [Ca(2+)](i) response induced by fMLF or WKYMVm, suggesting that the binding affinity of the inhibitors to the putative fMLF binding site may be lower than that of fMLF or WKYMVm.

Granulocyte colony-stimulating factor negatively regulates Toll-like receptor agonist-induced cytokine production in human neutrophils.

We studied the effect of G-CSF on TLR agonist-induced cytokine production in human neutrophils. Human neutrophils produced IL-8 and TNF-alpha in response to stimulation with TLR agonists such as LPS and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine. This response was dependent on activation of ERK, p38, and PI3K, but not JNK. TLR agonist-induced cytokine production in neutrophils was inhibited by G-CSF, whereas it was enhanced by GM-CSF, and GM-CSF-mediated enhancement was attenuated by G-CSF. G-CSF and GM-CSF did not affect TLR agonist-induced phosphorylation of ERK, p38, JNK, Akt, and IkappaBalpha. STAT3 activation was much greater in G-CSF-stimulated neutrophils than that in GM-CSF-stimulated cells. G-CSF-mediated STAT3 phosphorylation and inhibition of TLR agonist-induced cytokine production were prevented by pretreatment of cells with AG-490 (JAK2 inhibitor). These findings suggest that G-CSF and GM-CSF exert the opposite effects on TLR agonist-induced cytokine production, and G-CSF negatively regulates TLR agonist-induced cytokine production in neutrophils via activation of STAT3.

Calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of protein kinase A and protein kinase A-mediated stabilization of Mcl-1 and X-linked inhibitor of apoptosis (XIAP)

Human neutrophils underwent spontaneous apoptosis, which was accompanied with proteasome-mediated degradation of Mcl-1 and X-linked inhibitor of apoptosis (XIAP). Calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO) prevented spontaneous neutrophil apoptosis and degradation of Mcl-1 and XIAP, and the effects of calpain inhibitors on neutrophils were resistant to cycloheximide. Calpain inhibitors induced protein kinase A (PKA) activation, which was unaccompanied with an increase in intracellular cyclic AMP. Calpain inhibition-mediated delayed neutrophil apoptosis, stabilization of Mcl-1 and XIAP, and phosphorylation of PKA substrates were suppressed by H-89 (specific PKA inhibitor). These findings suggest that calpain inhibition delays neutrophil apoptosis via cyclic AMP-independent activation of PKA and PKA-mediated stabilization of Mcl-1 and XIAP.

Calpain inhibitors stimulate phagocyte functions via activation of human formyl peptide receptors.

Calpain inhibitors induce pertussis toxin (PTx)-sensitive chemotaxis in human neutrophils and monocytes. Here, we show that various calpain inhibitors (PD150606, PD151746, N-acetyl-Leu-Leu-Nle-CHO [ALLN], N-acetyl-Leu-Leu-Met-CHO [ALLM], and calpeptin) and γ-secretase inhibitor I induced PTx-sensitive increase in cytoplasmic free Ca(2+) ([Ca(2+)](i)) in human neutrophils and neutrophil migration. HEK-293 cells stably expressing human formyl peptide receptor (hFPR) or hFPR-like 1 (hFPRL1) displayed stimulus-specific increase in [Ca(2+)](i) in response to calpain inhibitors (PD150606, PD151746, ALLN, ALLM, MG-132, and calpeptin), γ-secretase inhibitor I, and N-formyl-Met-Leu-Phe. Parent HEK-293 cells also displayed PTx-sensitive increase in [Ca(2+)](i) in response to calpeptin and γ-secretase inhibitor I, whereas they displayed PTx-resistant increase in [Ca(2+)](i) in response to MG-132. MDL-28170 induced neither an increase in [Ca(2+)](i) in neutrophils and HEK-293 cells nor neutrophil migration. Ionomycin-induced cleavage of talin (a substrate of calpain) in neutrophils was inhibited by all inhibitors used here. These findings suggest that potent calpain inhibitors could stimulate phagocyte functions via activation of hFPR, hFPRL1 and/or other G-protein coupled receptors depending on the inhibitors used.

Prediction of treatment responses to neoadjuvant chemotherapy in triple-negative breast cancer by analysis of immune checkpoint protein expression

Background“Avoiding immune destruction” has recently been established as one of the hallmarks of cancer. The programmed cell death (PD)-1/programmed cell death-ligand (PD-L) 1 pathway is an important immunosuppression mechanism that allows cancer cells to escape host immunity. The present study investigated how the expressions of these immune checkpoint proteins affected responses to neo-adjuvant chemotherapy (NAC) in breast cancer.MethodsA total of 177 patients with resectable early-stage breast cancer were treated with NAC. Estrogen receptor, progesteron receptor, human epidermal growth factor receptor 2, Ki67, PD-L1, PDL-2 and PD-1 status were assessed by immunohistochemistry.ResultsThere were 37 (20.9%) patients with high PD-1 expression, 42 (23.7%) patients had high PD-L1 expression, and 52 (29.4%) patients had high PD-L2 expression. The patients with high PD-1 and PD-L1 expressions had a significantly higher rate of triple-negative breast cancer (TNBC) (p = 0.041) (p < 0.001). In TNBC, patients with high PD-1 and PD-L1 expressions had significantly higher rates of non-pCR (p = 0.003) (p < 0.001). Univariate analysis showed that PD-1 and PD-L1 expressions also significantly shortened disease free survival in TNBC (p = 0.048, HR = 3.318) (p = 0.007, HR = 8.375). However, multivariate analysis found that only PD-L1 expression was an independent prognostic factor (p = 0.041, HR = 9.479).ConclusionsPD-1 and PD-L1 expressions may be useful as biomarkers to predict treatment responses to NAC in breast cancer. Above all, PD-L1 expression may also be useful as biomarkers for more effective chemotherapy in TNBC.

Study on the progression types of cancer in patients with breast cancer undergoing eribulin chemotherapy and tumor microenvironment

BackgroundRecently, the concepts of progression due to pre-existing lesions (PPL) and progression due to new metastasis (PNM) have been proposed to differentiate the progression types of treatment-resistant cancers. Previously, the differences between these two progression types did not affect the determination of treatment strategies since both PPL and PNM are classified as progressive disease based on the response evaluation criteria in solid tumors (RECIST) diagnostic criteria. On the other hand, tumor infiltrating lymphocytes (TILs) are effective when used as indicators for monitoring the immune tumor microenvironment (iTME) in the cancer host, and TILs play an important role as biomarkers in predicting prognosis and therapeutic effects. This study focused on the progression types of cancer in patients undergoing eribulin chemotherapy. In addition, the iTME in individuals with PPL and PNM was evaluated using TILs as a marker.MethodsOf the 52 patients with locally advanced or metastatic breast cancer who underwent chemotherapy with eribulin, 40 remained in the study, and 12 patients were dropout cases. The antitumor effect was evaluated based on the RECIST criteria using version 1.1. TILs were defined as the infiltrating lymphocytes within tumor stroma and were expressed in proportion to the field investigated. In PPL cases, the high-TIL group was considered as type I and the low-TIL group was classified as type II. In PNM cases, the high-TIL group was considered as type III and the low-TIL group was classified as type IV.ResultsIn 19 cases, individuals with type I progression had significantly longer progression free survival and overall survival (OS) compared to those with type III progression (p = 0.040, p < 0.001, log-rank). Individuals with type I progression had significantly prolonged survival post progression compared to those with type II progression (p = 0.048, log-rank). A multivariate analysis that validate the effect of OS showed that these were independent factors of good prognosis (p = 0.003; hazard ratio [HR] = 0.065) (p = 0.006; HR = 0.105).ConclusionsThe effects of eribulin chemotherapy suggested that patients with progressive-type breast cancer that proliferates in a good iTME may have a good prognosis.