Neutrophil extracellular traps in CSF and serum of dogs with steroid-responsive meningitis-arteritis.

In steroid-responsive meningitis-arteritis (SRMA), inflammatory dysregulation is driven by neutrophilic granulocytes resulting in purulent leptomeningitis. Neutrophils can generate neutrophil extracellular traps (NET). Uncontrolled NET-formation or impaired NET-clearance evidently cause tissue and organ damage resulting in immune-mediated diseases. The aim of the study was to verify that NET-formation is detectable in ex vivo samples of acute diseased dogs with SRMA by visualizing and measuring NET-markers in serum and cerebrospinal fluid (CSF) samples. CSF-samples of dogs with acute SRMA (n = 5) and in remission (n = 4) were examined using immunofluorescence (IF)-staining of DNA-histone-1-complexes, myeloperoxidase and citrullinated Histone H3 (H3Cit). Immunogold-labeling of H3Cit and neutrophil elastase followed by transmission electron microscopy (TEM) were used to determine ultrastructural NET-formation in the CSF of one exemplary dog. H3Cit-levels and DNase-activity were measured in CSF and serum samples using an H3Cit-ELISA and a DNase-activity-assay, respectively in patients with the following diseases: acute SRMA (n = 34), SRMA in remission (n = 4), bacterial encephalitis (n = 3), meningioma with neutrophilic inflammation (n = 4), healthy dogs (n = 6). NET-formation was detectable with IF-staining in n = 3/5 CSF samples of dogs with acute SRMA but were not detectable during remission. Vesicular NET-formation was detectable in one exemplary dog using TEM. DNase-activity was significantly reduced in dogs suffering from acute SRMA compared to healthy control group (p < 0.0001). There were no statistical differences of H3Cit levels in CSF or serum samples of acute diseased dogs compared to dogs under treatment, dogs suffering from meningioma or bacterial encephalitis or the healthy control group. Our findings demonstrate that NET-formation and insufficient NET-clearance possibly drive the immunologic dysregulation and complement the pathogenesis of SRMA. The detection of NETs in SRMA offers many possibilities to explore the aetiopathogenetic influence of this defence mechanism of the innate immune system in infectious and non-infectious canine neuropathies.

Investigating Chemokine-Matrix Networks in Breast Cancer: Tenascin-C Sets the Tone for CCL2.

Bidirectional dialogue between cellular and non-cellular components of the tumor microenvironment (TME) drives cancer survival. In the extracellular space, combinations of matrix molecules and soluble mediators provide external cues that dictate the behavior of TME resident cells. Often studied in isolation, integrated cues from complex tissue microenvironments likely function more cohesively. Here, we study the interplay between the matrix molecule tenascin-C (TNC) and chemokine CCL2, both elevated in and associated with the progression of breast cancer and playing key roles in myeloid immune responses. We uncover a correlation between TNC/CCL2 tissue levels in HER2+ breast cancer and examine the physical and functional interactions of these molecules in a murine disease model with tunable TNC levels and in in vitro cellular and cell-free models. TNC supported sustained CCL2 synthesis, with chemokine binding to TNC via two distinct domains. TNC dominated the behavior of tumor-resident myeloid cells; CCL2 did not impact macrophage survival/activation whilst TNC facilitated an immune suppressive macrophage phenotype that was not dependent on or altered by CCL2 co-expression. Together, these data map new binding partners within the TME and demonstrate that whilst the matrix exerts transcriptional control over the chemokine, each plays a distinct role in subverting anti-tumoral immunity.

Mitochondrial complex III deficiency drives c-MYC overexpression and illicit cell cycle entry leading to senescence and segmental progeria.

Accumulating evidence suggests mitochondria as key modulators of normal and premature aging, yet whether primary oxidative phosphorylation (OXPHOS) deficiency can cause progeroid disease remains unclear. Here, we show that mice with severe isolated respiratory complex III (CIII) deficiency display nuclear DNA damage, cell cycle arrest, aberrant mitoses, and cellular senescence in the affected organs such as liver and kidney, and a systemic phenotype resembling juvenile-onset progeroid syndromes. Mechanistically, CIII deficiency triggers presymptomatic cancer-like c-MYC upregulation followed by excessive anabolic metabolism and illicit cell proliferation against lack of energy and biosynthetic precursors. Transgenic alternative oxidase dampens mitochondrial integrated stress response and the c-MYC induction, suppresses the illicit proliferation, and prevents juvenile lethality despite that canonical OXPHOS-linked functions remain uncorrected. Inhibition of c-MYC with the dominant-negative Omomyc protein relieves the DNA damage in CIII-deficient hepatocytes in vivo. Our results connect primary OXPHOS deficiency to genomic instability and progeroid pathogenesis and suggest that targeting c-MYC and aberrant cell proliferation may be therapeutic in mitochondrial diseases.

Competitive survival of clonal serial Pseudomonas aeruginosa isolates from cystic fibrosis airways in human neutrophils.

Chronic airway infections with Pseudomonas aeruginosa are the major co-morbidity in most people with cystic fibrosis (CF) sustained by neutrophils as the major drivers of lung inflammation, damage, and remodeling. Phagocytosis assays were performed with clonal consortia of longitudinal P. aeruginosa airway isolates collected from people with CF since the onset of lung colonization until patient's death or replacement by another clone. The extra- and intracellular abundance of individual strains was assessed by deep amplicon sequencing of strain-specific single nucleotide variants in the bacterial genome. The varied microevolution of the accessory genome of the P. aeruginosa clones during mild and severe courses of infection corresponded with a differential persistence of clonal progeny in the neutrophil phagosome. By simultaneously exposing the ancestor and its progeny to the same habitat, the study recapitulated the time lapse of the temporal change of the fitness of the clone to survive in neutrophils.

A redox-dependent thiol-switch and a Ca2+ binding site within the hinge region hierarchically depend on each other in α7ß1 integrin regulation.

Integrin-mediated cell contacts with the extracellular matrix (ECM) are essential for cellular adhesion, force transmission, and migration. Several effectors, such as divalent cations and redox-active compounds, regulate ligand binding activities of integrins and influence their cellular functions. To study the role of the Ca2+ binding site within the hinge region of the integrin α7 subunit, we genetically abrogated it in the α7hiΔCa mutant. This mutant folded correctly, associated with the ß1 subunit and was exposed on the cell surface, but showed reduced ligand binding and weaker cell adhesion to laminin-111. Thus, it resembles the α7hiΔSS mutant, in which the redox-regulated pair of cysteines, closeby to the Ca2+ binding site within the hinge, was abrogated. Comparing both mutants in adhesion strength and cell migration revealed that both Ca2+ complexation and redox-regulation within the hinge interdepend on each other. Moreover, protein-chemical analyses of soluble integrin ectodomains containing the same α7 hinge mutations suggest that integrin activation via the subunit α hinge is primed by the formation of the cysteine pair-based crosslinkage. Then, this allows Ca2+ complexation within the hinge, which is another essential step for integrin activation and ligand binding. Thus, the α hinge is an allosteric integrin regulation site, in which both effectors, Ca2+ and redox-active compounds, synergistically and hierarchically induce far-ranging conformational changes, such as the extension of the integrin ectodomain, resulting in integrin activation of ECM ligand binding and altered integrin-mediated cell functions.

Modulating tenascin-C functions by targeting the MAtrix REgulating MOtif, "MAREMO".

The extracellular matrix molecule Tenascin-C (TNC) promotes cancer and chronic inflammation by multiple mechanisms. Recently, TNC was shown to promote an immune suppressive tumor microenvironment (TME) through binding soluble chemoattracting factors, thus retaining leukocytes in the stroma. TNC also binds to fibronectin (FN) and other molecules, raising the question of a potential common TNC binding mechanism. By sequence comparison of two TNC-interacting domains in FN, the fifth (FN5) and thirteenth (FN13) fibronectin type III domains we identified a MAtrix REgulating MOtif "MAREMO" or M-motif that is highly conserved amongst vertebrates. By sequence analysis, structural modeling and functional analysis we found also putative M-motifs in TNC itself. We showed by negative staining electron microscopic imaging that the M-motif in FN mediates interactions with FN as well as with TNC. We generated two M-motif mimetic peptides P5 and P13 resembling the M-motif in FN5 and FN13, respectively. By using structural information we modelled binding of these M-motif mimetics revealing a putative MAREMO binding site MBS in FN5 and TN3, respectively overlapping with the M-motif. We further demonstrated that the M-motif mimetic peptides blocked several functions of TNC, such as binding of TNC to FN, cell rounding on a mixed FN/TNC substratum, FN matrix expression and subsequent assembly, TNC-induced signaling and gene expression, TNC chemokine binding and dendritic cell retention, thus providing novel opportunities to inhibit TNC actions. Our results suggest that targeting the MAREMO/MBS interaction could be exploited for reducing inflammation and matrix functions in cancer and fibrosis.

Collagen's primary structure determines collagen:HSP47 complex stoichiometry.

Collagens play important roles in development and homeostasis in most higher organisms. In order to function, collagens require the specific chaperone HSP47 for proper folding and secretion. HSP47 is known to bind to the collagen triple helix, but the exact positions and numbers of binding sites are not clear. Here, we employed a collagen II peptide library to characterize high-affinity binding sites for HSP47. We show that many previously predicted binding sites have very low affinities due to the presence of a negatively charged amino acid in the binding motif. In contrast, large hydrophobic amino acids such as phenylalanine at certain positions in the collagen sequence increase binding strength. For further characterization, we determined two crystal structures of HSP47 bound to peptides containing phenylalanine or leucine. These structures deviate significantly from previously published ones in which different collagen sequences were used. They reveal local conformational rearrangements of HSP47 at the binding site to accommodate the large hydrophobic side chain from the middle strand of the collagen triple helix and, most surprisingly, possess an altered binding stoichiometry in the form of a 1:1 complex. This altered stoichiometry is explained by steric collisions with the second HSP47 molecule present in all structures determined thus far caused by the newly introduced large hydrophobic residue placed on the trailing strand. This exemplifies the importance of considering all three sites of homotrimeric collagen as independent interaction surfaces and may provide insight into the formation of higher oligomeric complexes at promiscuous collagen-binding sites.

Complement inhibitor CSMD1 modulates epidermal growth factor receptor oncogenic signaling and sensitizes breast cancer cells to chemotherapy.

BACKGROUND: Human CUB and Sushi multiple domains 1 (CSMD1) is a large membrane-bound tumor suppressor in breast cancer. The current study aimed to elucidate the molecular mechanism underlying the effect of CSMD1 in highly invasive triple negative breast cancer (TNBC). METHODS: We examined the antitumor action of CSMD1 in three TNBC cell lines overexpressing CSMD1, MDA-MB-231, BT-20 and MDA-MB-486, in vitro using scanning electron microscopy, proteome array, qRT-PCR, immunoblotting, proximity ligation assay, ELISA, co-immunoprecipitation, immunofluorescence, tumorsphere formation assays and flow cytometric analysis. The mRNA expression pattern and clinical relevance of CSMD1 were evaluated in 3520 breast cancers from a modern population-based cohort. RESULTS: CSMD1-expressing cells had distinct morphology, with reduced deposition of extracellular matrix components. We found altered expression of several cancer-related molecules, as well as diminished expression of signaling receptors including Epidermal Growth Factor Receptor (EGFR), in CSMD1-expressing cells compared to control cells. A direct interaction of CSMD1 and EGFR was identified, with the EGF-EGFR induced signaling cascade impeded in the presence of CSMD1. Accordingly, we detected increased  ubiquitination levels of EGFR upon activation in CSMD1-expressing cells, as well as increased degradation kinetics and chemosensitivity. Accordingly, CSMD1 expression rendered tumorspheres pretreated with gefitinib more sensitive to chemotherapy. In addition, higher mRNA levels of CSMD1 tend to be associated with better outcome of triple negative breast cancer patients treated with chemotherapy. CONCLUSIONS: Our results indicate that CSMD1 cross-talks with the EGFR endosomal trafficking cascade in a way that renders highly invasive breast cancer cells sensitive to chemotherapy. Our study unravels one possible underlying molecular mechanism of CSMD1 tumor suppressor function and may provide novel avenues for design of better treatment.

Tenascin-C immobilizes infiltrating T lymphocytes through CXCL12 promoting breast cancer progression.

Immune checkpoint therapy, where CD8 tumor infiltrating T lymphocytes (TIL) are reactivated, is a promising anti-cancer treatment approach, yet with low response rates. The extracellular matrix, in particular tenascin-C, may generate barriers for TIL. To investigate this possibility, we used a MMTV-NeuNT and syngeneic mammary gland grafting model derived thereof with engineered tenascin-C levels and observed accumulation of CD8 TIL in tenascin-C-rich stroma. Inhibition studies revealed that tenascin-C induced CXCL12 through TLR4. By binding CXCL12, tenascin-C retained CD8 TIL in the stroma. Blockade of CXCR4, the receptor of CXCL12, enhanced macrophage and CD8 TIL infiltration and reduced tumor growth and subsequent metastasis. Retention of CD8 TIL by tenascin-C/CXCL12 was also observed in human breast cancer by tissue staining. Moreover, whereas high CD8 TIL numbers correlated with longer metastasis-free survival, this was not the case when also tenascin-C and CXCL12 levels were high. Altogether, these results may be useful for improving tumor immunity as diagnostic tool and to formulate a future "TIL-matrix-release-and-reactivate" strategy.

Novel Human Tenascin-C Function-Blocking Camel Single Domain Nanobodies.

The extracellular matrix (ECM) molecule Tenascin-C (TNC) is well-known to promote tumor progression by multiple mechanisms. However, reliable TNC detection in tissues of tumor banks remains limited. Therefore, we generated dromedary single-domain nanobodies Nb3 and Nb4 highly specific for human TNC (hTNC) and characterized the interaction with TNC by several approaches including ELISA, western blot, isothermal fluorescence titration and negative electron microscopic imaging. Our results revealed binding of both nanobodies to distinct sequences within fibronectin type III repeats of hTNC. By immunofluroescence and immunohistochemical imaging we observed that both nanobodies detected TNC expression in PFA and paraffin embedded human tissue from ulcerative colitis, solid tumors and liver metastasis. As TNC impairs cell adhesion to fibronectin we determined whether the nanobodies abolished this TNC function. Indeed, Nb3 and Nb4 restored adhesion of tumor and mesangial cells on a fibronectin/TNC substratum. We recently showed that TNC orchestrates the immune-suppressive tumor microenvironment involving chemoretention, causing tethering of CD11c+ myeloid/dendritic cells in the stroma. Here, we document that immobilization of DC2.4 dendritic cells by a CCL21 adsorbed TNC substratum was blocked by both nanobodies. Altogether, our novel TNC specific nanobodies could offer valuable tools for detection of TNC in the clinical practice and may be useful to inhibit the immune-suppressive and other functions of TNC in cancer and other diseases.

Complement inhibitor factor H expressed by breast cancer cells differentiates CD14+ human monocytes into immunosuppressive macrophages.

Macrophages are a major immune cell type in the tumor microenvironment, where they display a tumor-supporting phenotype. Factor H (FH) is a complement inhibitor that also plays a role in several cellular functions. To date, the phenotype of monocytes stimulated with FH has been unexplored. We discovered that FH is a survival factor for CD14+ primary human monocytes, promoting their differentiation into macrophages in serum-free medium. This activity was localized to the C-terminal domains of FH and it was inhibited in plasma, indicating that the phenomenon may be most relevant in tissues. FH-induced macrophages display characteristics of immunosuppressive cells including expression of CD163 and CD206, release of the anti-inflammatory cytokine IL-10 and changes in metabolism. Furthermore, FH-induced macrophages express low levels of HLA-DR but high levels of co-inhibitory molecule programmed death-ligand 1 (PD-L1), and accordingly, a reduced capacity for T-cell activation. Finally, we show that FH is expressed by human breast cancer cells and that this correlates with the presence of immunosuppressive macrophages, breast cancer recurrence and severity of the disease. We propose that the expression of FH by tumor cells and the promotion of an immunosuppressive cancer microenvironment by this protein should be taken into account when considering the effectiveness of immunotherapies against breast cancer.

Tenascin-C Orchestrates an Immune-Suppressive Tumor Microenvironment in Oral Squamous Cell Carcinoma.

Inherent immune suppression represents a major challenge in the treatment of human cancer. The extracellular matrix molecule tenascin-C promotes cancer by multiple mechanisms, yet the roles of tenascin-C in tumor immunity are incompletely understood. Using a 4NQO-induced oral squamous cell carcinoma (OSCC) model with abundant and absent tenascin-C, we demonstrated that tenascin-C enforced an immune-suppressive lymphoid stroma via CCL21/CCR7 signaling, leading to increased metastatic tumors. Through TLR4, tenascin-C increased expression of CCR7 in CD11c+ myeloid cells. By inducing CCL21 in lymphatic endothelial cells via integrin α9ß1 and binding to CCL21, tenascin-C immobilized CD11c+ cells in the stroma. Inversion of the lymph node-to-tumor CCL21 gradient, recruitment of T regulatory cells, high expression of anti-inflammatory cytokines, and matrisomal components were hallmarks of the tenascin-C-instructed lymphoid stroma. Ablation of tenascin-C or CCR7 blockade inhibited the lymphoid immune-suppressive stromal properties, reducing tumor growth, progression, and metastasis. Thus, targeting CCR7 could be relevant in human head and neck tumors, as high tenascin-C expression and an immune-suppressive stroma correlate to poor patient survival.

Affinity-Enhanced Multimeric VEGF (Vascular Endothelial Growth Factor) and PlGF (Placental Growth Factor) Variants for Specific Adsorption of sFlt-1 to Restore Angiogenic Balance in Preeclampsia.

Preeclampsia is a potentially life-threatening multisystem disease affecting 4% to 8% of pregnant women after the 20th week of gestation. An excess of placental expressed antiangiogenic soluble VEGF (vascular endothelial growth factor)-receptor 1 (soluble FMS-like tyrosine kinase 1) scavenges VEGF and PlGF (placental growth factor), causing generalized endothelial dysfunction. Interventions to restore the angiogenic balance in preeclamptic pregnancies are intensively studied and improve maternal and neonatal outcomes. Especially extracorporeal strategies to remove sFlt-1 are promising in human pregnancy. However, available apheresis systems adsorb sFlt-1 unspecifically and with low efficiency. Affinity-enhanced ligands are needed to improve performance and compatibility of apheresis treatments. Using computerized molecular modeling, we developed multimeric VEGF molecules comprised of single-chain VEGF165 dimers (scVEGF165). A short peptide linker hampers intrachain dimerization to induce assembly preferably as tetrameric molecules as visualized in negative staining electron microscopy. scVEGF165 multimers possess 1.2-fold higher affinity for sFlt-1 as compared to the available antibodies or monomeric VEGF. Consequently, scVEGF multimers have the ability to competitively release sFlt-1 bound PlGF and, in particular, VEGF. In ex vivo adsorption experiments using serum samples from patients with preeclampsia, scVEGF multimers reduce sFlt-1 levels by 85% and increase PlGF and VEGF levels by 20- and 9-fold, respectively. Finally, performance and stability of sFlt-1 capturing scVEGF165 multimers were scrutinized on different matrices of which biocompatible agarose matrix yielded optimal results. We introduce the first VEGF-based highly efficient sFlt-1 apheresis system that is directly applicable in vivo due to utilization of inert agarose matrix, using a homomultimeric form of VEGF165 to restore the angiogenic balance in preeclampsia.

Extracellular Redox Regulation of α7ß Integrin-Mediated Cell Migration Is Signaled via a Dominant Thiol-Switch.

While adhering to extracellular matrix (ECM) proteins, such as laminin-111, cells temporarily produce hydrogen peroxide at adhesion sites. To study the redox regulation of α7ß1 integrin-mediated cell adhesion to laminin-111, a conserved cysteine pair within the α-subunit hinge region was replaced for alanines. The molecular and cellular effects were analyzed by electron and atomic force microscopy, impedance-based migration assays, flow cytometry and live cell imaging. This cysteine pair constitutes a thiol-switch, which redox-dependently governs the equilibrium between an extended and a bent integrin conformation with high and low ligand binding activity, respectively. Hydrogen peroxide oxidizes the cysteines to a disulfide bond, increases ligand binding and promotes cell migration toward laminin-111. Inversely, extracellular thioredoxin-1 reduces the disulfide, thereby decreasing laminin binding. Mutation of this cysteine pair into the non-oxidizable hinge-mutant shows molecular and cellular effects similar to the reduced wild-type integrin, but lacks redox regulation. This proves the existence of a dominant thiol-switch within the α subunit hinge of α7ß1 integrin, which is sufficient to implement activity regulation by extracellular redox agents in a redox-regulatory circuit. Our data reveal a novel and physiologically relevant thiol-based regulatory mechanism of integrin-mediated cell-ECM interactions, which employs short-lived hydrogen peroxide and extracellular thioredoxin-1 as signaling mediators.

Platelet IP6K1 regulates neutrophil extracellular trap-microparticle complex formation in acute pancreatitis.

Platelet inositol hexakisphosphate kinase 1 (IP6K1) has been shown to control systemic inflammation. Herein, we examined if platelets and IP6K1 regulate pancreatic tissue injury via formation of NETs in experimental models of acute pancreatitis (AP) in mice. By use of electron microscopy abundant NET formation was observed in the inflamed pancreas. These NETs contained numerous microparticles (MP) expressing CD41 or Mac-1. Platelet depletion reduced deposition of NET-MP complexes in the inflamed pancreas. Circulating platelet-neutrophil aggregates (PNA) were increased and inhibition of P-selectin not only disrupted PNA formation but also reduced NETs formation in the inflamed pancreas. NETs depleted of MPs had lower capacity to provoke amylase secretion and STAT-3 phosphorylation in acinar cells. Taurocholate-induced NETs formation, inflammation and tissue damage in the pancreas were decreased in IP6K1-deficient mice. Thrombin stimulation of mixtures of wild-type platelets and neutrophils resulted in NETs formation but not when IP6K1-deficient platelets were incubated with wild-type neutrophils. Polyphosphate rescue restored thrombin-induced NET formation in mixtures of IP6K1-deficient platelets and wild-type neutrophils. Platelet IP6K1 regulates NET-MP complex formation in the pancreas of mice during induction of AP. Targeting platelet IP6K1 might useful to decrease NET-dependent pancreatic tissue inflammation and tissue injury in patients with AP.

C-terminal proteolysis of the collagen VI α3 chain by BMP-1 and proprotein convertase(s) releases endotrophin in fragments of different sizes.

The assembly of collagen VI microfibrils is a multistep process in which proteolytic processing within the C-terminal globular region of the collagen VI α3 chain plays a major role. However, the mechanisms involved remain elusive. Moreover, C5, the short and most C-terminal domain of the α3 chain, recently has been proposed to be released as an adipokine that enhances tumor progression, fibrosis, inflammation, and insulin resistance and has been named "endotrophin." Serum endotrophin could be a useful biomarker to monitor the progression of such disorders as chronic obstructive pulmonary disease, systemic sclerosis, and kidney diseases. Here, using biochemical and isotopic MS-based analyses, we found that the extracellular metalloproteinase bone morphogenetic protein 1 (BMP-1) is involved in endotrophin release and determined the exact BMP-1 cleavage site. Moreover, we provide evidence that several endotrophin-containing fragments are present in various tissues and body fluids. Among these, a large C2-C5 fragment, which contained endotrophin, was released by furin-like proprotein convertase cleavage. By using immunofluorescence microscopy and EM, we also demonstrate that these proteolytic maturations occur after secretion of collagen VI tetramers and during microfibril assembly. Differential localization of N- and C-terminal regions of the collagen VI α3 chain revealed that cleavage products are deposited in tissue and cell cultures. The detailed information on the processing of the collagen VI α3 chain reported here provides a basis for unraveling the function of endotrophin (C5) and larger endotrophin-containing fragments and for refining their use as biomarkers of disease progression.

c-Abl kinase regulates neutrophil extracellular trap formation, inflammation, and tissue damage in severe acute pancreatitis.

Neutrophil extracellular traps (NETs) are involved in acute pancreatitis (AP) but mechanisms controlling NET expulsion in AP are incompletely understood. Herein, we examined the role of c-Abelson (c-Abl) kinase in NET formation and tissue damage in severe AP. AP was induced by taurocholate infusion into pancreatic duct or intraperitoneal administration of l-arginine in mice. Pancreatic, lung, and blood samples were collected and levels of phosphorylated c-Abl kinase, citrullinated histone 3, DNA-histone complexes, myeloperoxidase, amylase, cytokines, and CXC chemokines were quantified. Citrullinated histone 3, reactive oxygen species (ROS), and NET formation were determined in bone marrow neutrophils. Taurocholate challenge increased phosphorylation of c-Abl kinase and levels of citrullinated histone 3 in the pancreas as well as DNA-histone complexes in the plasma. Administration of the c-Abl kinase inhibitor GZD824 not only abolished activation of c-Abl kinase but also decreased levels of citrullinated histone 3 in the pancreas and DNA-histone complexes in the plasma of animals with AP. Moreover, GZD824 decreased plasma levels of amylase, IL-6, and MMP-9 as well as edema, acinar cell necrosis, hemorrhage, CXC chemokine formation, and neutrophil infiltration in the inflamed pancreas. A beneficial effect of c-Abl kinase inhibition was confirmed in l-arginine-induced pancreatitis. In vitro, inhibition of c-Abl kinase reduced TNF-α-induced formation of ROS, histone 3 citrullination, and NETs in isolated bone marrow neutrophils. Our findings demonstrate that c-Abl kinase regulates NET formation in the inflamed pancreas. In addition, inhibition of c-Abl kinase reduced pancreatic tissue inflammation, and damage in AP. Thus, targeting c-Abl kinase might be a useful way to protect the pancreas in severe AP.

Targeting peptidylarginine deiminase reduces neutrophil extracellular trap formation and tissue injury in severe acute pancreatitis.

Recent evidence suggests that neutrophil extracellular traps (NETs) play an important role in the development of acute pancreatitis (AP). Herein, we examined the role of peptidylarginine deiminase (PAD), which has been shown to regulate NET formation, in severe AP. AP was induced by retrograde of taurocholate infusion into pancreatic duct in C57BL/6 mice. PAD was pharmacologically inhibited using Cl-amidine, a pan-PAD inhibitor. Pancreata were collected, and histones, citrullinated histone 3, chemokines, myeloperoxidase, and NETs were quantified. Chemokines, matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), and DNA-histone complexes were determined in plasma samples. Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice. Pretreatment with Cl-amidine markedly reduced the NET formation in the inflamed pancreas. Moreover, inhibition of PAD decreased the levels of blood amylase as well as edema, acinar cell necrosis, hemorrhage, and neutrophil infiltration in the pancreas of animals with AP. Administration of Cl-amidine attenuated the myeloperoxidase levels in the pancreas and lung of mice exposed to taurocholate. In addition, Cl-amidine decreased pancreatic levels of CXC chemokines, plasma levels of IL-6, and MMP-9 in mice with severe AP. This study shows that Cl-amidine is a potent inhibitor of NET formation in severe AP. Also, our results suggest that PAD regulates pathological inflammation and tissue damage in the inflamed pancreas. Thus, targeting PAD might be a useful strategy to treat patients with severe AP.

A novel derivative of the fungal antimicrobial peptide plectasin is active against Mycobacterium tuberculosis.

Tuberculosis has been reaffirmed as the infectious disease causing most deaths in the world. Co-infection with HIV and the increase in multi-drug resistant Mycobacterium tuberculosis strains complicate treatment and increases mortality rates, making the development of new drugs an urgent priority. In this study we have identified a promising candidate by screening antimicrobial peptides for their capacity to inhibit mycobacterial growth. This non-toxic peptide, NZX, is capable of inhibiting both clinical strains of M. tuberculosis and an MDR strain at therapeutic concentrations. The therapeutic potential of NZX is further supported in vivo where NZX significantly lowered the bacterial load with only five days of treatment, comparable to rifampicin treatment over the same period. NZX possesses intracellular inhibitory capacity and co-localizes with intracellular bacteria in infected murine lungs. In conclusion, the data presented strongly supports the therapeutic potential of NZX in future anti-TB treatment.