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Neutrophils are the most abundant circulating and first-responding innate myeloid cells and have so far been underestimated in the context of multiple sclerosis (MS). MS is the most frequent, immune-mediated, inflammatory disease of the central nervous system. MS is treatable but not curable and its cause(s) and pathogenesis remain elusive. The involvement of neutrophils in MS pathogenesis has been suggested by the use of preclinical animal disease models, as well as on the basis of patient sample analysis. In this review, we provide an overview of the possible mechanisms and functions by which neutrophils may contribute to the development and pathology of MS. Neutrophils display a broad variety of effector functions enabling disease pathogenesis, including (1) the release of inflammatory mediators and enzymes, such as interleukin-1β, myeloperoxidase and various proteinases, (2) destruction and phagocytosis of myelin (as debris), (3) release of neutrophil extracellular traps, (4) production of reactive oxygen species, (5) breakdown of the blood–brain barrier and (6) generation and presentation of autoantigens. An important question relates to the issue of whether neutrophils exhibit a predominantly proinflammatory function or are also implicated in the resolution of chronic inflammatory responses in MS.

Proteolysis is a crucial process in life, tightly controlled by numerous natural protease inhibitors. In human blood, alpha-2-macroglobulin is an emergency protease inhibitor preventing coagulation and damage to endothelia and leukocytes. With the use of a unique protease trapping mechanism, alpha-2-macroglobulin lures active proteases into its snap-trap, shields these from potential substrates and ‘flags’ their complex for elimination by receptor-mediated endocytosis. Matrix metalloprotease-9/gelatinase B is a secreted protease increased in blood of patients with inflammations, vascular disorders and cancers. Matrix metalloprotease-9 occurs as monomers and stable homotrimers, but the reason for their co-existence remains obscure. We discovered that matrix metalloprotease-9 homotrimers undergo reduced anti-proteolytic regulation by alpha-2-macroglobulin and are able to travel as a proteolytically active hitchhiker on alpha-2-macroglobulin. As a comparison, we revealed that monomeric active matrix metalloprotease-9 is efficiently trapped by human plasma alpha-2-macroglobulin and this masks the detection of activated matrix metalloprotease-9 with standard analysis techniques. In addition, we show that alpha-2-macroglobulin/trimer complexes escape clearance through the receptor low-density lipoprotein receptor-related protein 1, also known as the alpha-2-macroglobulin receptor. Thus, the biochemistry and biology of matrix metalloprotease-9 monomers and trimers are completely different as multimerization enables active matrix metalloprotease-9 to partially avoid alpha-2-macroglobulin regulation both by direct protease inhibition and by removal from the extracellular space by receptor-mediated endocytosis. Finally, for the biomarker field, the analysis of alpha-2-macroglobulin/protease complexes with upgraded technology is advocated as a quotum for protease activation in human plasma samples.

Neutrophil extracellular traps (NETs) composed of DNA decorated with histones and proteases trap and kill bacteria but also injure host tissue. Here we show that during a bloodstream infection with methicillin-resistant Staphylococcus aureus , the majority of bacteria are sequestered immediately by hepatic Kupffer cells, resulting in transient increases in liver enzymes, focal ischaemic areas and a robust neutrophil infiltration into the liver. The neutrophils release NETs into the liver vasculature, which remain anchored to the vascular wall via von Willebrand factor and reveal significant neutrophil elastase (NE) proteolytic activity. Importantly, DNase although very effective at DNA removal, and somewhat effective at inhibiting NE proteolytic activity, fails to remove the majority of histones from the vessel wall and only partly reduces injury. By contrast, inhibition of NET production as modelled by PAD4-deficiency, or prevention of NET formation and proteolytic activity as modelled in NE −/− mice prevent collateral host tissue damage. Neutrophil extracellular traps (NETs) released by neutrophils trap pathogens but may also cause tissue damage. Here the authors show that during systemic Staphylococcus aureus infection NETs anchoring to the vasculature are only partially DNase-sensitive, advocating for better anti-NET therapies.

A review of zymography techniques is presented. Zymography approaches yield valuable information about enzyme forms and localization of activity in tissues or in whole organisms. Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful, but often misinterpreted, tool yielding information on potential hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tissue sections with in situ zymography. In vivo zymography can pinpoint proteolytic activity to sites in an intact organism. Future development of novel substrate probes and improvement in detection and imaging methods will increase the applicability of zymography for (reverse) degradomics studies.

Crohn’s disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) with the involvement of immune cells and molecules, including cytokines, chemokines and proteases. A previous extensive review about the molecular biology of matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs), related to intestinal barrier destruction and restoration functions in IBD, is here complemented with the literature from the last five years. We also compare IBD as a prototypic mucosal inflammation of an epithelial barrier against microorganisms with inflammatory retinopathy as a disease with a barrier dysfunction at the level of blood vessels. Multiple reasons are at the basis of halting clinical trials with monoclonal antibodies against MMP-9 for IBD treatment. These include (i) the absence of a causative role of MMP-9 in the pathology in animal models of IBD, (ii) the fact that endotoxins, crossing the intestinal barrier, induce massive local release of both neutrophil collagenase (MMP-8) and gelatinase B (MMP-9), (iii) insufficient recognition that MMPs modify the activities of cytokines, chemokines and their receptors, (iv) ignorance that MMPs exist as mixtures of proteoforms with different posttranslational modifications and with different specific activities and (v) the fact that MMPs and TIMPs act in an interactive network, possibly having also beneficial effects on IBD evolution. Nevertheless, inhibition of MMPs may be a useful therapeutic approach during specific IBD disease phases or in specific sub-phenotypes. This temporary “window of opportunity” for MMP-9 inhibition may be complemented by a locoregional one, provided that the pharmacological agents are targeted in time to affected tissues, as is achieved in ophthalmological inflammation. Thus, in order to discover spatial and temporal windows of opportunity for MMP inhibition as treatment of IBD, more preclinical work including well controlled animal studies will be further needed. In this respect, MMP-9/NGAL complex analysis in various body compartments is helpful for better stratification of IBD patients who may benefit from anti-MMP-9.

This study describes the transcriptional programming of yolk sac–derived microglia specification in the brain, in which c-kit–positive erythromyeloid cells are further modified into three developmental subpools of microglia progenitors and their microglia differentiation is mediated by the transcription factors Pu.1 and IRF8. Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac has been recognized for some time, their precise precursors and the transcription program that is used are not known. We found that mouse microglia were derived from primitive c-kit + erythromyeloid precursors that were detected in the yolk sac as early as 8 d post conception. These precursors developed into CD45 + c-kit lo CX 3 CR1 − immature (A1) cells and matured into CD45 + c-kit − CX 3 CR1 + (A2) cells, as evidenced by the downregulation of CD31 and concomitant upregulation of F4/80 and macrophage colony stimulating factor receptor (MCSF-R). Proliferating A2 cells became microglia and invaded the developing brain using specific matrix metalloproteinases. Notably, microgliogenesis was not only dependent on the transcription factor Pu.1 (also known as Sfpi), but also required Irf8, which was vital for the development of the A2 population, whereas Myb, Id2, Batf3 and Klf4 were not required. Our data provide cellular and molecular insights into the origin and development of microglia.

Key Points Historically, testing matrix metalloproteinase inhibitors (MMPIs) for the therapy of invasive or metastatic cancers has not yielded the expected beneficial results, but has had a positive effect on the development of MMPIs. However, cancers are genetically unstable and more heterogeneous, which implies that the treatment outcomes are less predictable than in inflammation. In addition, it is not clear whether inflammatory cell infiltrations in cancers have beneficial or detrimental roles for the host. Seminal work on inflammation and vascular MMP biology is reviewed here and indicates a further need to test MMPIs in established and new animal models of inflammation, infection and ischaemia. In analogy with inducible cyclooxygenase as a drug target, inducible MMPs are preferred above constitutive or homeostatic enzymes to target with MMPIs in inflammatory and vascular diseases. In contrast to microbiological enzyme targets, MMPs are host enzymes. This implies that with MMPI treatment, normal physiological functions will be blocked with subsequent side effects. Such side effects are, however, tolerable in life-saving situations. In acute life-threatening inflammation, oral use and high selectivity of MMPIs — set as unreached double challenges in the development of MMPIs for cancer treatment — are not necessary. This implies that obligately parenteral drugs, including recombinant proteins (for example, interferon affecting the tissue inhibitor of matrix metalloproteinase (TIMP)/MMP balance) and monoclonal antibodies that inhibit MMPs and peptide MMPIs, are worthwhile to develop and to combine. For chronic inflammatory diseases, the picture is completely different to acute inflammation. High selectivity and oral availability are in demand. These diseases constitute important targets for the pharmaceutical industry. Therefore, major basic and animal research efforts need to be continued and extended. For vascular diseases, the primary question of whether to use MMPIs relates to the aetiology of the pathology and the localization of the injury. Acute ischaemic insults might benefit from MMPI treatment by the rescue of tissue in the penumbra zones. Long-term treatment of chronic ischaemia with MMPIs will necessitate the development of novel oral, highly selective MMPIs. Studies with Mmp -knockout mice contribute to novel insights and are a reasonable indicator to discover whether selective MMPIs have potential benefit. However, acute and chronic inflammatory and vascular animal models have not been sufficiently studied, yielded sometimes discrepant data and are prone to compensatory mechanisms and strain-specific differences. The development of inducible and multiple Mmp -knockout mice, studies of various strain backcrosses and better complementation of data might overcome some of these problems. From animal models of organ-specific and tissue-specific acute inflammation and ischaemia the general view is that specific MMPs dominate in the pathogenesis, for example, MMP12 in lung emphysema and MMP9 in acute ischaemia and multiple sclerosis. MMP9 has been studied most as an inducible drug target and many (unspecific) inhibitors against MMP9 have been synthesized and are reviewed. Efforts to generate the complete, rather than partial, MMP crystal structures, molecular modelling and empirical inhibitor screening studies need to be combined with extensive animal experiments for the discovery of novel highly specific and therapeutically active inhibitors. For development of synthetic MMPIs, high-affinity zinc-binding groups, mainly hydroxamates, have been preferred. With the discovery of zinc-containing ADAMs (a disintegrin and metalloproteinases) and ADAMTSs (ADAMs with a thrombospondin motif) and their evolving roles in biology and pathology, other determinants for the binding of inhibitors to MMPs, including the active-site pockets and exosites, need to be further investigated. Protein domain specificity, pH dependence of carboxylates and better substrate-based design might be exploited in future directions for MMPI development. Although clinical trials using matrix metalloproteinase inhibitors (MMPIs) for cancer therapy were disappointing, Opdenakker and colleagues discuss how the use of selective MMPIs might lead to new treatments for acute and chronic inflammatory and vascular diseases. Matrix metalloproteinases (MMPs) have outgrown the field of extracellular-matrix biology and have progressed towards being important regulatory molecules in cancer and inflammation. This rise in status was accompanied by the development of various classes of inhibitors. Although clinical trials with synthetic inhibitors for the treatment of cancer were disappointing, recent data indicate that the use of selective inhibitors might lead to new therapies for acute and chronic inflammatory and vascular diseases. In this Review, we compare the major classes of MMP inhibitors and advocate that future drug discovery should be based on crucial insights into the differential roles of specific MMPs in pathophysiology obtained with animal models, including knockout studies.

Herpes simplex virus 2 (HSV-2) remains a significant cause of morbidity and mortality in immunocompromised individuals, despite the availability of effective antivirals. Infections caused by drug-resistant isolates are an emerging concern among these patients. Understanding evolutionary aspects of HSV-2 resistance is crucial for designing improved therapeutic strategies. Here, we characterized 11 HSV-2 isolates recovered from various body sites of a single immunocompromised patient suffering from a primary HSV-2 infection unresponsive to acyclovir and foscarnet. The isolates were analyzed phenotypically and genotypically (Sanger sequencing of viral thymidine kinase and DNA polymerase genes). Viral clone isolations, deep sequencing, viral growth kinetics, and dual infection competition assays were performed retrospectively to assess viral heterogeneity and fitness. Sanger sequencing identified mixed populations of DNA polymerase mutant variants. Viral clones were plaque-purified and genotyped, revealing 17 DNA polymerase mutations (K533E, A606V, C625R, R628C, A724V, S725G, S729N, I731F, Q732R, M789T/K, Y823C, V842M, R847C, F923L, T934A, and R964H) associated with acyclovir and foscarnet resistance. Deep-sequencing of the DNA polymerase detected drug-resistant variants ranging between 1 and 95%, although the first two isolates had a wild-type DNA polymerase. Some mutants showed reduced fitness, evidenced by (i) the frequency of variants identified by deep-sequencing not correlating with the proportion of mutants found by plaque-purification, (ii) loss of the variants upon passaging in cell culture, or (iii) reduced frequencies in competition assays. This study reveals the rapid evolution of heterogeneous drug-resistant HSV-2 populations under antiviral therapy, highlighting the need for alternative treatment options and resistance surveillance, especially in severe infections.

Approximately 80% of neuromyelitis optica spectrum disorder (NMOSD) patients harbor serum anti-aquaporin-4 autoantibodies targeting astrocytes in the CNS. Crucial for NMOSD lesion initiation is disruption of the blood-brain barrier (BBB), which allows the entrance of Abs and serum complement into the CNS and which is a target for new NMOSD therapies. Astrocytes have important functions in BBB maintenance; however, the influence of their loss and the role of immune cell infiltration on BBB permeability in NMOSD have not yet been investigated. Using an experimental model of targeted NMOSD lesions in rats, we demonstrate that astrocyte destruction coincides with a transient disruption of the BBB and a selective loss of occludin from tight junctions. It is noteworthy that BBB integrity is reestablished before astrocytes repopulate. Rather than persistent astrocyte loss, polymorphonuclear leukocytes (PMNs) are the main mediators of BBB disruption, and their depletion preserves BBB integrity and prevents astrocyte loss. Inhibition of PMN chemoattraction, activation, and proteolytic function reduces lesion size. In summary, our data support a crucial role for PMNs in BBB disruption and NMOSD lesion development, rendering their recruitment and activation promising therapeutic targets.

Inflammation and fibrosis are key features of proliferative vitreoretinal disorders. We aimed to define the macrophage phenotype and investigate the role of macrophage-myofibroblast transition (MMT) in the contribution to myofibroblast populations present in epiretinal membranes. Vitreous samples from proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and nondiabetic control patients, epiretinal fibrovascular membranes from PDR patients and fibrocellular membranes from PVR patients, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by ELISA, immunohistochemistry and flow cytometry analysis. Myofibroblasts expressing α-SMA, fibroblast activation protein-α (FAP-α) and fibroblast-specific protein-1 (FSP-1) were present in all membranes. The majority of CD68+ monocytes/macrophages co-expressed the M2 macrophage marker CD206. In epiretinal membranes, cells undergoing MMT were identified by co-expression of the macrophage marker CD68 and myofibroblast markers α-SMA and FSP-1. Further analysis revealed that CD206+ M2 macrophages co-expressed α-SMA, FSP-1, FAP-α and ß-catenin. Soluble (s) CD206 and sFAP-α levels were significantly higher in vitreous samples from PDR and PVR patients than in nondiabetic control patients. The proinflammatory cytokine TNF-α and the hypoxia mimetic agent cobalt chloride induced upregulation of sFAP-α in culture media of Müller cells but not of HRMECs. The NF-ĸß inhibitor BAY11-7085 significantly attenuated TNF-α-induced upregulation of sFAP-α in Müller cells. Our findings suggest that the process of MMT might contribute to myofibroblast formation in epiretinal membranes, and this transition involved macrophages with a predominant M2 phenotype. In addition, sFAP-α as a vitreous biomarker may be derived from M2 macrophages transitioned to myofibroblasts and from Müller cells.