Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Lund University

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

Many bacterial pathogens have developed methods to overcome the defences of the host innate immune system. One such defence is the release of antimicrobial peptides (AMPs). Histones have been found to function as AMPs, in addition to their main biological function of packaging and organising DNA into nucleosomes. In this study, the Gram-positive anaerobic coccus Finegoldia magna was found to bind histones by Western blot and immunoprecipitation analysis. F. magna, which is normally a commensal of the skin and mucous membranes, is also known to act as an opportunistic pathogen and has been isolated from various clinical infection sites. It was found to bind to histones extracted from human skin epidermis through its surface and extracellular adhesion protein FAF. Through FAF binding, F. magna was protected from histone bactericidal activity. Furthermore, the histones were found to be degraded by SufA, a subtilisin-like extracellular serine protease of F. magna. Hence, the results of the present study will give more insight into how F. magna persists both as a commensal organism at the basement membrane of the skin and as an opportunistic pathogen during infection.

FAF and SufA: proteins of Finegoldia magna that modulate the antibacterial activity of histones.

The complement system is activated in response to tissue injury. During wound healing, complement activation seems beneficial in acute wounds but may be detrimental in chronic wounds. We found that the epidermal expression of many complement components was only increased to a minor extent in skin wounds in vivo and in cultured keratinocytes after exposure to supernatant from stimulated mononuclear cells. In contrast, the epidermal expression of complement components was downregulated in ex vivo injured skin lacking the stimulation from infiltrating inflammatory cells but with intact injury-induced epidermal growth factor receptor (EGFR)-mediated growth factor response. In cultured primary keratinocytes, stimulation with the potent EGFR ligand, TGF-α, yielded a significant downregulation of complement component expression. Indeed, EGFR inhibition significantly enhanced the induction of complement components in keratinocytes and epidermis following stimulation with proinflammatory cytokines. Importantly, EGFR inhibition of cultured keratinocytes either alone or in combination with proinflammatory stimulus promoted activation of the complement system after incubation with serum. In keratinocytes treated solely with the EGFR inhibitor, complement activation was dependent on serum-derived C1q, whereas in keratinocytes stimulated with a combination of proinflammatory cytokines and EGFR inhibition, complement activation was found even with C1q-depleted serum. In contrast to human keratinocytes, EGFR inhibition did not enhance complement component expression or cause complement activation in murine keratinocytes. These data demonstrate an important role for EGFR in regulating the expression of complement components and complement activation in human epidermis and keratinocytes and, to our knowledge, identify for the first time a pathway important for the epidermal regulation of complement activation. 

The epidermal growth factor receptor is a regulator of epidermal complement component expression and complement activation.

Neutrophils are essential for host defense at the oral mucosa and neutropenia or functional neutrophil defects lead to disordered oral homeostasis. We found that neutrophils from the oral mucosa harvested from morning saliva had released neutrophil extracellular traps (undergone NETosis) in vivo. The NETosis was mediated through intracellular signals elicited by binding of sialyl Lewis(X) present on salival mucins to l-selectin on neutrophils. This led to rapid loss of nuclear membrane and intracellular release of granule proteins with subsequent neutrophil extracellular trap (NET) release independent of elastase and reduced NAD phosphate-oxidase activation. The saliva-induced NETs were more DNase-resistant and had higher capacity to bind and kill bacteria than NETs induced by bacteria or by phorbol-myristate acetate. Furthermore, saliva/sialyl Lewis(X) mediated signaling enhanced intracellular killing of bacteria by neutrophils. Saliva from patients with aphthous ulcers and Behçet disease prone to oral ulcers failed to induce NETosis, but for different reasons it demonstrated that disordered homeostasis in the oral cavity may result in deficient saliva-mediated NETosis. 

A novel mechanism for NETosis provides antimicrobial defense at the oral mucosa.

Sepsis 2016 Agra, India. Agra, India. 5-6 February 2016.

Streptococcal pharyngitis is among the most common bacterial infections, but the molecular mechanisms involved remain poorly understood. Here we investigate the interactions among three major players in streptococcal pharyngitis: streptococci, plasma, and saliva. We find that saliva activates the plasma coagulation system through both the extrinsic and the intrinsic pathways, entrapping the bacteria in fibrin clots. The bacteria escape the clots by activating host plasminogen. Our results identify a potential function for the intrinsic pathway of coagulation in host defense and a corresponding role for fibrinolysis in streptococcal immune evasion.

Saliva-Induced Clotting Captures Streptococci: Novel Roles for Coagulation and Fibrinolysis in Host Defense and Immune Evasion.

Neutrophil extracellular traps (NETs) that are extensive webs of DNA covered with antimicrobial proteins into the extracellular environment during infection or inflammation as a part of their defense arsenal. Image acquisition of fluorescently labeled NETs and subsequent image-based quantification is frequently used to analyze NET formation (NETosis) in response to various stimuli. However, there are important limitations in the present methods for quantification. Manual methods tend to be error-prone, tedious, and often quite subjective, whereas the software-rooted options are either semi-automatic or difficult to operate. Here, we present an automated and uncomplicated approach for quantifying NETs from fluorescence images, built as a freely available app for MATLAB. It is based on detection of a set of clearly defined parameters, all related to the biological manifestation of NETs and allowing for single-cell resolution quantification and analysis.

NETQUANT: Automated Quantification of Neutrophil Extracellular Traps.

Neutrophils are crucial mediators of host defense that are recruited to the central nervous system (CNS) in large numbers during acute bacterial meningitis caused by Streptococcus pneumoniae. Neutrophils release neutrophil extracellular traps (NETs) during infections to trap and kill bacteria. Intact NETs are fibrous structures composed of decondensed DNA and neutrophil-derived antimicrobial proteins. Here we show NETs in the cerebrospinal fluid (CSF) of patients with pneumococcal meningitis, and their absence in other forms of meningitis with neutrophil influx into the CSF caused by viruses, Borrelia and subarachnoid hemorrhage. In a rat model of meningitis, a clinical strain of pneumococci induced NET formation in the CSF. Disrupting NETs using DNase I significantly reduces bacterial load, demonstrating that NETs contribute to pneumococcal meningitis pathogenesis in vivo. We conclude that NETs in the CNS reduce bacterial clearance and degrading NETs using DNase I may have significant therapeutic implications.

Tirthankar Mohanty

Department of Clinical Sciences,
Division of Infection Medicine,
Department of Clinical Sciences, Division of Infection Medicine

Lund University

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Tirthankar Mohanty

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Clinical Sciences,Division of Infection Medicine,Department of Clinical Sciences, Division of Infection Medicine

Lund University

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Department of Clinical Sciences,
Division of Infection Medicine,
Department of Clinical Sciences, Division of Infection Medicine