No abstract

Neutrophil heterogeneity was described decades ago, but it could not be elucidated at the time whether the existence of different neutrophil subsets had any biological relevance. It has been corroborated in recent years that neutrophil subsets, defined by differential expression of various markers, are indeed present in human blood, calling for renewed attention to this question. The expression of the granule protein olfactomedin 4 (OLFM4) has been suggested to define two such neutrophil subsets. We confirm the simultaneous presence of one OLFM4-positive and one OLFM4-negative neutrophil subpopulation as well as the localization of the protein to specific granules. In vitro, these neutrophil subsets displayed equal tendency to undergo apoptosis and phagocytose bacteria. In addition, the subpopulations were recruited equally to inflammatory sites in vivo, and this was true both in an experimental model of acute inflammation and in naturally occurring pathological joint inflammation. In line with its subcellular localization, only limited OLFM4 release was seen upon in vivo transmigration, and release through conventional degranulation required strong secretagogues. However, extracellular release of OLFM4 could be achieved upon formation of neutrophil extracellular traps (NETs) where it was detected only in a subset of the NETs. Although we were unable to demonstrate any functional differences between the OLFM4-defined subsets, our data show that different neutrophil subsets are present in inflamed tissue in vivo. Furthermore, we demonstrate NETs characterized by different markers for the first time, and our results open up for functions of OLFM4 itself in the extracellular space through exposure in NETs.

Staphylococcus aureus biofilms, a leading cause of persistent infections, are highly resistant to immune defenses and antimicrobial therapies. In the present study, we investigated the contribution of fibrin and staphylokinase (Sak) to biofilm formation. In both clinical S. aureus isolates and laboratory strains, high Sak-producing strains formed less biofilm than strains that lacked Sak, suggesting that Sak prevents biofilm formation. In addition, Sak induced detachment of mature biofilms. This effect depended on plasminogen activation by Sak. Host-derived fibrin, the main substrate cleaved by Sak-activated plasminogen, was a major component of biofilm matrix, and dissolution of this fibrin scaffold greatly increased susceptibility of biofilms to antibiotics and neutrophil phagocytosis. Sak also attenuated biofilm-associated catheter infections in mouse models. In conclusion, our results reveal a novel role for Sak-induced plasminogen activation that prevents S. aureus biofilm formation and induces detachment of existing biofilms through proteolytic cleavage of biofilm matrix components.

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O2). The consumed O2 is utilized by an NADPH-oxidase to generate highly reactive oxygen species (ROS) by a one electron reduction, initially generating superoxide anion (O2 (-)) that then dismutates to hydrogen peroxide (H2O2). The ROS are strongly bactericidal molecules but may also cause tissue destruction, and are capable of driving immune competent cells of both the innate and the adaptive immune systems into apoptosis. The development of basic techniques to measure/quantify ROS generation by phagocytes during activation of the respiratory burst is of great importance, and a large number of methods have been used for this purpose. A selection of methods, including chemiluminescence amplified by luminol or isoluminol, the absorbance change following reduction of cytochrome c, and the fluorescence increase upon oxidation of PHPA, are described in detail in this chapter with special emphasis on how to distinguish between ROS that are released extracellularly, and those that are retained within intracellular organelles. These techniques can be valuable tools in research spanning from basic phagocyte biology to more clinically oriented research on innate immune mechanisms and inflammation.

Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin α (PSMα) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMα-induced NETs contained the typical protein markers and were able to capture microbes. The PSMα-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMα-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMα peptides, a process that may be of importance for CA-MRSA virulence.