The Neutrophil.

Neutrophils are immune cells with unusual biological features that furnish potent antimicrobial properties. These cells phagocytose and subsequently kill prokaryotic and eukaryotic organisms very efficiently. Importantly, it is not only their ability to attack microbes within a constrained intracellular compartment that endows neutrophils with antimicrobial function. They can unleash their effectors into the extracellular space, where, even post-mortem, their killing machinery can endure and remain functional. The antimicrobial activity of neutrophils must not be misconstrued as being microbe specific and should be viewed more generally as biotoxic. Outside of fighting infections, neutrophils can harness their noxious machinery in other contexts, like cancer. Inappropriate or dysregulated neutrophil activation damages the host and contributes to autoimmune and inflammatory disease. Here we review a number of topics related to neutrophil biology based on contemporary findings.

Extracellular Acidity Reprograms Macrophage Metabolism and Innate Responsiveness.

Although organ hypofunction and immunosuppression are life-threatening features of severe sepsis, the hypofunctioning organs and immune cells usually regain normal functionality if patients survive. Because tissue interstitial fluid can become acidic during the septic response, we tested the hypothesis that low extracellular pH (pHe) can induce reversible metabolic and functional changes in peritoneal macrophages from C57BL/6J mice. When compared with macrophages cultured at normal pHe, macrophages living in an acidic medium used less glucose and exogenous fatty acid to produce ATP. Lactate, glutamine, and de novo-synthesized fatty acids supported ATP production by mitochondria that gained greater mass, maximal oxygen consumption rate, and spare respiratory capacity. The cells transitioned to an M2-like state, with altered immune responses to LPS and slightly decreased phagocytic ability, yet they regained basal energy production, normal mitochondrial function, and proinflammatory responsiveness when neutral pHe was restored. Low pHe induces changes that support macrophage survival while rendering the cells less proinflammatory (more "tolerant") and less able to phagocytose bacteria. Macrophage responses to low interstitial pH may contribute to the reversible organ hypofunction and immunoparalysis noted in many patients with sepsis.

Intracellular and Extracellular Roles of Granzyme K.

Granzymes are a family of serine proteases stored in granules inside cytotoxic cells of the immune system. Granzyme K (GrK) has been only limitedly characterized and knowledge on its molecular functions is emerging. Traditionally GrK is described as a granule-secreted, pro-apoptotic serine protease. However, accumulating evidence is redefining the functions of GrK by the discovery of novel intracellular (e.g. cytotoxicity, inhibition of viral replication) and extracellular roles (e.g. endothelial activation and modulation of a pro-inflammatory immune cytokine response). Moreover, elevated GrK levels are associated with disease, including viral and bacterial infections, airway inflammation and thermal injury. This review aims to summarize and discuss the current knowledge of i) intracellular and extracellular GrK activity, ii) cytotoxic and non-cytotoxic GrK functioning, iii) the role of GrK in disease, and iv) GrK as a potential therapeutic target.

Autoantibodies Targeting Intracellular and Extracellular Proteins in Autoimmunity.

Detecting autoantibodies provides foundational information for the diagnosis of most autoimmune diseases. An important pathophysiological distinction is whether autoantibodies are directed against extracellular or intracellular proteins. Autoantibodies targeting extracellular domains of proteins, such as membrane receptors, channels or secreted molecules are often directly pathogenic, whereby autoantibody binding to the autoantigen disrupts the normal function of a critical protein or pathway, and/or triggers antibody-dependent cell surface complement killing. By comparison, autoantibodies directed against intracellular proteins are recognized as useful diagnostic biomarkers of abnormal autoimmune activity, but the link between antigenicity and pathogenicity is less straightforward. Because intracellular autoantigens are generally inaccessible to autoantibody binding, for the most part, they do not directly contribute to pathogenesis. In a few diseases, autoantibodies to intracellular targets cause damage indirectly by immune complex formation, immune activation, and other processes. In this review, the general features of and differences between autoimmune diseases segregated on the basis of intracellular or extracellular autoantigens are explored using over twenty examples. Expression profiles of autoantigens in relation to the tissues targeted by autoimmune disease and the temporal appearance of autoantibodies before clinical diagnosis often correlate with whether the respective autoantibodies mostly recognize either intracellular or extracellular autoantigens. In addition, current therapeutic strategies are discussed from this vantage point. One drug, rituximab, depletes CD20+ B-cells and is highly effective for autoimmune disorders associated with autoantibodies against extracellular autoantigens. In contrast, diseases associated with autoantibodies directed predominately against intracellular autoantigens show much more complex immune cell involvement, such as T-cell mediated tissue damage, and require different strategies for optimal therapeutic benefit. Understanding the clinical ramifications of autoimmunity derived by autoantibodies against either intracellular or extracellular autoantigens, or a spectrum of both, has practical implications for guiding drug development, generating monitoring tools, stratification of patient interventions, and designing trials based on predictive autoantibody profiles for autoimmune diseases.

Mechanisms of toxicity mediated by neutrophil and eosinophil granule proteins.

Neutrophils and eosinophils are granulocytes which are characterized by the presence of granules in the cytoplasm. Granules provide a safe storage site for granule proteins that play important roles in the immune function of granulocytes. Upon granulocytes activation, diverse proteins are released from the granules into the extracellular space and contribute to the fight against infections. In this article, we describe granule proteins of both neutrophils and eosinophils able to kill pathogens and review their anticipated mechanism of antimicrobial toxicity. It should be noted that an excess of granules protein release can lead to tissue damage of the host resulting in chronic inflammation and organ dysfunction.

Emerging Roles of Blood-Borne Intact and Respiring Mitochondria as Bidirectional Mediators of Pro- and Anti-Inflammatory Processes.

Over the past two decades, a major goal of our research group has been elucidation of the functional roles of several key regulatory molecules in proinflammatory preconditioning involved in the pathophysiology of seemingly diverse human disease states. By necessity, operational definitions of proinflammation must be intrinsically fluid based on recent advances in our understanding of complex regulation of innate and adaptive immune processes. Similar to systemic acute stress, a physiological proinflammatory state appears to be a key autoregulatory mechanism for maintaining optimal immune surveillance against potentially infective microorganisms, viruses, and toxic xenobiotics. Perturbation of normative biochemical and molecular mosaics of ongoing proinflammatory tone, exemplified by altered expression of pro- and anti-inflammatory cytokines and their respective protein complexes, is hypothesized to be a common modality for initiation and full expression of various autoimmune diseases and comorbid syndromes evolving from metabolic and metastatic diseases. The newly reported presence of "free" (extracellular) mitochondria exponentially adds to our hypothesis that in conditions of acute stress, a new source of potential ATP producers may be recruited and present to deal with such an acute process. Furthermore, given this phenomenon, an early surveillance role and a dysfunctional chronic inflammation-prolonging component may also be surmised.

A gastric cancer cell derived extracellular compounds suppresses CD161+CD3- lymphocytes and aggravates tumor formation in a syngeneic mouse model.

Evasion of the immune system is often associated with malignant tumors. The cancer cell microenvironment plays an important role in tumor progression, but its mechanism is largely unknown. Here we show that an extracellular compound derived from gastric cancer (GC-EC) selectively suppresses CD161+CD3- natural killer (NK) cells. Splenocytes treated with GC-EC showed considerable proliferation and the CD161+CD3- NK cell population was time-dependently suppressed. Intracellular staining of IFN-γ was shown to be down-regulated in concert with granzyme B and perforin. A cytotoxicity assay of splenocytes treated with GC-EC against K-562 cells showed a significant reduction in cytolytic activity. Further, the immune-suppressive effect of GC-EC was more evident in a syngeneic tumor model in C57BL/6 mice. Animals treated with B16 F10 and GC-EC exhibited more aggravated tumor formation than animals treated with B16 F10 only. We demonstrated that inhibition of apoptosis while increasing PI3 K/AKT levels may provoke tumor formation by GC-EC. A cytokine array revealed the presence of several cytokines in GC-EC that negatively regulate immune cytolytic activity and could be potential candidates for immune-suppressive effects.

House dust mite allergens induce interleukin 33 (IL-33) synthesis and release from keratinocytes via ATP-mediated extracellular signaling.

In atopic diseases, the epithelium releases cytokines and chemokines that initiate skin inflammation. Atopic dermatitis (AD) is characterized by a disrupted epidermal barrier and is triggered or exacerbated by environmental stimuli such as house dust mite (HDM) allergens. The proinflammatory cytokine interleukin 33 (IL-33) plays an important role in the pathogenesis of AD, but how IL-33 production in keratinocytes is elicited by HDM is unknown. To that end, here we stimulated monolayer-cultured human keratinocytes and human living skin equivalents with Dermatophagoides pteronyssinus HDM extract to investigate its effects on IL-33 production from keratinocytes. The HDM extract induced intracellular expression of IL-33 and modulated its processing and maturation, triggering rapid IL-33 release from keratinocytes. Group 1 HDM allergen but not group 2 HDM allergen elicited IL-33 production. An ATP assay of keratinocyte culture supernatants revealed an acute and transient accumulation of extracellular ATP immediately after the HDM extract stimulation. Using the broad-spectrum P2 antagonist suramin, the specific purinergic receptor P2Y2 (P2RY2) antagonist AR-C118925XX, and P2RY2-specific siRNA, we discovered that the HDM extract-induced IL-33 expression was mainly dependent on extracellular ATP/P2Y2 signaling mediated by transactivation of epidermal growth factor receptor, followed by activation of the ERK kinase signaling pathway. Moreover, HDM extract-induced release of 25-kDa IL-33 from the keratinocytes depended on an extracellular ATP/P2 signaling-mediated intracellular Ca2+ increase. Our study demonstrates the new mechanism controlling the induction and maturation of keratinocyte-produced IL-33 by HDM allergens, an innate immune process that might play a role in AD development or severity.

A self-assembled peptide hydrogel for cytokine sequestration.

Cytokine-directed monocyte infiltration is involved in multiple pathological processes. Immuno-isolating matrices that can sequester cell-released chemokines in a microenvironment may prolong the viability and functionality of implanted materials. We describe a self-assembling peptide-based hydrogel that can capture the cytokine CCL2 released in the extracellular space by immune cells and stromal cells. The shear-responsive matrix can absorb and retain this signaling molecule needed for the chemotaxis of the infiltrating monocytes and their differentiation into phagocytic macrophages. Such cytokine-sequestering biomaterials may be useful as adjunctive materials with the delivery of exogenous implants or cell suspensions for tissue regeneration, without the administration of systemic immunosuppressants. Our work highlights the versatility of nanofibrous peptide hydrogels for modulating the biological response in tissue niches.

Different measures of HMGB1 location in cancer immunology.

HMGB1 is the most abundant non-histone nuclear protein. It regulates transcriptional access to open areas of chromatin and limits release of DNA with apoptotic death, serving to both inhibit apoptosis and promote DNA repair. When HMGB1 is translocated to the cytosol with many types of cellular stress, it is a powerful inducer of autophagy. It can also be released by activated immune cells and damaged or dying cells into the extracellular space, where it acts as a damage associated molecular pattern (DAMP) molecule, contributing to the pathogenesis and progression of cancer. Here, the most common methodologies to not only measure HMGB1 but also to effectively determine its subcellular localization, which dictates many of HMGB1's different functions, are reviewed.

Assessment of annexin A1 release during immunogenic cell death.

The protein annexin A1 (ANXA1) belongs to the danger-associated molecular patterns (DAMPs) that alert the innate immune system about tissue perturbations. In the context of immunogenic cell death (ICD), ANXA1 is released from the cytoplasm of dying cells and, once extracellular, acts on formyl peptide receptor 1 (FPR1) expressed on dendritic cells to favor long-term interactions between dying and dendritic cells. As a result, the accumulation of extracellular ANXA1 constitutes one of the hallmarks of ICD. In the past, the detection of ANXA1 was based on semiquantitative immunoblots. More recently, a commercial enzyme-linked immunosorbent assay (ELISA) has been developed to measure ANXA1 in an accurate fashion. Here, we detail the protocol to measure the concentration of ANXA1 in the supernatants of cancer cells treated with chemotherapy.

Luciferase-assisted detection of extracellular ATP and ATP metabolites during immunogenic death of cancer cells.

The efficacy of cancer chemotherapy is enhanced by induction of sustainable anti-tumor immune responses. Such responses involve accumulation of immunogenic mediators, such as extracellular ATP and ATP metabolites, within the tumor microenvironment. Recent studies have identified nucleotide-permeable plasma membrane channels or pores that are activated as early downstream consequences of different regulated cell death pathways: pannexin-1 channels in apoptosis, MLKL pores in necroptosis, and gasdermin-family pores in pyroptosis. This chapter describes the use of highly quantitative and semi-high-throughput methods based on the ATP sensor luciferase to measure dynamic changes in extracellular ATP, ADP, and AMP in tissue/cell culture models of cancer cells during various modes of regulated cell death in response to chemotherapeutic drugs, death receptors, or metabolic perturbation.

Mesothelial cell CSF1 sustains peritoneal macrophage proliferation.

Macrophages play a central role during infection, inflammation and tissue homeostasis maintenance. Macrophages have been identified in all organs and their core transcriptomic signature and functions differ from one tissue to another. Interestingly, macrophages have also been identified in the peritoneal cavity and these cells have been extensively used as a model for phagocytosis, efferocytosis and polarization. Peritoneal macrophages are involved in B-cell IgA production, control of inflammation and wound healing following thermal-induced liver surface injury. These cells presumably require and interact with the omentum, where milky spot stromal cells have been proposed to secrete CSF1 (colony stimulating factor 1). Peritoneal macrophages depend on CSF1 for their generation and survival, but the identity of CSF1 producing cells inside the large peritoneal cavity remains unknown. Here we investigated peritoneal macrophage localization and their interaction with mesothelial cells, the major cell type predicted to secrete CSF1. Our data revealed that mesothelial cells produce membrane bound and secreted CSF1 that both sustain peritoneal macrophage growth.

IL-26, a Cytokine With Roles in Extracellular DNA-Induced Inflammation and Microbial Defense.

Interleukin 26 (IL-26) is the most recently identified member of the IL-20 cytokine subfamily, and is a novel mediator of inflammation overexpressed in activated or transformed T cells. Novel properties have recently been assigned to IL-26, owing to its non-conventional cationic, and amphipathic features. IL-26 binds to DNA released from damaged cells and, as a carrier molecule for extracellular DNA, links DNA to inflammation. This observation suggests that IL-26 may act both as a driver and an effector of inflammation, leading to the establishment of a deleterious amplification loop and, ultimately, sustained inflammation. Thus, IL-26 emerges as an important mediator in local immunity/inflammation. The dysregulated expression and extracellular DNA carrier capacity of IL-26 may have profound consequences for the chronicity of inflammation. IL-26 also exhibits direct antimicrobial properties. This review summarizes recent advances on the biology of IL-26 and discusses its roles as a novel kinocidin.

Alarmins of the extracellular space.

The ability of the immune system to discriminate between healthy-self, abnormal-self, and non-self has been attributed mainly to alarmins signaling as "danger signals". It is now evident, however, that alarmins are much more complex and can perform specialized functions that can regulate a wide spectrum of processes ranging from propagation of disease to tissue homeostasis. As such, alarmins and their signaling mechanisms are now actively pursued as therapeutic targets. The clinical utility of alarmins requires an understanding of their specific localization. Specifically, many alarmins can function paradoxically depending upon their localization, intra or extracellular. The present review focuses upon alarmin presence and differential expression in the extracellular space versus within the cell and how variation of the localization of alarmins can reveal important mechanistic insights into alarmin functions and their efficacy as biomarkers of disease and therapeutic targets.

Biology, role and therapeutic potential of circulating histones in acute inflammatory disorders.

Histones are positively charged nuclear proteins that facilitate packaging of DNA into nucleosomes common to all eukaryotic cells. Upon cell injury or cell signalling processes, histones are released passively through cell necrosis or actively from immune cells as part of extracellular traps. Extracellular histones function as microbicidal proteins and are pro-thrombotic, limiting spread of infection or isolating areas of injury to allow for immune cell infiltration, clearance of infection and initiation of tissue regeneration and repair. Histone toxicity, however, is not specific to microbes and contributes to tissue and end-organ injury, which in cases of systemic inflammation may lead to organ failure and death. This review details the processes of histones release in acute inflammation, the mechanisms of histone-related tissue toxicity and current and future strategies for therapy targeting histones in acute inflammatory diseases.

Immune Modulation by Human Secreted RNases at the Extracellular Space.

The ribonuclease A superfamily is a vertebrate-specific family of proteins that encompasses eight functional members in humans. The proteins are secreted by diverse innate immune cells, from blood cells to epithelial cells and their levels in our body fluids correlate with infection and inflammation processes. Recent studies ascribe a prominent role to secretory RNases in the extracellular space. Extracellular RNases endowed with immuno-modulatory and antimicrobial properties can participate in a wide variety of host defense tasks, from performing cellular housekeeping to maintaining body fluid sterility. Their expression and secretion are induced in response to a variety of injury stimuli. The secreted proteins can target damaged cells and facilitate their removal from the focus of infection or inflammation. Following tissue damage, RNases can participate in clearing RNA from cellular debris or work as signaling molecules to regulate the host response and contribute to tissue remodeling and repair. We provide here an overall perspective on the current knowledge of human RNases' biological properties and their role in health and disease. The review also includes a brief description of other vertebrate family members and unrelated extracellular RNases that share common mechanisms of action. A better knowledge of RNase mechanism of actions and an understanding of their physiological roles should facilitate the development of novel therapeutics.

Advances, challenges, and opportunities in extracellular RNA biology: insights from the NIH exRNA Strategic Workshop.

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.

Extracellular Vesicles: Packages Sent With Complement.

Cells communicate with other cells in their microenvironment by transferring lipids, peptides, RNA, and sugars in extracellular vesicles (EVs), thereby also influencing recipient cell functions. Several studies indicate that these vesicles are involved in a variety of critical cellular processes including immune, metabolic, and coagulatory responses and are thereby associated with several inflammatory diseases. Furthermore, EVs also possess anti-inflammatory properties and contribute to immune regulation, thus encouraging an emerging interest in investigating and clarifying mechanistic links between EVs and innate immunity. Current studies indicate complex interactions of the complement system with EVs, with a dramatic influence on local and systemic inflammation. During inflammatory conditions with highly activated complement, including after severe tissue trauma and during sepsis, elevated numbers of EVs were found in the circulation of patients. There is increasing evidence that these shed vesicles contain key complement factors as well as complement regulators on their surface, affecting inflammation and the course of disease. Taken together, interaction of EVs regulates complement activity and contributes to the pro- and anti-inflammatory immune balance. However, the molecular mechanisms behind this interaction remain elusive and require further investigation. The aim of this review is to summarize the limited current knowledge on the crosstalk between complement and EVs. A further aspect is the clinical relevance of EVs with an emphasis on their capacity as potential therapeutic vehicles in the field of translational medicine.

Neutrophil killing of Mycobacterium abscessus by intra- and extracellular mechanisms.

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, are increasingly present in soft tissue infections and chronic lung diseases, including cystic fibrosis, and infections are characterized by growth in neutrophil-rich environments. M. abscessus is observed as two distinct smooth and rough morphotypes. The environmental smooth morphotype initiates infection and has a relatively limited ability to activate neutrophils. The rough morphotype has increased virulence and immunogenicity. However, the neutrophil response to the rough morphotype has not been explored. Killing of the smooth and rough strains, including cystic fibrosis clinical isolates, was equivalent. Neutrophil uptake of M. abscessus was similar between morphotypes. Mechanistically, both rough and smooth morphotypes enhanced neutrophil reactive oxygen species generation but inhibition of NADPH oxidase activity did not affect M. abscessus viability. However, inhibition of phagocytosis and extracellular traps reduced killing of the smooth morphotype with lesser effects against the rough morphotype. Neutrophils treated with M. abscessus released a heat-labile mycobactericidal activity against the rough morphotype, but the activity was heat-tolerant against the smooth morphotype. Overall, M. abscessus stimulates ineffective neutrophil reactive oxygen species generation, and key mechanisms differ in killing of the smooth (phagocytosis-dependent, extracellular traps, and heat-tolerant secreted factor) and rough (extracellular traps and a heat-labile secreted factor) morphotypes. These studies represent an essential advancement in understanding the host response to M. abscessus, and help explain the recalcitrance of infection.