Professional Phagocytes Research Articles

Galectin-3 (MAC-2) controls phagocytosis and macropinocytosis through intracellular and extracellular mechanisms.

Galectin-3 (Gal-3; formally named MAC-2) is a β-galactoside-binding lectin. Various cell types produce Gal-3 under either normal conditions and/or pathological conditions. Gal-3 can be present in cells' nuclei and cytoplasm, secreted from producing cells, and associated with cells' plasma membranes. This review focuses on how Gal-3 controls phagocytosis and macropinocytosis. Intracellular and extracellular Gal-3 promotes the phagocytosis of phagocytic targets/cargo (e.g., tissue debris and apoptotic cells) in “professional phagocytes” (e.g., microglia and macrophages) and “non-professional phagocytes” (e.g., Schwann cells and astrocytes). Intracellularly, Gal-3 promotes phagocytosis by controlling the “eat me” signaling pathways that phagocytic receptors generate, directing the cytoskeleton to produce the mechanical forces that drive the structural changes on which phagocytosis depends, protrusion and then retraction of filopodia and lamellipodia as they, respectively, engulf and then internalize phagocytic targets. Extracellularly, Gal-3 promotes phagocytosis by functioning as an opsonin, linking phagocytic targets to phagocytic receptors, activating them to generate the “eat me” signaling pathways. Macropinocytosis is a non-selective endocytic mechanism that various cells use to internalize the bulk of extracellular fluid and included materials/cargo (e.g., dissolved nutrients, proteins, and pathogens). Extracellular and intracellular Gal-3 control macropinocytosis in some types of cancer. Phagocytosed and macropinocytosed targets/cargo that reach lysosomes for degradation may rupture lysosomal membranes. Damaged lysosomal membranes undergo either repair or removal by selective autophagy (i.e., lysophagy) that intracellular Gal-3 controls.

SYK coordinates neuroprotective microglial responses in neurodegenerative disease

SUMMARYRecent studies have begun to reveal critical roles for the brain’s professional phagocytes, microglia, and their receptors in the control of neurotoxic amyloid beta (Aβ) and myelin debris accumulation in neurodegenerative disease. However, the critical intracellular molecules that orchestrate neuroprotective functions of microglia remain poorly understood. In our studies, we find that targeted deletion of SYK in microglia leads to exacerbated Aβ deposition, aggravated neuropathology, and cognitive defects in the 5xFAD mouse model of Alzheimer’s disease (AD). Disruption of SYK signaling in this AD model was further shown to impede the development of disease-associated microglia (DAM), alter AKT/GSK3β-signaling, and restrict Aβ phagocytosis by microglia. Conversely, receptor-mediated activation of SYK limits Aβ load. We also found that SYK critically regulates microglial phagocytosis and DAM acquisition in demyelinating disease. Collectively, these results broaden our understanding of the key innate immune signaling molecules that instruct beneficial microglial functions in response to neurotoxic material.

INDICATORS OF THE SKIN MICROBIOTA AND THE PHAGOCYTIC ACTIVITY IN MEAT AND EGG PRODUCTION WORKERS

Aim. The analysis of the indicators of skin microbiota and phagocytic activity of neutrophils and monocytes in employees of the "Peremoga Nova" poultry farm. Methods. The presence of sanitary and epidemiologically important groups of bacteria and the number of mesophilic aerobic and facultative anaerobic microorganisms (MAFAnM) on the skin surface, leukogram parameters and phagocytic activity of professional phagocytes were determined. The indicators of students of the Bohdan Khmelnytsky National University of Cherkasy were used as a control. Results. It was found that the MAFAnM index in the experimental group (3.2×103 CFU/cm3) is significantly lower than in the control group (2.7×103 CFU/cm3), however, the percentage of Staphylococcus spp. carriers is higher (67.5% versus 40.0%). In the experimental group, the relative and total number of monocytes is significantly higher compared to the control group. There is a positive correlation between the phagocytic number and the phagocytic index of monocytes and the value of MAFAnM. Conclusions. There was an increase of the level of monocytes in meat and egg products workers against the background of the presence of bacteria of the Staphylococcus spp. group on the skin. It may indicate the activation of pro-inflammatory factors at the level of peripheral blood. An increased percentage of staphylococcal carriers is a sign of adaptation of Staphylococcus spp. bacteria to the antibiotics used in the manufacturing process.

Consequences and mechanisms of myelin debris uptake and processing by cells in the central nervous system

Central nervous system (CNS) disorders and trauma involving changes to the neuronal myelin sheath have long been a topic of great interest. One common pathological change in these diseases is the generation of myelin debris resulting from the breakdown of the myelin sheath. Myelin debris contains many inflammatory and neurotoxic factors that inhibit remyelination and make its clearance a prerequisite for healing in CNS disorders. Many professional and semiprofessional phagocytes participate in the clearance of myelin debris in the CNS. These cells use various mechanisms for the uptake of myelin debris, and each cell type produces its own unique set of pathologic consequences resulting from the debris uptake. Examining these cells’ phagocytosis of myelin debris will contribute to a more complete understanding of CNS disease pathogenesis and help us conceptualize how the necessary clearance of myelin debris must be balanced with the detrimental consequences brought about by its clearance.

Receptor for advanced glycation end-products (RAGE) mediates phagocytosis in nonprofessional phagocytes

In mammals, both professional phagocytes and nonprofessional phagocytes (NPPs) can perform phagocytosis. However, limited targets are phagocytosed by NPPs, and thus, the mechanism remains unclear. We find that spores of the yeast Saccharomyces cerevisiae are internalized efficiently by NPPs. Analyses of this phenomenon reveals that RNA fragments derived from cytosolic RNA species are attached to the spore wall, and these fragments serve as ligands to induce spore internalization. Furthermore, we show that a multiligand receptor, RAGE (receptor for advanced glycation end-products), mediates phagocytosis in NPPs. RAGE-mediated phagocytosis is not uniquely induced by spores but is an intrinsic mechanism by which NPPs internalize macromolecules containing RAGE ligands. In fact, artificial particles labeled with polynucleotides, HMGB1, or histone (but not bovine serum albumin) are internalized in NPPs. Our findings provide insight into the molecular basis of phagocytosis by NPPs, a process by which a variety of macromolecules are targeted for internalization.

Immunotoxicity of Carbon-Based Nanomaterials, Starring Phagocytes.

In the field of science, technology and medicine, carbon-based nanomaterials and nanoparticles (CNMs) are becoming attractive nanomaterials that are increasingly used. However, it is important to acknowledge the risk of nanotoxicity that comes with the widespread use of CNMs. CNMs can enter the body via inhalation, ingestion, intravenously or by any other route, spread through the bloodstream and penetrate tissues where (in both compartments) they interact with components of the immune system. Like invading pathogens, CNMs can be recognized by large numbers of receptors that are present on the surface of innate immune cells, notably monocytes and macrophages. Depending on the physicochemical properties of CNMs, i.e., shape, size, or adsorbed contamination, phagocytes try to engulf and process CNMs, which might induce pro/anti-inflammatory response or lead to modulation and disruption of basic immune activity. This review focuses on existing data on the immunotoxic potential of CNMs, particularly in professional phagocytes, as they play a central role in processing and eliminating foreign particles. The results of immunotoxic studies are also described in the context of the entry routes, impacts of contamination and means of possible elimination. Mechanisms of proinflammatory effect depending on endocytosis and intracellular distribution of CNMs are highlighted as well.

Hematopoietic plasticity mapped in Drosophila and other insects.

Hemocytes, similar to vertebrate blood cells, play important roles in insect development and immunity, but it is not well understood how they perform their tasks. New technology, in particular single-cell transcriptomic analysis in combination with Drosophila genetics, may now change this picture. This review aims to make sense of recently published data, focusing on Drosophila melanogaster and comparing to data from other drosophilids, the malaria mosquito, Anopheles gambiae, and the silkworm, Bombyx mori. Basically, the new data support the presence of a few major classes of hemocytes: (1) a highly heterogenous and plastic class of professional phagocytes with many functions, called plasmatocytes in Drosophila and granular cells in other insects. (2) A conserved class of cells that control melanin deposition around parasites and wounds, called crystal cells in D. melanogaster, and oenocytoids in other insects. (3) A new class of cells, the primocytes, so far only identified in D. melanogaster. They are related to cells of the so-called posterior signaling center of the larval hematopoietic organ, which controls the hematopoiesis of other hemocytes. (4) Different kinds of specialized cells, like the lamellocytes in D. melanogaster, for the encapsulation of parasites. These cells undergo rapid evolution, and the homology relationships between such cells in different insects are uncertain. Lists of genes expressed in the different hemocyte classes now provide a solid ground for further investigation of function.

Intracellular Habitation of Staphylococcus aureus: Molecular Mechanisms and Prospects for Antimicrobial Therapy.

Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a global health threat, especially with the continuous development of antibiotic resistance. As an opportunistic pathogen, MRSA infections have a high mortality rate worldwide. Although classically described as an extracellular pathogen, many studies have shown over the past decades that MRSA also has an intracellular aspect to its infectious cycle, which has been observed in vitro in both non-professional as well as professional phagocytes. In vivo, MRSA has been shown to establish an intracellular niche in liver Kupffer cells upon bloodstream infection. The staphylococci have evolved various evasion strategies to survive the antimicrobial environment of phagolysosomes and use these compartments to hide from immune cells and antibiotics. Ultimately, the host cells get overwhelmed by replicating bacteria, leading to cell lysis and bacterial dissemination. In this review, we describe the different intracellular aspects of MRSA infection and briefly mention S. aureus evasion strategies. We discuss how this intracellular niche of bacteria may assist in antibiotic tolerance development, and lastly, we describe various new antibacterial strategies that target the intracellular bacterial niche.

521 Development of 3D reconstructed skin models with PBMC-derived immune cells

Skin immune system is composed of specialized immune cells that plays an important role in skin homeostasis and protection against injury. Among these cells, Langerhans cells and macrophages are key elements of both the innate and adaptive immune response. In epidermal compartment, Langerhans cells (LCs) are immature dendritic cells involved in antigen presentation to T Cells whereas in the dermal compartment, macrophages are tissue resident professional phagocytes. Under inflammatory context, epidermis and dermis are depleted from resident Langerhans cells and macrophages through migration or cell death respectively.

The Ubiquitin-Proteasome System in Apoptosis and Apoptotic Cell Clearance.

Ubiquitination, a critical post-translational modification of proteins, refers to the covalent attachment of ubiquitin to the substrate and is involved in various biological processes such as protein stability regulation, DNA damage repair, and apoptosis, among others. E3 ubiquitin ligases are essential enzymes of the ubiquitin pathway with high substrate specificity and precisely regulate specific proteins’ turnover. As one of the most well-studied forms of programmed cell death, apoptosis is substantially conserved across the evolutionary tree. The final critical stage in apoptosis is the removal of apoptotic cells by professional and non-professional phagocytes. Apoptosis and apoptotic cell clearance are crucial for the normal development, differentiation, and growth of multicellular organisms, as well as their association with a variety of inflammatory and immune diseases. In this review, we discuss the role of ubiquitination and deubiquitination in apoptosis and apoptotic cell clearance.

RIP2 Contributes to Expanded CD4+ T Cell IFN-γ Production during Efferocytosis of Streptococcus pneumoniae–Infected Apoptotic Cells

Apoptotic cell clearance by professional and nonprofessional phagocytes in the process of efferocytosis is critical to preserve tissue homeostasis. Uptake of apoptotic cells by dendritic cells generates regulatory T cells and induces immunologic tolerance against self-antigens. In contrast, ingestion of infected apoptotic cells promotes activation of TLR4/MyD88-dependent bone marrow-derived dendritic cells (BMDCs) and triggers Th17 cell differentiation. In this study, we evaluated the impact of Streptococcus pneumoniae-infected apoptotic cell efferocytosis by BMDCs derived from C57BL/6 mice on differentiation and expansion of CD4+ T cell subsets, as well as the role of TLR2/4 and receptor-interacting protein 2 (RIP2) receptors in recognizing intracellular pathogens during efferocytosis. We demonstrated that BMDC-mediated efferocytosis of S. pneumoniae-infected apoptotic cells induced Th1 cell differentiation and expansion. Although TLR2/4 and RIP2 deficiency in BMDCs did not affect Th1 cell differentiation during efferocytosis, the absence of RIP2 decreased IFN-γ production by CD4 T cells during the expansion phase. These findings suggest that RIP2-mediated IL-1β production during efferocytosis of S. pneumoniae-infected apoptotic cells partially supports a Th1-mediated IFN-γ production microenvironment.

Phagocytic microglia and macrophages in brain injury and repair.

AimsPhagocytosis is the cellular digestion of extracellular particles, such as pathogens and dying cells, and is a key element in the evolution of central nervous system (CNS) disorders. Microglia and macrophages are the professional phagocytes of the CNS. By clearing toxic cellular debris and reshaping the extracellular matrix, microglia/macrophages help pilot the brain repair and functional recovery process. However, CNS resident and invading immune cells can also magnify tissue damage by igniting runaway inflammation and phagocytosing stressed—but viable—neurons.DiscussionMicroglia/macrophages help mediate intercellular communication and react quickly to the “find‐me” signals expressed by dead/dying neurons. The activated microglia/macrophages then migrate to the injury site to initiate the phagocytic process upon encountering “eat‐me” signals on the surfaces of endangered cells. Thus, healthy cells attempt to avoid inappropriate engulfment by expressing “do not‐eat‐me” signals. Microglia/macrophages also have the capacity to phagocytose immune cells that invade the injured brain (e.g., neutrophils) and to regulate their pro‐inflammatory properties. During brain recovery, microglia/macrophages engulf myelin debris, initiate synaptogenesis and neurogenesis, and sculpt a favorable extracellular matrix to support network rewiring, among other favorable roles. Here, we review the multilayered nature of phagocytotic microglia/macrophages, including the molecular and cellular mechanisms that govern microglia/macrophage‐induced phagocytosis in acute brain injury, and discuss strategies that tap into the therapeutic potential of this engulfment process.ConclusionIdentification of biological targets that can temper neuroinflammation after brain injury without hindering the essential phagocytic functions of microglia/macrophages will expedite better medical management of the stroke recovery stage.

Understanding the Phagocytosis of Particles: the Key for Rational Design of Vaccines and Therapeutics.

A robust comprehension of phagocytosis is crucial for understanding its importance in innate immunity. A detailed description of the molecular mechanisms that lead to the uptake and clearance of endogenous and exogenous particles has helped elucidate the role of phagocytosis in health and infectious or autoimmune diseases. Furthermore, knowledge about this cellular process is important for the rational design and development of particulate systems for the administration of vaccines or therapeutics. Depending on these specific applications and the required biological responses, particles must be designed to encourage or avoid their phagocytosis and prolong their circulation time. Functionalization with specific polymers or ligands and changes in the size, shape, or surface of particles have important effects on their recognition and internalization by professional and nonprofessional phagocytes and have a major influence on their fate and safety. Here, we review the phagocytosis of particles intended to be used as carrier or delivery systems for vaccines or therapeutics, the cells involved in this process depending on the route of administration, and the strategies employed to obtain the most desirable particles for each application through the manipulation of their physicochemical characteristics. We also offer a view of the challenges and potential opportunities in the field and give some recommendations that we expect will enable the development of improved approaches for the rational design of these systems.

Human macrophage polarization determines bacterial persistence of Staphylococcus aureus in a liver-on-chip-based infection model

Infections with Staphylococcus aureus (S. aureus) have been reported from various organs ranging from asymptomatic colonization to severe infections and sepsis. Although considered an extracellular pathogen, S. aureus can invade and persist in professional phagocytes such as monocytes and macrophages. Its capability to persist and manipulate macrophages is considered a critical step to evade host antimicrobial reactions. We leveraged a recently established human liver-on-chip model to demonstrate that S. aureus specifically targets macrophages as essential niche facilitating bacterial persistence and phenotype switching to small colony variants (SCVs). In vitro, M2 polarization was found to favor SCV-formation and was associated with increased intracellular bacterial loads in macrophages, increased cell death, and impaired recruitment of circulating monocytes to sites of infection. These findings expand the knowledge about macrophage activation in the liver and its impact on bacterial persistence and dissemination in the course of infection.

Phagosomal Acidification Is Required to Kill Streptococcus pneumoniae in a Zebrafish Model

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen causing invasive disease, including community-acquired bacteraemia, and remains a leading cause of global mortality. Understanding the role of phagocytes in killing bacteria is still limited, especially in vivo. In this study, we established a zebrafish model to study the interaction between intravenously administered pneumococci and professional phagocytes such as macrophages and neutrophils, to unravel bacterial killing mechanisms employed by these immune cells. Our model confirmed the key role of polysaccharide capsule in promoting pneumococcal virulence through inhibition of phagocytosis. Conversely, we show pneumococci lacking a capsule are rapidly internalised by macrophages. Low doses of encapsulated S. pneumoniae cause near 100% mortality within 48 hours postinfection (hpi), while 50 times higher doses of unencapsulated pneumococci are easily cleared. Time course analysis of in vivo bacterial numbers reveals that while encapsulated pneumococcus proliferates to levels exceeding 105 CFU at the time of host death, unencapsulated bacteria are unable to grow and are cleared within 20 hpi. Using genetically induced macrophage depletion, we confirmed an essential role for macrophages in bacterial clearance. Additionally, we show that upon phagocytosis by macrophages, phagosomes undergo rapid acidification. Genetic and chemical inhibition of vacuolar ATPase (v-ATPase) prevents intracellular bacterial killing and induces host death indicating a key role of phagosomal acidification in immunity to invading pneumococci. We also show that our model can be used to study the efficacy of antimicrobials against pneumococci in vivo. Collectively, our data confirm that larval zebrafish can be used to dissect killing mechanisms during pneumococcal infection in vivo and highlight key roles for phagosomal acidification in macrophages for pathogen clearance.

NADPH oxidase family proteins: signaling dynamics to disease management.

Reactive oxygen species (ROS)are pervasive signaling molecules in biological systems. In humans, a lack of ROS causes chronic and extreme bacterial infections, while uncontrolled release of these factors causes pathologies due to excessive inflammation. Professional phagocytes such as neutrophils (PMNs), eosinophils, monocytes, and macrophages use superoxide-generating NADPH oxidase (NOX) as part of their arsenal of antimicrobial mechanisms to produce high levels of ROS. NOXis a multisubunit enzyme complex composed of five essential subunits, two of which are localized in the membrane, while three are localized in the cytosol. In resting phagocytes, the oxidase complex is unassembled and inactive; however, it becomes activated after cytosolic components translocate to the membrane and are assembled into a functional oxidase. The NOX isoforms play a variety of roles in cellular differentiation, development, proliferation, apoptosis, cytoskeletal control, migration, and contraction. Recent studies have identified NOX as a major contributor to disease pathologies, resulting in a shift in focus on inhibiting the formation of potentially harmful free radicals. Therefore, a better understanding of the molecular mechanisms and the transduction pathways involved in NOX-mediated signaling is essential for the development of new therapeutic agents that minimize the hyperproduction of ROS. The current review provides a thorough overview of the various NOX enzymes and their roles in disease pathophysiology, highlights pharmacological strategies, and discusses the importance of computational modeling for future NOX-related studies.

The Impact of the CTHRSSVVC Peptide Upon Experimental Models of Trypanosoma cruzi Infection.

Chagas disease (CD), caused by the hemoflagellate protozoan Trypanosoma cruzi, affects more than six million people worldwide and presents an unsatisfactory therapy, based on two nitroderivatives, introduced in clinical medicine for decades. The synthetic peptide, with CTHRSSVVC sequence (PepA), mimics the CD163 and TNF-α tripeptide “RSS” motif and binds to atheromatous plaques in carotid biopsies of human patients, spleen tissues, and a low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis. CD163 receptor is present on monocytes, macrophages, and neutrophils, acting as a regulator of acute-phase processes and modulating aspects of the inflammatory response and the establishment of infections. Due to the potential theranostic role of PepA, our aim was to investigate its effect upon T. cruzi infection in vitro and in vivo. PepA and two other peptides with shuffled sequences were assayed upon different binomials of host cell/parasite, including professional [as peritoneal mouse macrophages (PMM)] and non-professional phagocytes [primary cultures of cardiac cells (CM)], under different protocols. Also, their impact was further addressed in vivo using a mouse model of acute experimental Chagas disease. Our in-vitro findings demonstrate that PepA and PepB (the peptide with random sequence retaining the “RS” sequence) reduced the intracellular parasitism of the PMM but were inactive during the infection of cardiac cells. Another set of in-vitro and in-vivo studies showed that they do not display a trypanocidal effect on bloodstream trypomastigotes nor exhibit in-vivo efficacy when administered after the parasite inoculation. Our data report the in-vitro activity of PepA and PepB upon the infection of PMM by T. cruzi, possibly triggering the microbicidal arsenal of the host professional phagocytes, capable of controlling parasitic invasion and proliferation.

Iron metabolism in the social amoeba Dictyostelium discoideum: A role for ferric chelate reductases

Iron is the most abundant transition metal in all living organisms and is essential for several cellular activities, including respiration, oxygen transport, energy production and regulation of gene expression. Iron starvation is used by professional phagocytes, from Dictyostelium to macrophages, as a form of defense mechanism against intracellular pathogens. Previously, we showed that Dictyostelium cells express the proton-driven iron transporter Nramp1 (Natural Resistance-Associated Macrophage Protein 1) and the homolog NrampB (Nramp2) in membranes of macropinosomes and phagosomes or of the contractile vacuole network, respectively. The Nramp-driven transport of iron across membranes is selective for ferrous ions. Since iron is mostly present as ferric ions in growth media and in engulfed bacteria, we have looked for proteins with ferric reductase activity. The Dictyostelium genome does not encode for classical STEAP (Six-Transmembrane Epithelial Antigen of Prostate) ferric reductases, but harbors three genes encoding putative ferric chelate reductase belonging to the Cytochrome b561 family containing a N terminus DOMON domain (DOpamine β-MONooxygenase N-terminal domain). We have cloned the three genes, naming them fr1A, fr1B and fr1C. fr1A and fr1B are mainly expressed in the vegetative stage while fr1C is highly expressed in the post aggregative stage. All three reductases are localized in the endoplasmic reticulum, but Fr1A is also found in endolysosomal vesicles, in the Golgi and, to a much lower degree, in the plasma membrane, whereas Fr1C is homogeneously distributed in the plasma membrane and in macropinosomal and phagosomal membranes. To gain insight in the function of the three genes we generated KO mutants, but gene disruption was successful only for two of them (fr1A and fr1C), being very likely lethal for fr1B. fr1A- shows a slight delay in the aggregation stage of development, while fr1C- gives rise to large multi-tipped streams during aggregation and displays a strong delay in fruiting body formation. The two single mutants display altered cell growth under conditions of ferric ions overloading and, in the ability to reduce Fe3+, confirming a role of these putative ferric reductases in iron reduction and transport from endo-lysosomal vesicles to the cytosol.

The Influence of Antibiotic Resistance on Innate Immune Responses to Staphylococcus aureus Infection.

Staphylococcus aureus (S. aureus) causes a broad range of infections and is associated with significant morbidity and mortality. S. aureus produces a diverse range of cellular and extracellular factors responsible for its invasiveness and ability to resist immune attack. In recent years, increasing resistance to last-line anti-staphylococcal antibiotics daptomycin and vancomycin has been observed. Resistant strains of S. aureus are highly efficient in invading a variety of professional and nonprofessional phagocytes and are able to survive inside host cells. Eliciting immune protection against antibiotic-resistant S. aureus infection is a global challenge, requiring both innate and adaptive immune effector mechanisms. Dendritic cells (DC), which sit at the interface between innate and adaptive immune responses, are central to the induction of immune protection against S. aureus. However, it has been observed that S. aureus has the capacity to develop further antibiotic resistance and acquire increased resistance to immunological recognition by the innate immune system. In this article, we review the strategies utilised by S. aureus to circumvent antibiotic and innate immune responses, especially the interaction between S. aureus and DC, focusing on how this relationship is perturbed with the development of antibiotic resistance.

Mycobacterium avium-intracellulare complex promote release of pro-inflammatory enzymes matrix metalloproteinases by inducing neutrophil extracellular trap formation

The prevalence of and mortality from non-tuberculous mycobacteria (NTM) infections have been steadily increasing worldwide. Most NTM infections are caused by Mycobacterium avium-intracellulare complex (MAC). MAC can escape from killing by neutrophils, which are professional phagocytes. However, the involvement of neutrophils in the pathogenesis of MAC infection is poorly understood. The present study assessed the roles of neutrophil extracellular trap (NET) formation in neutrophil defense mechanisms against infection with MAC strains, including M. avium isolated from patients with severe or mild lung tissue destruction. Although all MAC induced NET formation, non-pathogenic mycobacteria (M. gordonae and M. smegmatis) slightly but not significantly induced NET formation. Peptidylarginine deiminase 4 (PAD4) inhibitor reduced MAC-induced NET formation but did not affect MAC escape from neutrophils. PAD4 inhibition attenuated the MAC-induced matrix metalloproteinase (MMP)-8 and 9 release to the levels of MMPs from non-pathogenic mycobacteria. MAC also induced interleukin (IL)-8 release by neutrophils, a process independent of MAC-induced NET formation. Taken together, these findings suggest that MAC induce NET formation, IL-8 release and NETs-dependent release of MMP-8 and -9 from neutrophils, leading to neutrophil accumulation and further inflammation, thereby enhancing the progression of infection in the lungs.