Mycoplasma pneumoniae Mpn133 is a cytotoxic nuclease with a glutamic acid‐, lysine‐ and serine‐rich region essential for binding and internalization but not enzymatic activity

We identified Mpn133 as a Ca²⁺‐dependent cytotoxic nuclease of Mycoplasma pneumoniae. Flow cytometry analysis and immunofluorescence studies revealed the binding and internalization of recombinant Mpn133 (rMpn133) in human airway A549 cells. Amino acid sequence comparisons of Mpn133 with other mycoplasma nucleases demonstrated the presence of a unique glutamic acid‐, lysine‐ and serine‐rich region (EKS region; amino acids 72–110). Deletion of this EKS peptide (rMpn133^Δ72–110) abrogated its binding and internalization but not its nuclease activity. The function of the EKS region in host cell trafficking and nuclear localization was reinforced by the successful delivery of EKS‐conjugated mCherry protein into A549 cells. rMpn133, but not rMpn133^Δ72–110, induced apoptosis‐like death in A549 cells. This observation suggested a unique role of Mpn133 as an important contributor to M. pneumoniae‐associated life cycle events and as a virulence factor in host‐associated cytopathologies. In addition, the distinct property of the EKS peptide in delivery of proteins, like mCherry, into target cells opens new avenues to the establishment of novel concepts of drug delivery and therapy.     

Neutrophil extracellular Taps play an important role in clearance of Streptococcus suis in vivo

Streptococcus suis infection induces formation of neutrophil extracellular traps (NETs) in vitro; however, the contribution of NETs‐mediated killing to the pathogenesis of S. suis in vivo is yet to be elicited. The findings of the present study indicated that extracellular DNA fiber can be induced in a murine model in response to S. suis infection. A nuclease that destroys their structure was used to evaluate the role of NETs on S. suis infection. Treatment with nuclease resulted in a greater bacteria load and higher serum TNF‐α concentrations in response to S. suis infection, indicating that NETs structure played an essential role in S. suis clearance and inflammation. Furthermore, nuclease treatment resulted in more severe clinical signs during and higher mortality from S. suis infection. These findings indicated that NETs structure contributes to protection against S. suis infection.     

Vibrio cholerae Evades Neutrophil Extracellular Traps by the Activity of Two Extracellular Nucleases

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.     

In vitro resistance mechanisms of Neisseria meningitidis against neutrophil extracellular traps

Neisseria meningitidis (Nm) is a leading cause of septicemia in childhood. Nm septicemia is unique with respect to very quick disease progression, high in vivo bacterial replication rate and its considerable mortality. Nm circumvents major mechanisms of innate immunity such as complement system and phagocytosis. Neutrophil extracellular traps (NETs) are formed from neutrophils during systemic infection and are suggested to contain invading microorganisms. Here, we investigated the interaction of Nm with NETs. Both, meningococci and spontaneously released outer membrane vesicles (SOMVs) were potent NET inducers. NETs were unable to kill NET bound meningococci, but slowed down their proliferation rate. Using Nm as model organism we identified three novel mechanisms how bacteria can evade NET‐mediated killing: (i) modification of lipid A of meningococcal LPS with phosphoethanolamine protected Nm from NET‐bound cathepsin G; (ii) expression of the high‐affinity zinc uptake receptor ZnuD allowed Nm to escape NET‐mediated nutritional immunity; (iii) binding of SOMVs to NETs saved Nm from NET binding and the consequent bacteriostatic effect. Escape from NETs may contribute to the most rapid progression of meningococcal disease. The induction of NET formation by Nm in vivo might aggravate thrombosis in vessels ultimately directing to disseminated intravascular coagulation (DIC).     

Mycoplasma pneumoniae protects infected epithelial cells from hydrogen peroxide‐induced cell detachment

Epithelial cell shedding is a defence mechanism against infectious microbes that use these cells as an infection foothold and that eliminate microbes from infection foci by removing infected cells. Mycoplasma pneumoniae, a causative agent of respiratory infections, is known to adhere to and colonise the surface of ciliated airway epithelial cells; it produces a large amount of hydrogen peroxide, indicating its capability of regulating hydrogen peroxide‐induced infected cell detachment. In this study, we found that M. pneumoniae reduces exogenous hydrogen peroxide‐induced detachment of the infected cells from culture plates. This cell detachment occurred dependently of DNA damage‐initiated, poly (ADP‐ribose) polymerase 1 (PARP1)‐mediated cell death, or parthanatos. In cells infected with M. pneumoniae, exogenous hydrogen peroxide failed to induce DNA damage‐initiated poly (ADP‐ribose) (PAR) synthesis and concomitant increased cytoplasmic membrane rupture, both of which are biochemical hallmarks of parthanatos. The impairment of PAR synthesis was attributed to a reduction in the amount of cytosolic nicotinamide adenine dinucleotide (NAD), a substrate of PARP1, caused by M. pneumoniae. On the other hand, nonadherent mutant strains of M. pneumoniae showed a lower ability to reduce cell detachment than wild‐type strains, but the extent to which NAD was decreased in infected cells was comparable to that seen in the wild‐type strain. We found that NAD depletion could induce PARP1‐independent cell detachment pathways following stimulation with hydrogen peroxide and that M. pneumoniae could also regulate PARP1‐independent cell detachment in a cytoadhesion‐dependent manner. These results suggest that M. pneumoniae might regulate infected cell detachment induced by hydrogen peroxide that it produces itself, and such a mechanism may contribute to sustaining the bacterial infection.     

Induction of neutrophil extracellular traps by Campylobacter jejuni

Campylobacter jejuni is the leading cause of bacterial‐derived gastroenteritis worldwide and can lead to several post‐infectious inflammatory disorders. Despite the prevalence and health impacts of the bacterium, interactions between the host innate immune system and C. jejuni remain poorly understood. To expand on earlier work demonstrating that neutrophils traffic to the site of infection in an animal model of campylobacteriosis, we identified significant increases in several predominantly neutrophil‐derived proteins in the faeces of C. jejuni‐infected patients, including lipocalin‐2, myeloperoxidase and neutrophil elastase. In addition to demonstrating that these proteins significantly inhibited C. jejuni growth, we determined they are released during formation of C. jejuni‐induced neutrophil extracellular traps (NETs). Using quantitative and qualitative methods, we found that purified human neutrophils are activated by C. jejuni and exhibit signatures of NET generation, including presence of protein arginine deiminase‐4, histone citrullination, myeloperoxidase, neutrophil elastase release and DNA extrusion. Production of NETs correlated with C. jejuni phagocytosis/endocytosis and invasion of neutrophils suggesting that host‐ and bacterial‐mediated activities are responsible for NET induction. Further, NET‐like structures were observed within intestinal tissue of C. jejuni‐infected ferrets. Finally, induction of NETs significantly increased human colonocyte cytotoxicity, indicating that NET formation during C. jejuni infection may contribute to observed tissue pathology. These findings provide further understanding of C. jejuni–neutrophil interactions and inflammatory responses during campylobacteriosis.     

Neutrophil extracellular trap (NET)-mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the CF airway, independent of CFTR

The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa.     

Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis

Neutrophils are multifaceted cells that are often the immune system's first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system.     

PSGL-1 on Leukocytes is a Critical Component of the Host Immune Response against Invasive Pneumococcal Disease

Bacterial uptake by phagocytic cells is a vital event in the clearance of invading pathogens such as Streptococcus pneumoniae. A major role of the P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes against invasive pneumococcal disease is described in this study. Phagocytosis experiments using different serotypes demonstrated that PSGL-1 is involved in the recognition, uptake and killing of S. pneumoniae. Co-localization of several clinical isolates of S. pneumoniae with PSGL-1 was demonstrated, observing a rapid and active phagocytosis in the presence of PSGL-1. Furthermore, the pneumococcal capsular polysaccharide and the main autolysin of the bacterium ―the amidase LytA― were identified as bacterial ligands for PSGL-1. Experimental models of pneumococcal disease including invasive pneumonia and systemic infection showed that bacterial levels were markedly increased in the blood of PSGL-1 ^−/− mice. During pneumonia, PSGL-1 controls the severity of pneumococcal dissemination from the lung to the bloodstream. In systemic infection, a major role of PSGL-1 in host defense is to clear the bacteria in the systemic circulation controlling bacterial replication. These results confirmed the importance of this receptor in the recognition and clearance of S. pneumoniae during invasive pneumococcal disease. Histological and cellular analysis demonstrated that PSGL-1 ^−/− mice have increased levels of T cells migrating to the lung than the corresponding wild-type mice. In contrast, during systemic infection, PSGL-1 ^−/− mice had increased numbers of neutrophils and macrophages in blood, but were less effective controlling the infection process due to the lack of this functional receptor. Overall, this study demonstrates that PSGL-1 is a novel receptor for S. pneumoniae that contributes to protection against invasive pneumococcal disease.     

Histoplasma capsulatum‐induced extracellular DNA trap release in human neutrophils

Neutrophils are leukocytes that are capable of eliminating both intra‐ and extracellular pathogens by mechanisms such as phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs). Histoplasma capsulatum var. capsulatum (H. capsulatum) is a dimorphic fungus with a global distribution that causes histoplasmosis, a disease that is endemic in different geographic areas and is spreading worldwide. The release of NETs has been described as an important host defense mechanism against different fungi; however, there are no reports demonstrating that this process is implicated in neutrophil response to H. capsulatum infection. Therefore, the aim of this work is to investigate whether isolated human neutrophils release NETs in response to H. capsulatum and the potential mechanisms involved, as well as delineate the NETs antifungal activity. Using both confocal fluorescence and scanning electron microscopy techniques, we determined that NETs are released in vitro in response to H. capsulatum via an oxidative mechanism that is downstream of activation of the Syk and Src kinase pathways and is also dependent on CD18. NETs released in response to H. capsulatum yeasts involve the loss of neutrophil viability and are associated with elastase and citrullinated histones, however also can occur in a PAD4 histone citrullination independent pathway. This NETs also presented fungicidal activity against H. capsulatum yeasts. Our findings may contribute to the understanding of how neutrophils recognize and respond as immune effector cells to H. capsulatum, which may lead to better knowledge of histoplasmosis pathophysiology and treatment.     

A1 adenosine receptor signaling reduces Streptococcus pneumoniae adherence to pulmonary epithelial cells by targeting expression of platelet‐activating factor receptor

Extracellular adenosine production is crucial for host resistance against Streptococcus pneumoniae (pneumococcus) and is thought to affect antibacterial immune responses by neutrophils. However, whether extracellular adenosine alters direct host–pathogen interaction remains unexplored. An important determinant for lung infection by S. pneumoniae is its ability to adhere to the pulmonary epithelium. Here we explored whether extracellular adenosine can directly impact bacterial adherence to lung epithelial cells. We found that signaling via A1 adenosine receptor significantly reduced the ability of pneumococci to bind human pulmonary epithelial cells. A1 receptor signaling blocked bacterial binding by reducing the expression of platelet‐activating factor receptor, a host protein used by S. pneumoniae to adhere to host cells. In vivo, A1 was required for control of pneumococcal pneumonia as inhibiting it resulted in increased host susceptibility. As S. pneumoniae remain a leading cause of community‐acquired pneumonia in the elderly, we explored the role of A1 in the age‐driven susceptibility to infection. We found no difference in A1 pulmonary expression in young versus old mice. Strikingly, triggering A1 signaling boosted host resistance of old mice to S. pneumoniae pulmonary infection. This study demonstrates a novel mechanism by which extracellular adenosine modulates resistance to lung infection by targeting bacterial–host interactions.     

Defining a pro‐inflammatory neutrophil phenotype in response to schistosome eggs

Neutrophils contribute to the pathological processes of a number of inflammatory disorders, including rheumatoid arthritis, sepsis and cystic fibrosis. Neutrophils also play prominent roles in schistosomiasis japonica liver fibrosis, being central mediators of inflammation following granuloma formation. In this study, we investigated the interaction between Schistosoma japonicum eggs and neutrophils, and the effect of eggs on the inflammatory phenotype of neutrophils. Our results showed significant upregulated expression of pro‐inflammatory cytokines (IL‐1α, IL‐1β and IL‐8) and chemokines (CCL3, CCL4 and CXCL2) in neutrophils after 4 h in vitro stimulation with S. japonicum eggs. Furthermore, mitochondrial DNA was released by stimulated neutrophils, and induced the production of matrix metalloproteinase 9 (MMP‐9), a protease involved in inflammation and associated tissue destruction. We also found that intact live eggs and isolated soluble egg antigen (SEA) triggered the release of neutrophil extracellular traps (NETs), but, unlike those reported in bacterial or fungal infection, NETs did not kill schistosome eggs in vitro. Together these show that S. japonicum eggs can induce the inflammatory phenotype of neutrophils, and further our understanding of the host–parasite interplay that takes place within the in vivo microenvironment of schistosome‐induced granuloma. These findings represent novel findings in a metazoan parasite, and confirm characteristics of NETs that have until now, only been observed in response to protozoan pathogens.     

Pyocyanin-Enhanced Neutrophil Extracellular Trap Formation Requires the NADPH Oxidase

Beyond intracellular killing, a novel neutrophil-based antimicrobial mechanism has been recently discovered: entrapment and killing by neutrophil extracellular traps (NETs). NETs consist of extruded nuclear DNA webs decorated with granule proteins. Although NET formation is an important innate immune mechanism, uncontrolled NET release damages host tissues and has been linked to several diseases including cystic fibrosis (CF). The major CF airway pathogen Pseudomonas aeruginosa establishes chronic infection. Pseudomonas imbedded within biofilms is protected against the immune system, but maintains chronic inflammation that worsens disease symptoms. Aberrant NET release from recruited neutrophils was found in CF, but the underlying mechanisms remain unclear. One of the most important Pseudomonas virulence factors is pyocyanin, a redox-active pigment that has been associated with diminished lung function in CF. Here we show that pyocyanin promotes NET formation in a time- and dose-dependent manner. Most CF Pseudomonas clinical isolates tested produce pyocyanin in vitro. Pyocyanin-derived reactive oxygen species are required for its NET release. Inhibitor experiments demonstrated involvement of Jun N-terminal Kinase (JNK) and phosphatidylinositol 3-Kinase (PI3K) in pyocyanin-induced NET formation. Pyocyanin-induced NETs also require the NADPH oxidase because NET release in chronic granulomatous disease neutrophils was greatly reduced. Comparison of neutrophils from gp91phox- and p47phox-deficient patients revealed that pyocyanin-triggered NET formation is proportional to their residual superoxide production. Our studies identify pyocyanin as the first secreted bacterial toxin that enhances NET formation. The involvement of NADPH oxidase in pyocyanin-induced NET formation represents a novel mechanism of pyocyanin toxicity.     

Kaempferol blocks neutrophil extracellular traps formation and reduces tumour metastasis by inhibiting ROS‐PAD4 pathway

Kaempferol (kaem) is a dietary flavonoid found in a variety of fruits and vegetables. The inhibitory effects of kaem on primary tumour growth have been extensively investigated; however, its effects on tumour metastasis are largely unknown. In the present study, we found that kaem significantly suppresses both primary tumour growth and lung metastasis in mouse breast tumour model. Furthermore, decreased expression of citrullinated histone H3 (H3‐cit), a biomarker of neutrophil extracellular traps (NETs), had been founded in metastatic lung upon treated with kaem. The reduction of H3‐cit is not, however, due to the cytotoxicity of kaem on neutrophils since the frequency of CD11b⁺Ly6G⁺ neutrophils did not change in lung, tumour or blood in the presence of kaem. We then confirm the anti‐NETs effects of kaem in vitro by co‐culturing mouse neutrophils and kaem. Supplementing the neutrophils with GSK484, a potent NET inhibitor, totally abrogated the inhibitory effects of kaem on tumour metastasis while having little or no impact on primary tumour growth, indicating the specificity of kaem acting on NET formation and tumour metastasis. We also found that kaem suppressed ROS production in mouse bone‐marrow derived neutrophils. Supplementing with the ROS scavenger DPI abrogated kaem's effects on NET formation, suggesting the involvement of kaempferol in NADPH/ROS‐NETs signalling. Finally, we applied the kaem on NET‐deficient PAD4^‐/‐ mice and found decreased primary tumour volume and weight but similar lung metastatic tumour with kaempferol treatment. Therefore, our findings reveal a novel mechanism of kaem in breast cancer development by targeting NETs induced tumour metastasis.     

Neutrophil Extracellular Traps Contain Calprotectin,a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.     

Bcl10 synergistically links CEACAM3 and TLR‐dependent inflammatory signalling

The neutrophil‐specific innate immune receptor CEACAM3 functions as a decoy to capture Gram‐negative pathogens, such as Neisseria gonorrhoeae, that exploit CEACAM family members to adhere to the epithelium. Bacterial binding to CEACAM3 results in their efficient engulfment and triggers activation of an nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB)‐dependent inflammatory response by human neutrophils. Herein, we report that CEACAM3 cross‐linking is not sufficient for induction of cytokine production and show that the inflammatory response induced by Neisseria gonorrhoeae infection is elicited by an integration of signals from CEACAM3 and toll‐like receptors. Using neutrophils from a human CEACAM‐expressing mouse line (CEABAC), we use a genetic approach to reveal a molecular bifurcation of the CEACAM3‐mediated antimicrobial and inflammatory responses. Ex vivo experiments with CEABAC‐Rac2^?/?, CEABAC‐Bcl10^?/?, and CEABAC‐Malt1^?/? neutrophils indicate that these effectors are not necessary for gonococcal engulfment, yet all 3 effectors contribute to CEACAM3‐mediated cytokine production. Interestingly, although Bcl10 and Malt1 are often inextricably linked, Bcl10 enabled synergy between toll‐like receptor 4 and CEACAM3, whereas Malt1 did not. Together, these findings reveal an integration of the specific innate immune receptor CEACAM3 into the network of more conventional pattern recognition receptors, providing a mechanism by which the innate immune system can unleash its response to a relentless pathogen.     

Integrin β3 is not critical for neutrophil recruitment in a mouse model of pneumococcal pneumonia

Streptococcus pneumoniae is one of the most common causes of bacterial pneumonias in humans. Neutrophil migration into lungs infected with S. pneumoniae is central to the host defense but the mechanisms of neutrophil recruitment, as mediated by S. pneumoniae, into lungs are incompletely understood. Therefore, we have assessed the role of integrin αvβ3 by evaluating its subunit β3 in a mouse model of lung inflammation induced by S. pneumonia. Integrin subunit β3 knockout (β3^-/-) and wild-type (WT) mice were intratracheally instilled with either S. pneumoniae or saline. Other groups of WT mice were treated intraperitoneally with 25 μg or 50 μg of antibody against integrin β3 or with isotype-matched antibody at 1 h before instillation of S. pneumoniae. Mice were killed 24 h after infection. Flow cytometry confirmed the absence or presence of integrin subunit β3 on peripheral blood neutrophils in β3^-/- or WT mice, respectively. Neutrophil numbers in bronchoalveolar lavage (BAL) from infected β3^-/- and WT mice showed no differences. Neutrophil numbers in BAL of infected WT mice treated with β3 antibody were lower compared with those without antibody but similar to those of mice administered isotype-matched antibody. Many neutrophils were present in the perivascular spaces of the lungs in β3^-/- mice. Lungs from infected β3^-/- mice had negligible mitogen-activated protein kinase expression compared with those of infected WT mice. Thus, integrin β3 or its heterodimer αvβ3 is not critical for neutrophil migration into lungs infected with S. pneumoniae.     

Trypanosoma cruzi and Its Soluble Antigens Induce NET Release by Stimulating Toll-Like Receptors

Neutrophils release fibrous traps of DNA, histones, and granule proteins known as neutrophil extracellular traps (NETs), which contribute to microbicidal killing and have been implicated in autoimmunity. The role of NET formation in the host response to nonbacterial pathogens is not well-understood. In this study, we investigated the release of NETs by human neutrophils upon their interaction with Trypanosoma cruzi (Y strain) parasites. Our results showed that human neutrophils stimulated by T. cruzi generate NETs composed of DNA, histones, and elastase. The release occurred in a dose-, time-, and reactive oxygen species-dependent manner to decrease trypomastigote and increase amastigote numbers of the parasites without affecting their viability. NET release was decreased upon blocking with antibodies against Toll-like receptors 2 and 4. In addition, living parasites were not mandatory in the release of NETs induced by T. cruzi, as the same results were obtained when molecules from its soluble extract were tested. Our results increase the understanding of the stimulation of NETs by parasites, particularly T. cruzi. We suggest that contact of T. cruzi with NETs during Chagas’s disease can limit infection by affecting the infectivity/pathogenicity of the parasite.