Regulation of human neutrophil IL-1ß secretion induced by Escherichia coli O157:H7 responsible for hemolytic uremic syndrome.

Shiga-toxin producing Escherichia coli (STEC) infections can cause from bloody diarrhea to Hemolytic Uremic Syndrome. The STEC intestinal infection triggers an inflammatory response that can facilitate the development of a systemic disease. We report here that neutrophils might contribute to this inflammatory response by secreting Interleukin 1 beta (IL-1ß). STEC stimulated neutrophils to release elevated levels of IL-1ß through a mechanism that involved the activation of caspase-1 driven by the NLRP3-inflammasome and neutrophil serine proteases (NSPs). Noteworthy, IL-1ß secretion was higher at lower multiplicities of infection. This secretory profile modulated by the bacteria:neutrophil ratio, was the consequence of a regulatory mechanism that reduced IL-1ß secretion the higher were the levels of activation of both caspase-1 and NSPs, and the production of NADPH oxidase-dependent reactive oxygen species. Finally, we also found that inhibition of NSPs significantly reduced STEC-triggered IL-1ß secretion without modulating the ability of neutrophils to kill the bacteria, suggesting NSPs might represent pharmacological targets to be evaluated to limit the STEC-induced intestinal inflammation.

Transient Receptor Potential Vanilloid-1 Channels Facilitate Axonal Degeneration of Corneal Sensory Nerves in Dry Eye.

Corneal nerve impairment contributes significantly to dry eye disease (DED) symptoms and is thought to be secondary to corneal epithelial damage. Transient receptor potential vanilloid-1 (TRPV1) channels abound in corneal nerve fibers and respond to inflammation-derived ligands, which increase in DED. TRPV1 overactivation promotes axonal degeneration in vitro, but whether it participates in DED-associated corneal nerve dysfunction is unknown. To explore this, DED was surgically induced in wild-type and TRPV1-knockout mice, which developed comparable corneal epithelial damage and reduced tear secretion. However, corneal mechanosensitivity decreased progressively only in wild-type DED mice. Sensitivity to capsaicin (TRPV1 agonist) increased in wild-type DED mice, and consistently, only this strain displayed DED-induced pain signs. Wild-type DED mice exhibited nerve degeneration throughout the corneal epithelium, whereas TRPV1-knockout DED mice only developed a reduction in the most superficial nerve endings that failed to propagate to the deeper subbasal corneal nerves. Pharmacologic TRPV1 blockade reproduced these findings in wild-type DED mice, whereas CD4+ T cells from both strains were equally pathogenic when transferred, ruling out a T-cell-mediated effect of TRPV1 deficiency. These data show that ocular desiccation triggers superficial corneal nerve damage in DED, but proximal propagation of axonal degeneration requires TRPV1 expression. Local inflammation sensitized TRPV1 channels, which increased ocular pain. Thus, ocular TRPV1 overactivation drives DED-associated corneal nerve impairment.

An ocular Th1 immune response promotes corneal nerve damage independently of the development of corneal epitheliopathy.

Proper sight is not possible without a smooth, transparent cornea, which is highly exposed to environmental threats. The abundant corneal nerves are interspersed with epithelial cells in the anterior corneal surface and are instrumental to corneal integrity and immunoregulation. Conversely, corneal neuropathy is commonly observed in some immune-mediated corneal disorders but not in others, and its pathogenesis is poorly understood. Here we hypothesized that the type of adaptive immune response may influence the development of corneal neuropathy. To test this, we first immunized OT-II mice with different adjuvants that favor T helper (Th)1 or Th2 responses. Both Th1-skewed mice (measured by interferon-γ production) and Th2-skewed (measured by interleukin-4 production) developed comparable ocular surface inflammation and conjunctival CD4+ T cell recruitment but no appreciable corneal epithelial changes upon repeated local antigenic challenge. Th1-skewed mice showed decreased corneal mechanical sensitivity and altered corneal nerve morphology (signs of corneal neuropathy) upon antigenic challenge. However, Th2-skewed mice also developed milder corneal neuropathy immediately after immunization and independently of ocular challenge, suggestive of adjuvant-induced neurotoxicity. All these findings were confirmed in wild-type mice. To circumvent unwanted neurotoxicity, CD4+ T cells from immunized mice were adoptively transferred to T cell-deficient mice. In this setup, only Th1-transferred mice developed corneal neuropathy upon antigenic challenge. To further delineate the contribution of each profile, CD4+ T cells were polarized in vitro to either Th1, Th2, or Th17 cells and transferred to T cell-deficient mice. Upon local antigenic challenge, all groups had commensurate conjunctival CD4+ T cell recruitment and macroscopic ocular inflammation. However, none of the groups developed corneal epithelial changes and only Th1-transferred mice showed signs of corneal neuropathy. Altogether, the data show that corneal nerves, as opposed to corneal epithelial cells, are sensitive to immune-driven damage mediated by Th1 CD4+ T cells in the absence of other pathogenic factors. These findings have potential therapeutic implications for ocular surface disorders.

Interplay between neutrophils and trophoblast cells conditions trophoblast function and triggers vascular transformation signals.

Normal placentation entails highly regulated interactions of maternal leukocytes with vascular and trophoblast cells to favor vascular transformation. Neutrophil activation and neutrophil extracellular trap (NET) formation associate with poor placentation and severe pregnancy complications. To deepen into the mechanisms of trophoblast-neutrophil interaction, we explored the effects of NETs on trophoblast cell function and, conversely, whether trophoblast cell-derived factors condition neutrophils to favor angiogenesis and anti-inflammatory signals required for fetal growth. NETs isolated from activated neutrophils hindered trophoblast cell migration. Trophoblast conditioned media prevented the effect as well as the vasoactive intestinal peptide (VIP) known to regulate trophoblast and neutrophil function. On the other hand, factors released by trophoblast cells and VIP shaped neutrophils to a proangiogenic profile with increased vascular endothelial growth factor synthesis and increased capacity to promote vascular transformation. Results presented here provide novel clues to reconstruct the interaction of trophoblast cells and neutrophils in vivo during placentation in humans.

Transient tear hyperosmolarity disrupts the neuroimmune homeostasis of the ocular surface and facilitates dry eye onset.

Dry eye disease (DED) is a highly prevalent ocular surface disorder with neuroimmune pathophysiology. Tear hyperosmolarity (THO), a frequent finding in affected patients, is considered a key element in DED pathogenesis, yet existing animal models are based on subjecting the ocular surface to the more complex desiccating stress - decreased tear production and/or increased evaporation - instead of strict hyperosmolar stress. Here we characterized a murine model of THO that does not involve desiccating stress, thus allowing us to dissect the contribution of THO to DED. Our results showed that THO is sufficient to disrupt neuroimmune homeostasis of the ocular surface in mice, and thus reproduce many sub-clinical DED findings. THO activated nuclear factor-κB signalling in conjunctival epithelial cells and increased dendritic cell recruitment and maturation, leading to more activated (CD69+ ) and memory (CD62lo CD44hi) CD4+ T-cells in the eye-draining lymph nodes. Ultimately, THO impaired the development of ocular mucosal tolerance to a topical surrogate antigen in a chain of events that included epithelial nuclear factor-κB signalling and activation of transient receptor potential vanilloid 1 as the probable hypertonicity sensor. Also, THO reduced the density of corneal intraepithelial nerves and terminals, and sensitized the ocular surface to hypertonicity. Finally, the adoptive transfer of T-cells from THO mice to naïve recipients under mild desiccating stress favoured DED development, showing that THO is enough to trigger an actual pathogenic T-cell response. Our results altogether demonstrate that THO is a critical initiating factor in DED development.

Modulation of γδ T-cell activation by neutrophil elastase.

γδ T cells are non-conventional, innate-like T cells, characterized by a restricted T-cell receptor repertoire. They participate in protective immunity responses against extracellular and intracellular pathogens, tumour surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up-regulation of CD69 expression, and the production of interferon-γ and tumour necrosis factor-α induced by anti-CD3 antibodies was potentiated by neutrophils. We found that inhibition of caspase-1 and neutralization of interleukin-18 did not affect neutrophil-mediated modulation. By contrast, the treatment with serine protease inhibitors prevented the potentiation of γδ T-cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T-cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T-cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the protease-activated receptor, PAR1, because the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T-cell activation.

A Brucella spp. Protease Inhibitor Limits Antigen Lysosomal Proteolysis, Increases Cross-Presentation, and Enhances CD8+ T Cell Responses.

In this study, we demonstrate that the unlipidated (U) outer membrane protein (Omp) 19 from Brucella spp. is a competitive inhibitor of human cathepsin L. U-Omp19 inhibits lysosome cathepsins and APC-derived microsome activity in vitro and partially inhibits lysosomal cathepsin L activity within live APCs. Codelivery of U-Omp19 with the Ag can reduce intracellular Ag digestion and increases Ag half-life in dendritic cells (DCs). U-Omp19 retains the Ag in Lamp-2(+) compartments after its internalization and promotes a sustained expression of MHC class I/peptide complexes in the cell surface of DCs. Consequently, U-Omp19 enhances Ag cross-presentation by DCs to CD8(+) T cells. U-Omp19 s.c. delivery induces the recruitment of CD11c(+)CD8α(+) DCs and monocytes to lymph nodes whereas it partially limits in vivo Ag proteolysis inside DCs. Accordingly, this protein is able to induce CD8(+) T cell responses in vivo against codelivered Ag. Antitumor responses were elicited after U-Omp19 coadministration, increasing survival of mice in a murine melanoma challenge model. Collectively, these results indicate that a cysteine protease inhibitor from bacterial origin could be a suitable component of vaccine formulations against tumors.

Neutrophils from chronic lymphocytic leukemia patients exhibit an increased capacity to release extracellular traps (NETs).

Chronic lymphocytic leukemia (CLL) is characterized by immune defects that contribute to a high rate of infections and autoimmune cytopenias. Neutrophils are the first line of innate immunity and respond to pathogens through multiple mechanisms, including the release of neutrophil extracellular traps (NETs). These web-like structures composed of DNA, histones, and granular proteins are also produced under sterile conditions and play important roles in thrombosis and autoimmune disorders. Here we show that neutrophils from CLL patients are more prone to release NETs compared to those from age-matched healthy donors (HD). Increased generation of NETs was not due to higher levels of elastase, myeloperoxidase, or reactive oxygen species production. Instead, we found that plasma from CLL patients was able to prime neutrophils from HD to generate higher amounts of NETs upon activation. Plasmatic IL-8 was involved in the priming effect since its depletion reduced plasma capacity to enhance NETs release. Finally, we found that culture with NETs delayed spontaneous apoptosis and increased the expression of activation markers on leukemic B cells. Our study provides new insights into the immune dysregulation in CLL and suggests that the chronic inflammatory environment typical of CLL probably underlies this inappropriate neutrophil priming.

Trophoblast cells inhibit neutrophil extracellular trap formation and enhance apoptosis through vasoactive intestinal peptide-mediated pathways.

STUDY QUESTION: Do human trophoblast cells modulate neutrophil extracellular trap (NET) formation, reactive oxygen species (ROS) synthesis and neutrophil apoptosis through mechanisms involving vasoactive intestinal peptide (VIP)? SUMMARY ANSWER: Trophoblast cells inhibited NET formation and ROS synthesis and enhanced neutrophil apoptosis through VIP-mediated pathways in a model of maternal-placental interaction. WHAT IS KNOWN ALREADY: Immune homeostasis maintenance at the maternal-placental interface is mostly coordinated by trophoblast cells. Neutrophil activation and NET formation increases in pregnancies complicated by exacerbated pro-inflammatory responses. VIP has anti-inflammatory and immunosuppressant effects and is synthesized by trophoblast cells. STUDY DESIGN, SIZE, DURATION: This is a laboratory-based observational study that sampled circulating neutrophils from 50 healthy volunteers to explore their response in vitro to factors derived from human trophoblast cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Peripheral blood neutrophils were isolated from healthy volunteers and tested in vitro with first trimester trophoblast cell line (Swan-71 and HTR8) conditioned media (CM) or with VIP. The effect of VIP and trophoblast CM on NET formation was assessed by co-localization of elastase and DNA by confocal microscopy, DNA release and elastase activity measurement. Neutrophil apoptosis was determined by flow cytometry or fluorescence microscopy. ROS formation was assessed by flow cytometry with a fluorescent probe. VIP silencing was performed by siRNA transfection. For phagocytosis of apoptotic neutrophils, autologous monocytes were sampled, and engulfment and cytokines were assessed by flow cytometry and ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: Trophoblast CM and 10 nM VIP promoted neutrophil deactivation by preventing phorbol myristate acetate-induced NET formation and ROS synthesis while they increased neutrophil spontaneous apoptosis and reversed the anti-apoptotic effect of lipopolysaccharide (all P < 0.05 versus control). The effects of trophoblast CM were prevented by a VIP antagonist or when VIP knocked-down trophoblast cells were used (P < 0.05 versus control). Neutrophils driven to apoptosis by trophoblast CM could be rapidly engulfed by monocytes without increasing IL-12 production. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The mechanisms of neutrophil deactivation by trophoblast VIP are based on the results obtained with neutrophils drawn from peripheral blood of healthy individuals interacting with trophoblast cell lines in vitro. These studies were designed to investigate biological processes at the cellular and molecular level; therefore, they have the limitations of studies in vitro and it is not possible to ascertain if these mechanisms operate similarly in vivo. We tested 50 neutrophil samples from healthy volunteers that have a normal variability in their responses. Cell lines derived from human trophoblast were used, and we cannot rule out a differential behavior of trophoblast cells in contact with neutrophils in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Results presented here are consistent with an active mechanism through which neutrophils in contact with trophoblast cells would be deactivated and silently cleared by decidual macrophages throughout pregnancy. They support a novel immunomodulatory role of trophoblast VIP on neutrophils at the placenta, providing new clues for pharmacological targeting of immune and trophoblast cells in pregnancy complications associated with exacerbated inflammation. STUDY FUNDING/COMPETING INTERESTS: This work was funded by the National Agency of Sciences and Technology (PICT 2011-0144, 2014-0657 and 2013-2177) and University of Buenos Aires (UBACyT 20020130100040BA, 20020150100161BA and 20020130100744BA). The authors declare no competing interests.

Mucosal tolerance disruption favors disease progression in an extraorbital lacrimal gland excision model of murine dry eye.

Dry eye is a highly prevalent immune disorder characterized by a dysfunctional tear film and a Th1/Th17 T cell response at the ocular surface. The specificity of these pathogenic effector T cells remains to be determined, but auto-reactivity is considered likely. However, we have previously shown that ocular mucosal tolerance to an exogenous antigen is disrupted in a scopolamine-induced murine dry eye model and that it is actually responsible for disease progression. Here we report comparable findings in an entirely different murine model of dry eye that involves resection of the extraorbital lacrimal glands but no systemic muscarinic receptor blockade. Upon ocular instillation of ovalbumin, a delayed breakdown in mucosal tolerance to this antigen was observed in excised but not in sham-operated mice, which was mediated by interferon γ- and interleukin 17-producing antigen-specific T cells. Consistently, antigen-specific regulatory T cells were detectable in sham-operated but not in excised mice. As for other models of ocular surface disorders, epithelial activation of the NF-κB pathway by desiccating stress was determinant in the mucosal immune outcome. Underscoring the role of mucosal tolerance disruption in dry eye pathogenesis, its prevention by a topical NF-κB inhibitor led to reduced corneal damage in excised mice. Altogether these results show that surgically originated desiccating stress also initiates an abnormal Th1/Th17 T cell response to harmless exogenous antigens that reach the ocular surface. This event might actually contribute to corneal damage and challenges the conception of dry eye as a strictly autoimmune disease.

Neutrophils suppress γδ T-cell function.

γδ T cells have been shown to stimulate the recruitment and activation of neutrophils through the release of a range of cytokines and chemokines. Here, we investigated the reverse relationship, showing that human neutrophils suppress the function of human blood γδ T cells. We show that the upregulation of CD25 and CD69 expression, the production of IFN-γ, and the proliferation of γδ T cells induced by (E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate are inhibited by neutrophils. Spontaneous activation of γδ T cells in culture is also suppressed by neutrophils. We show that inhibitors of prostaglandin E2 and arginase I do not exert any effect, although, in contrast, catalase prevents the suppression of γδ T cells induced by neutrophils, suggesting the participation of neutrophil-derived ROS. We also show that the ROS-generating system xanthine/xanthine oxidase suppresses γδ T cells in a similar fashion to neutrophils, while neutrophils from chronic granulomatous disease patients only weakly inhibit γδ T cells. Our results reveal a bi-directional cross-talk between γδ T cells and neutrophils: while γδ T cells promote the recruitment and the activation of neutrophils to fight invading pathogens, neutrophils in turn suppress the activation of γδ T cells to contribute to the resolution of inflammation.

NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL-1ß secretion but not in inflammasome activation.

Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin-1 beta (IL-1ß) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL-1ß processing is still controversial and has not been previously studied in neutrophils. We report here that IL-1ß processing in human neutrophils is dependent on caspase-1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase-1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL-1ß; whereas ROS negatively controlled caspase-1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL-1ß out of the cell, a role never previously described. The complex ROS-mediated regulation of neutrophil IL-1ß secretion might constitute a physiological mechanism to control IL-1ß-dependent inflammatory processes where neutrophils play a crucial role.

Poly(allylamine)-tripolyphosphate polymeric nanoparticle as an NLRP3-dependent systemic adjuvant for the vaccine development.

Nanotechnology plays a crucial role in vaccine development and provides the opportunity to design functional nanoparticles (Np) of different compositions, sizes, charges and surface properties for biomedical applications. The present study aims to evaluate a complex coacervate-like Np composed of poly(allylamine hydrochloride) (PAH) and tripolyphosphate (Tpp) as a safe vehicle and adjuvant for systemic vaccines. We investigated the activation of different antigen-presenting cells (APCs) with Np-PAH and its adjuvanticity in Balbc/c and different KO mice that were intraperitoneally immunized with Np-OVA. We found that Np-PAH increased the expression of CD86 and MHCII and promoted the production and secretion of interleukin-1ß (IL-1ß) and IL-18 through the inflammasome NLRP3 when macrophages and dendritic cells were co-incubated with LPS and Np-PAH. We evidenced an unconventional IL-1ß release through the autophagosome pathway. The inhibition of autophagy with 3-methyladenine reduced the LPS/Np-PAH-induced IL-1ß secretion. Additionally, our findings showed that the systemic administration of mice with Np-OVA triggered a significant induction of serum OVA-specific IgG and IgG2a, an increased secretion of IFN-γ by spleen cells, and high frequencies of LT CD4 + IFN-γ + and LT CD8 + IFN-γ + . In conclusion, our findings show that PAH-based Np promoted the inflammasome activation of innate cells with Th1-dependent adjuvant properties, making them valuable for formulating of novel preventive or therapeutic vaccines for infectious and non-infectious diseases.

Low extracellular pH stimulates the production of IL-1ß by human monocytes.

The development of acidic environments is a hallmark of inflammatory processes of different etiology. We have previously shown that transient exposure to acidic conditions, similar to those encountered in vivo, induces the activation of neutrophils and the phenotypic maturation of dendritic cells. We here report that extracellular acidosis (pH 6.5) selectively stimulates the production and the secretion of IL-1ß by human monocytes without affecting the production of TNF-α, IL-6 and the expression of CD40, CD80, CD86, and HLA-DR. Stimulation of IL-1ß production by pH 6.5-treated monocytes was shown to be dependent on caspase-1 activity, and it was also observed using peripheral blood mononuclear cells instead of isolated monocytes. Contrasting with the results in monocytes, we found that pH 6.5 did not stimulate any production of IL-1ß by macrophages. Changes in intracellular pH seem to be involved in the stimulation of IL-1ß production. In fact, monocytes cultured at pH 6.5 undergo a fall in the values of intracellular pH while the inhibitor of the Na+/H+ exchanger, 5-(N-ethyl-N-isopropyl)amiloride induced both, a decrease in the values of intracellular pH and the stimulation of IL-1ß production. Real time quantitative PCR assays indicated that monocytes cultured either at pH 6.5 or in the presence of 5-(N-ethyl-N-isopropyl)amiloride expressed higher levels of pro-IL-1ß mRNA suggesting that low values of intracellular pH enhance the production of IL-1ß, at least in part, by stimulating the synthesis of its precursor.

Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms.

We previously demonstrated that extracellular bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Biofilms are microbial communities enclosed in a polymeric matrix that play a critical role in the pathogenesis of many infectious diseases. Because extracellular DNA is a key component of biofilms of different bacterial species, the aim of this study was to determine whether it plays a role in the ability of biofilms to induce human neutrophil activation. We found that degradation of matrix extracellular DNA with DNase I markedly reduced the capacity of Pseudomonas aeruginosa biofilms to induce the release of the neutrophil proinflammatory cytokines IL-8 and IL-1beta (>75%); reduced the upregulation of neutrophil activation markers CD18, CD11b, and CD66b (p < 0.001); reduced the number of bacteria phagocytosed per neutrophil contacting the biofilm; and reduced the production of neutrophil extracellular traps. Consistent with these findings, we found that biofilms formed by the lasI rhlI P. aeruginosa mutant strain, exhibiting a very low content of matrix extracellular DNA, displayed a lower capacity to stimulate the release of proinflammatory cytokines by neutrophils, which was not decreased further by DNase I treatment. Together, our findings support that matrix extracellular DNA is a major proinflammatory component of P. aeruginosa biofilms.

The interplay between serine proteases and caspase-1 regulates the autophagy-mediated secretion of Interleukin-1 beta in human neutrophils.

Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1ß) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1ß) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1ß is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1ß processing in these cells. We found that although caspase-1 is required for IL-1ß secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1ß processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1ß secretion.

In Vitro Identification and Isolation of Human Neutrophil Extracellular Traps.

Neutrophils release web like-structures known as neutrophil extracellular traps (NETs) that ensnare and kill microorganisms. These networks are constituted of a DNA scaffold with associated antimicrobial proteins, which are released to the extracellular space as an effective mechanism to fight against invading microorganisms. In parallel with this beneficial role to avoid microbial dissemination and wall off infections, accumulating evidence supports that under certain circumstances, NETs can exert deleterious effects in inflammatory, autoimmune, and thrombotic pathologies. Research on NET properties and their role in pathophysiological processes is a rapidly evolving and expanding field. Here, we describe a combination of methods to achieve a successful in vitro NET visualization, semiquantification, and isolation.

Neutrophil autophagy during human active tuberculosis is modulated by SLAMF1.

Neutrophils infected with Mycobacterium tuberculosis (Mtb) predominate in tuberculosis patients' lungs. Neutrophils phagocytose the pathogen, but the mechanism of pathogen elimination is controversial. Macroautophagy/autophagy, a crucial mechanism for several neutrophil functions, can be modulated by immunological mediators. The costimulatory molecule SLAMF1 can act as a microbial sensor in macrophages being also able to interact with autophagy-related proteins. Here, we demonstrate for the first time that human neutrophils express SLAMF1 upon Mtb-stimulation. Furthermore, SLAMF1 was found colocalizing with LC3B+ vesicles, and activation of SLAMF1 increased neutrophil autophagy induced by Mtb. Finally, tuberculosis patients' neutrophils displayed reduced levels of SLAMF1 and lower levels of autophagy against Mtb as compared to healthy controls. Altogether, these results indicate that SLAMF1 participates in neutrophil autophagy during active tuberculosis.Abbreviations: AFB: acid-fast bacilli; BafA1: bafilomycin A1; CLL: chronic lymphocytic leukemia; DPI: diphenyleneiodonium; EVs: extracellular vesicles; FBS: fetal bovine serum; HD: healthy donors; HR: high responder (tuberculosis patient); IFNG: interferon gamma; IL1B: interleukin 1 beta; IL17A: interleukin 17A; IL8: interleukin 8; LR: low responder (tuberculosis patient); mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK14/p38: mitogen-activated protein kinase 14; Mtb: Mycobacterium tuberculosis; Mtb-Ag: Mycobacterium tuberculosis, Strain H37Rv, whole cell lysate; NETs: neutrophils extracellular traps; PPD: purified protein derivative; ROS: reactive oxygen species; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; SLAMF1: signaling lymphocytic activation molecule family member 1; TB: tuberculosis; TLR: toll like receptor.

Flagellin delays spontaneous human neutrophil apoptosis.

Neutrophils are short-lived cells that rapidly undergo apoptosis. However, their survival can be regulated by signals from the environment. Flagellin, the primary component of the bacterial flagella, is known to induce neutrophil activation. In this study we examined the ability of flagellin to modulate neutrophil apoptosis. Neutrophils cultured for 12 and 24 h in the presence of flagellin from Salmonella typhimurium at concentrations found in pathological situations underwent a marked prevention of apoptosis. In contrast, Helicobacter pylori flagellin did not affect neutrophil survival, suggesting that Salmonella flagellin exerts the antiapoptotic effect by interacting with TLR5. The delaying in apoptosis mediated by Salmonella flagellin was coupled to higher expression levels of the antiapoptotic protein Mcl-1 and lower levels of activated caspase-3. Analysis of the signaling pathways indicated that Salmonella flagellin induced the activation of the p38 and ERK1/2 MAPK pathways as well as the PI3K/Akt pathway. Furthermore, it also stimulated IkappaBalpha degradation and the phosphorylation of the p65 subunit, suggesting that Salmonella flagellin also triggers NF-kappaB activation. Moreover, the pharmacological inhibition of ERK1/2 pathway and NF-kappaB activation partially prevented the antiapoptotic effects exerted by flagellin. Finally, the apoptotic delaying effect exerted by flagellin was also evidenced when neutrophils were cultured with whole heat-killed S. typhimurium. Both a wild-type and an aflagellate mutant S. typhimurium strain promoted neutrophil survival; however, when cultured in low bacteria/neutrophil ratios, the flagellate bacteria showed a higher capacity to inhibit neutrophil apoptosis, although both strains showed a similar ability to induce neutrophil activation. Taken together, our results indicate that flagellin delays neutrophil apoptosis by a mechanism partially dependent on the activation of ERK1/2 MAPK and NF-kappaB. The ability of flagellin to delay neutrophil apoptosis could contribute to perpetuate the inflammation during infections with flagellated bacteria.

Protein A Modulates Neutrophil and Keratinocyte Signaling and Survival in Response to Staphylococcus aureus.

The type 1 TNF-α receptor (TNFR1) has a central role in initiating both pro-inflammatory and pro-apoptotic signaling cascades in neutrophils. Considering that TNFR1 signals Staphylococcus aureus protein A (SpA), the aim of this study was to explore the interaction of this bacterial surface protein with neutrophils and keratinocytes to underscore the signaling pathways that may determine the fate of these innate immune cells in the infected tissue during staphylococcal skin infections. Using human neutrophils cultured in vitro and isogenic staphylococcal strains expressing or not protein A, we demonstrated that SpA is a potent inducer of IL-8 in neutrophils and that the induction of this chemokine is dependent on the SpA-TNFR1 interaction and p38 activation. In addition to IL-8, protein A induced the expression of TNF-α and MIP-1α highlighting the importance of SpA in the amplification of the inflammatory response. Protein A contributed to reduce neutrophil mortality prolonging their lifespan upon the encounter with S. aureus. Signaling initiated by SpA modulated the type of neutrophil cell death in vitro and during skin and soft tissue infections (SSTI) in vivo triggering the apoptotic pathway instead of necrosis. Moreover, SpA induced pro-inflammatory cytokines in keratinocytes, modulating their survival in vitro and preventing the exacerbated necrosis and ulceration of the epithelium during SSTI in vivo. Taken together, these results highlight the importance of the inflammatory signaling induced by protein A in neutrophils and skin epithelial cells. The ability of protein A to modulate the neutrophil/epithelial cell death program in the skin is of clinical relevance considering that lysis of neutrophils and epithelial cells will promote an intense inflammatory response and contribute to tissue damage, a non-desirable feature of complicated SSTI.