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.

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.

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.

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.

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.

The effect of ibrutinib on neutrophil and γδ T cell functions.

Ibrutinib is a BTK/ITK inhibitor with efficacy for the treatment of various lymphoid cancers, including CLL. Considering that innate and adaptative immune defects are a dominant feature of CLL patients, we evaluated whether in vitro ibrutinib affects the survival and function of neutrophils and γδ T cells, key players of the early immune response against microbes. Neutrophils and γδ T cells were obtained from peripheral blood of healthy donors and CLL patients. We found that ibrutinib reduces the production of reactive oxygen species (ROS) and bacteria killing capacity, and slightly impairs neutrophil extracellular traps (NETs) production without affecting bacteria-uptake and CD62L-downregulation induced by fMLP or aggregated IgG. In addition, ibrutinib reduces γδ T cell activation and CD107a degranulation induced by phosphoantigens or anti-CD3. These findings are in agreement with previous data suggesting that ibrutinib interferes with the protective immune response to pathogens, particularly Mycobacteria and Aspergillus.

Role of Shiga Toxins in Cytotoxicity and Immunomodulatory Effects of Escherichia coli O157:H7 during Host-Bacterial Interactions in vitro.

Enterohemorrhagic Escherichia coli (EHEC) strains are food-borne pathogens that can cause different clinical conditions. Shiga toxin 2a and/or 2c (Stx2)-producing E. coli O157:H7 is the serotype most frequently associated with severe human disease. In this work we analyzed the hypothesis that host cells participate in Stx2 production, cell damage, and inflammation during EHEC infection. With this aim, macrophage-differentiated THP-1 cells and the intestinal epithelial cell line HCT-8 were incubated with E. coli O157:H7. A time course analysis of cellular and bacterial survival, Stx2 production, stx2 transcription, and cytokine secretion were analyzed in both human cell lines. We demonstrated that macrophages are able to internalize and kill EHEC. Simultaneously, Stx2 produced by internalized bacteria played a major role in macrophage death. In contrast, HCT-8 cells were completely resistant to EHEC infection. Besides, macrophages and HCT-8 infected cells produce IL-1ß and IL-8 inflammatory cytokines, respectively. At the same time, bacterial stx2-specific transcripts were detected only in macrophages after EHEC infection. The interplay between bacteria and host cells led to Stx production, triggering of inflammatory response and cell damage, all of which could contribute to a severe outcome after EHEC infections.

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.

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.

Short-Term Fever-Range Hyperthermia Accelerates NETosis and Reduces Pro-inflammatory Cytokine Secretion by Human Neutrophils.

Fever is a hallmark of infections and inflammatory diseases, represented by an increase of 1-4°C in core body temperature. Fever-range hyperthermia (FRH) has been shown to increase neutrophil recruitment to local sites of infection. Here, we evaluated the impact of a short period (1 h) of FRH (STFRH) on pro-inflammatory and bactericidal human neutrophil functions. STFRH did not affect neutrophil spontaneous apoptosis but reverted the lipopolysaccharide (LPS)-induced anti-apoptotic effect compared with that under normothermic conditions. Furthermore, STFRH accelerated phorbol myristate acetate (PMA)-induced NETosis evaluated either by the nuclear DNA decondensation at 2 h post-stimulation or by the increase in extracellular DNA that colocalized with myeloperoxidase (MPO) at 4 h post-stimulation. Increased NETosis upon STFRH was associated with an increase in reactive oxygen species (ROS) production but not in autophagy levels. STFRH also increased NETosis in response to Pseudomonas aeruginosa challenge but moderately reduced its phagocytosis. However, these STFRH-induced effects did not influence the ability of neutrophils to kill bacteria after 4 h of co-culture. STFRH also significantly reduced neutrophil capacity to release the pro-inflammatory cytokines chemokine (C-X-C motif) ligand 8/interleukin 8 (CXCL8/IL-8) and IL-1ß in response to LPS and P. aeruginosa challenge. Altogether, these results indicate that a short and mild hyperthermal period is enough to modulate neutrophil responses to bacterial encounter. They also suggest that fever spikes during bacterial infections might lead neutrophils to trigger an emergency response promoting neutrophil extracellular trap (NET) formation to ensnare bacteria in order to wall off the infection and to reduce their release of pro-inflammatory cytokines in order to limit the inflammatory response.

The mucosal surfaces of both eyes are immunologically linked by a neurogenic inflammatory reflex involving TRPV1 and substance P.

Immunological interdependence between the two eyes has been reported for the cornea and the retina but not for the ocular mucosal surface. Intriguingly, patients frequently report ocular surface-related symptoms in the other eye after unilateral ocular surgery. Here we show how unilateral eye injuries in mice affect the mucosal immune response of the opposite ocular surface. We report that, despite the lack of lymphatic cross-drainage, a neurogenic inflammatory reflex in the contralateral conjunctiva is sufficient to increase, first, epithelial nuclear factor kappa B signaling, then, dendritic cell maturation, and finally, expansion of effector, instead of regulatory, T cells in the draining lymph node, leading to disrupted ocular mucosal tolerance. We also show that damage to ocular surface nerves is required. Using pharmacological inhibitors and agonists, we identified transient receptor potential vanilloid 1 (TRPV1) channel as the receptor sensing tissue damage in the injured eye and substance P released in the opposite ocular surface as the effector of the sympathetic response. Finally, blocking either step prevented subsequent ocular allergic reactions in the opposite eye in a unilateral corneal alkali burn model. This study demonstrates that both ocular surfaces are immunologically linked and suggests potential therapeutic targets for intervention.

Autophagy Mediates Interleukin-1ß Secretion in Human Neutrophils.

Interleukin-1ß (IL-1ß), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1ß in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1ß by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1ß secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1ß secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1ß secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1ß secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1ß was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1ß-LC3B in a vesicular compartment peaked before IL-1ß increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1ß and LC3B and then promoted neutrophil IL-1ß secretion. In addition, specific ELISAs indicated that although both IL-1ß and pro-IL-1ß are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1ß secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1ß secretion. Moreover, IL-1ß could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1ß intersect azurophil granules content and that serine proteases also regulate IL-1ß secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1ß secretion in human neutrophils.

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.

Neutrophil Extracellular Traps Stimulate Proinflammatory Responses in Human Airway Epithelial Cells.

Tissue injury leads to the release of uric acid (UA). At high local concentrations, UA can form monosodium urate crystals (MSU). MSU and UA stimulate neutrophils to release extracellular traps (NET). Here, we investigated whether these NET could be involved in the development of inflammation by stimulating cytokine release by airway epithelial cells. We found that NET significantly increased the secretion of CXCL8/IL-8 and IL-6 by alveolar and bronchial epithelial cells. These effects were not observed when NETosis was inhibited by Diphenyleneiodonium, elastase inhibitor, or Cl-amidine. Similar findings were made with NET induced by cigarette smoke extract, suggesting that NET proinflammatory capacity is independent of the inducing stimulus. Furthermore, NET affected neither the viability and morphology of epithelial cells nor the barrier integrity of polarized cells. The epithelial stimulatory capacity of NET was not affected by degradation of DNA with micrococcal nuclease, treatment with heparin, or inhibition of the elastase immobilized to DNA, but it was significantly reduced by pretreatment with an anti-HMGB-1 blocking antibody. Altogether, our findings indicate that NET exert direct proinflammatory effects on airway epithelial cells that might contribute in vivo to the further recruitment of neutrophils and the perpetuation of inflammation upon lung tissue damage.

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.

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.

Fever-range hyperthermia improves the anti-apoptotic effect induced by low pH on human neutrophils promoting a proangiogenic profile.

Neutrophils have the shortest lifespan among leukocytes and usually die via apoptosis, limiting their deleterious potential. However, this tightly regulated cell death program can be modulated by pathogen-associated molecular patterns (PAMPs), danger-associated molecular pattern (DAMPs), and inflammatory cytokines. We have previously reported that low pH, a hallmark of inflammatory processes and solid tumors, moderately delays neutrophil apoptosis. Here we show that fever-range hyperthermia accelerates the rate of neutrophil apoptosis at neutral pH but markedly increases neutrophil survival induced by low pH. Interestingly, an opposite effect was observed in lymphocytes; hyperthermia plus low pH prevents lymphocyte activation and promotes the death of lymphocytes and lymphoid cell lines. Analysis of the mechanisms through which hyperthermia plus low pH increased neutrophil survival revealed that hyperthermia further decreases cytosolic pH induced by extracellular acidosis. The fact that two Na+/H+ exchanger inhibitors, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and amiloride, reproduced the effects induced by hyperthermia suggested that it prolongs neutrophil survival by inhibiting the Na+/H+ antiporter. The neutrophil anti-apoptotic effect induced by PAMPs, DAMPs, and inflammatory cytokines usually leads to the preservation of the major neutrophil effector functions such as phagocytosis and reactive oxygen species (ROS) production. In contrast, our data revealed that the anti-apoptotic effect induced by low pH and hyperthermia induced a functional profile characterized by a low phagocytic activity, an impairment in ROS production and a high ability to suppress T-cell activation and to produce the angiogenic factors VEGF, IL-8, and the matrix metallopeptidase 9 (MMP-9). These results suggest that acting together fever and local acidosis might drive the differentiation of neutrophils into a profile able to promote both cancer progression and tissue repair during the late phase of inflammation, two processes that are strongly dependent on the local production of angiogenic factors by infiltrating immune cells.