Safety and effectiveness of COVID-19 SPUTNIK V vaccine in dialysis patients.

INTRODUCTION: Given the vulnerability of chronic kidney disease individuals to SARS-CoV-2, nephrology societies have issued statements calling for prioritization of these patients for vaccination. It is not yet known whether COVID-19 vaccines grant the same high level of protection in patients with kidney disease compared to the non-dialysis population. The aims of this study were to evaluate the safety - measured by the adverse events potentially attributed to vaccines (ESAVI) - and the effectiveness - evaluated by the presence of antibodies - in dialysis patients immunized with the COVID-19 Sputnik V vaccine. METHODS: multicenter, observational and analytical study of a prospective cohort of hemodialysis patients from the Ciudad Autónoma de Buenos Aires participating in an official vaccination program. Dialysis requiring individuals older than 18 years, who received both components of the COVID-19 vaccine were included. RESULTS: Data from 491 patients were included in the safety analysis. ESAVI with either the first or second component was detected in 186 (37.9%, 95% CI 33.6%-42.3%). Effectiveness analysis measuring antibodies levels against SARS-CoV-2 were performed in 102 patients; 98% presented these IgG antibodies at day 21 after the second component. In patients with COVID-19 prior to vaccination, antibodies at day 21 after the first component reached almost the highest levels compared to patients without previous COVID-19, but IgG rise among patients with previous COVID-19 was lower than in those without this previous disease. CONCLUSION: The Sputnik V vaccine has been shown to be safe and effective in this patient's population.

Introducción: Dada la vulnerabilidad al SARS-CoV-2 de las personas con enfermedad renal crónica, las sociedades de nefrología han emitido declaraciones pidiendo priorizar a estos pacientes para la vacunación. Aún no se sabe si las vacunas COVID-19 confieren el mismo nivel de protección en pacientes con enfermedad renal. Los objetivos de este estudio fueron evaluar la seguridad, medida por eventos supuestamente atribuidos a las vacunas (ESAVI) y la efectividad, evaluada por la presencia de anticuerpos en pacientes en diálisis inmunizados con la vacuna COVID-19 Sputnik V. Métodos: estudio multicéntrico, observacional y analítico de una cohorte prospectiva de pacientes en hemodiálisis, en la Ciudad Autónoma de Buenos Aires, con plan de vacunación. Se incluyeron pacientes mayores de 18 años en diálisis que recibieron ambos componentes de la vacuna COVID-19. Resultados: 491 pacientes fueron incluidos en el análisis de seguridad. Se detectó ESAVI con el primer o el segundo componente en 186 (37.9% IC 95%: 33.6%-42.3%). La efectividad medida por presencia de anticuerpos IgG contra SARS-Cov-2 se realizó en 102 pacientes, 98% presentaba IgG contra SARS-CoV-2, 21 días después del segundo componente. En pacientes con COVID-19 previo a la vacunación, los anticuerpos al día 21 del primer componente alcanzaron niveles casi mayores que en aquellos que no habían sufrido COVID-19, aunque el aumento de los niveles a los 21 días del segundo componente fue menor que en los pacientes sin COVID-19 previo. Conclusión: Los pacientes en diálisis constituyen una población vulnerable para la infección por SARS-CoV-2, por lo tanto, más allá de las recomendaciones implementadas por las unidades de diálisis, la vacunación completa es mandatoria. Se ha demostrado que la vacuna Sputnik V es segura y eficaz en esta población de pacientes.

Safety and effectiveness of COVID-19 SPUTNIK V vaccine in dialysis patients / Seguridad y efectividad de vacuna COVID-19 SPUTNIK V en pacientes en diálisis

Abstract Introduction: Given the vulnerability of chronic kidney disease individuals to SARS-CoV-2, nephrology societies have issued statements calling for prioritization of these patients for vaccination. It is not yet known whether COVID-19 vaccines grant the same high level of protection in patients with kidney disease compared to the non-dialysis population. The aims of this study were to evaluate the safety - measured by the adverse events potentially attributed to vaccines (ESAVI) - and the effectiveness - evaluated by the presence of antibodies - in dialysis patients immunized with the COVID-19 Sputnik V vaccine. Methods: multicenter, ob servational and analytical study of a prospective cohort of hemodialysis patients from the Ciudad Autónoma de Buenos Aires participating in an official vaccination program. Dialysis requiring individuals older than 18 years, who received both components of the COVID-19 vaccine were included. Results: Data from 491 patients were included in the safety analysis. ESAVI with either the first or second component was detected in 186 (37.9%, 95% CI 33.6%-42.3%). Effectiveness analysis measuring antibodies levels against SARS-CoV-2 were performed in 102 patients; 98% presented these IgG antibodies at day 21 after the second component. In patients with COVID-19 prior to vaccination, antibodies at day 21 after the first component reached almost the highest levels compared to patients without previous COVID-19, but IgG rise among patients with previous COVID-19 was lower than in those without this previous disease. Conclusion: The Sputnik V vaccine has been shown to be safe and effective in this patient's population.

Resumen Introducción: Dada la vulnerabilidad al SARS-CoV-2 de las personas con enfermedad renal crónica, las sociedades de nefrología han emitido declaraciones pidiendo priorizar a estos pacientes para la vacunación. Aún no se sabe si las vacunas COVID-19 confieren el mismo nivel de protección en pacientes con enfermedad renal. Los objetivos de este estudio fueron evaluar la seguridad, medida por eventos supuestamente atribuidos a las vacunas (ESAVI) y la efectividad, evaluada por la presencia de anticuerpos en pacientes en diálisis inmuniza dos con la vacuna COVID-19 Sputnik V. Métodos: estudio multicéntrico, observacional y analítico de una cohorte prospectiva de pacientes en hemodiálisis, en la Ciudad Autónoma de Buenos Aires, con plan de vacunación. Se incluyeron pacientes mayores de 18 años en diálisis que recibieron ambos componentes de la vacuna COVID-19. Resultados: 491 pacientes fueron incluidos en el análisis de seguridad. Se detectó ESAVI con el primer o el segundo componente en 186 (37.9% IC 95%: 33.6%-42.3%). La efectividad medida por presencia de anticuerpos IgG contra SARS-Cov-2 se realizó en 102 pacientes, 98% presentaba IgG contra SARS-CoV-2, 21 días después del segundo componente. En pacientes con COVID-19 previo a la vacunación, los anticuerpos al día 21 del primer componente alcanzaron niveles casi mayores que en aquellos que no habían sufrido COVID-19, aunque el aumento de los niveles a los 21 días del segundo componente fue menor que en los pacientes sin COVID-19 previo. Conclusión: Los pacientes en diálisis constituyen una población vulnerable para la infección por SARS-CoV-2, por lo tanto, más allá de las recomendaciones implementadas por las unidades de diálisis, la vacunación completa es mandatoria. Se ha demostrado que la vacuna Sputnik V es segura y eficaz en esta población de pacientes.

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.

CD32 Ligation Promotes the Activation of CD4+ T Cells.

Low affinity receptors for the Fc portion of IgG (FcγRs) represent a critical link between innate and adaptive immunity. Immune complexes (ICs) are the natural ligands for low affinity FcγRs, and high levels of ICs are usually detected in both, chronic viral infections and autoimmune diseases. The expression and function of FcγRs in myeloid cells, NK cells and B cells have been well characterized. By contrast, there are controversial reports about the expression and function of FcγRs in T cells. Here, we demonstrated that ~2% of resting CD4+ T cells express cell surface FcγRII (CD32). Analysis of CD32 expression in permeabilized cells revealed an increased proportion of CD4+CD32+ T cells (~9%), indicating that CD4+ T cells store a CD32 cytoplasmic pool. Activation of CD4+ T cells markedly increased the expression of CD32 either at the cell surface or intracellularly. Analysis of CD32 mRNA transcripts in activated CD4+ T cells revealed the presence of both, the stimulatory FcγRIIa (CD32a) and the inhibitory FcγRIIb (CD32b) isoforms of CD32, being the CD32a:CD32b mRNA ratio ~5:1. Consistent with this finding, we found not only that CD4+ T cells bind aggregated IgG, used as an IC model, but also that CD32 ligation by specific mAb induced a strong calcium transient in CD4+ T cells. Moreover, we found that pretreatment of CD4+ T cells with immobilized IgG as well as cross-linking of CD32 by specific antibodies increased both, the proliferative response of CD4+ T cells and the release of a wide pattern of cytokines (IL-2, IL-5, IL-10, IL-17, IFN-γ, and TNF-α) triggered by either PHA or anti-CD3 mAb. Collectively, our results indicate that ligation of CD32 promotes the activation of CD4+ T cells. These findings suggest that ICs might contribute to the perpetuation of chronic inflammatory responses by virtue of its ability to directly interact with CD4+ T cells through CD32a, promoting the activation of T cells into different inflammatory profiles.

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.

Galectin-1: A Jack-of-All-Trades in the Resolution of Acute and Chronic Inflammation.

Regulatory signals provide negative input to immunological networks promoting resolution of acute and chronic inflammation. Galectin-1 (Gal-1), a member of a family of evolutionarily conserved glycan-binding proteins, displays broad anti-inflammatory and proresolving activities by targeting multiple immune cell types. Within the innate immune compartment, Gal-1 acts as a resolution-associated molecular pattern by counteracting the synthesis of proinflammatory cytokines, inhibiting neutrophil trafficking, targeting eosinophil migration and survival, and suppressing mast cell degranulation. Likewise, this lectin controls T cell and B cell compartments by modulating receptor clustering and signaling, thus serving as a negative-regulatory checkpoint that reprograms cellular activation, differentiation, and survival. In this review, we discuss the central role of Gal-1 in regulatory programs operating during acute inflammation, autoimmune diseases, allergic inflammation, pregnancy, cancer, and infection. Therapeutic strategies aimed at targeting Gal-1-glycan interactions will contribute to overcome cancer immunosuppression and reinforce antimicrobial immunity, whereas stimulation of Gal-1-driven immunoregulatory circuits will help to mitigate exuberant 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.

AT1 receptor blockade delays postlactational mammary gland involution: a novel role for the renin angiotensin system.

Angiotensin II (AngII), the main effector peptide of the renin-angiotensin system (RAS), participates in multiple biological processes, including cell growth, apoptosis, and tissue remodeling. Since AngII activates, in different cell types, signal transducing pathways that are critical for mammary gland postlactational regression, we investigated the role of the RAS during this process. We found that exogenous administration of AngII in mammary glands of lactating Balb/c mice induced epithelium apoptosis [2.9±0.5% (control) vs. 9.6±1.1% (AngII); P < 0.001] and activation of the proapoptotic factor STAT3, an effect inhibited by irbesartan, an AT(1) receptor blocker. Subsequently, we studied the expression kinetics of RAS components during involution. We found that angiotensin-converting enzyme (ACE) mRNA expression peaked 6 h after weaning (5.7-fold; P<0.01), while induction of angiotensinogen and AT(1) and AT(2) receptors expression was detected 96 h after weaning (6.2-, 10-, and 6.2-fold increase, respectively; P<0.01). To assess the role of endogenously generated AngII, mice were treated with losartan, an AT(1) receptor blocker, during mammary involution. Mammary glands from losartan-treated mice showed activation of the survival factors AKT and BCL-(XL), significantly lower LIF and TNF-α mRNA expression (P<0.05), reduced apoptosis [12.1±2.1% (control) vs. 4.8±0.7% (losartan); P<0.001] and shedding of epithelial cells, inhibition of MMP-9 activity in a dose-dependent manner (80%; P<0.05; with losartan IC(50) value of 6.9 mg/kg/d] and lower collagen deposition and adipocyte invasion causing a delayed involution compared to vehicle-treated mice. Furthermore, mammary glands of forced weaned AT(1A)- and/or AT(1B)-deficient mice exhibited retarded apoptosis of epithelial cells [6.3±0.95% (WT) vs. 3.3±0.56% (AT(1A)/AT(1B) DKO); P<0.05] with remarkable delayed postlactational regression compared to wild-type animals. Taken together, these results strongly suggest that AngII, via the AT(1) receptor, plays a major role in mouse mammary gland involution identifying a novel role for the RAS. angiotensin system.

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.

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.

Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.

In recent years it has become clear that the therapeutic properties of bone marrow-derived mesenchymal stromal cells (MSC) are related not only to their ability to differentiate into different lineages but also to their capacity to suppress the immune response. We here studied the influence of MSC on macrophage function. Using mouse thioglycolate-elicited peritoneal macrophages (M) stimulated with LPS, we found that MSC markedly suppressed the production of the inflammatory cytokines TNF-alpha, IL-6, IL-12p70 and interferon-gamma while increased the production of IL-10 and IL-12p40. Similar results were observed using supernatants from MSC suggesting that factor(s) constitutively released by MSC are involved. Supporting a role for PGE(2) we observed that acetylsalicylic acid impaired the ability of MSC to inhibit the production of inflammatory cytokines and to stimulate the production of IL-10 by LPS-stimulated M. Moreover, we found that MSC constitutively produce PGE2 at levels able to inhibit the production of TNF-alpha and IL-6 by activated M. MSC also inhibited the up-regulation of CD86 and MHC class II in LPS-stimulated M impairing their ability to activate antigen-specific T CD4+ cells. On the other hand, they stimulated the uptake of apoptotic thymocytes by M. Of note, MSC turned M into cells highly susceptible to infection with the parasite Trypanosoma cruzi increasing more than 5-fold the rate of M infection. Using a model of inflammation triggered by s.c. implantation of glass cylinders, we found that MSC stimulated the recruitment of macrophages which showed a low expression of CD86 and the MHC class II molecule Ia(b) and a high ability to produce IL-10 and IL-12p40, but not IL-12 p70. In summary, our results suggest that MSC switch M into a regulatory profile characterized by a low ability to produce inflammatory cytokines, a high ability to phagocyte apoptotic cells, and a marked increase in their susceptibility to infection by intracellular pathogens.

Tolerogenic signals delivered by dendritic cells to T cells through a galectin-1-driven immunoregulatory circuit involving interleukin 27 and interleukin 10.

Despite their central function in orchestrating immunity, dendritic cells (DCs) can respond to inhibitory signals by becoming tolerogenic. Here we show that galectin-1, an endogenous glycan-binding protein, can endow DCs with tolerogenic potential. After exposure to galectin-1, DCs acquired an interleukin 27 (IL-27)-dependent regulatory function, promoted IL-10-mediated T cell tolerance and suppressed autoimmune neuroinflammation. Consistent with its regulatory function, galectin-1 had its highest expression on DCs exposed to tolerogenic stimuli and was most abundant from the peak through the resolution of autoimmune pathology. DCs lacking galectin-1 had greater immunogenic potential and an impaired ability to halt inflammatory disease. Our findings identify a tolerogenic circuit linking galectin-1 signaling, IL-27-producing DCs and IL-10-secreting T cells, which has broad therapeutic implications in immunopathology.

GM-CSF enhances a CpG-independent pathway of neutrophil activation triggered by bacterial DNA.

We have previously demonstrated that bacterial DNA induces neutrophil activation through a CpG- and TLR9-independent but MyD88-dependent-pathway. In this study we determined that GM-CSF enhances the activation of neutrophils by bacterial DNA. Granulocyte-macrophage colony-stimulating factor increased IL-8 and IL-1beta secretion, and CD11b-upregulation induced by single-stranded bacterial DNA. It also enhanced neutrophil IL-8 production induced by double-stranded bacterial DNA, methylated single-stranded DNA, plasmid DNA, and phosphorothioated-CpG and non-CpG-oligodeoxynucleotides. Together these observations indicated that GM-CSF enhances neutrophil responses triggered by bacterial DNA in a CpG-independent fashion. We also found that GM-CSF enhanced the activation of the MAPKs p38 and ERK1/2 induced by bacterial DNA. Moreover, the pharmacological inhibition of these pathways significantly diminished GM-CSF ability to increase neutrophil activation by bacterial DNA. Finally, we observed that GM-CSF was unable to increase the activation of MyD88(-/-) neutrophils by bacterial DNA. Our findings suggest that GM-CSF modulates the CpG-independent, MyD88-dependent neutrophil response to bacterial DNA, by increasing the activation of the MAPKs p38 and ERK1/2.

Characterization of bacterial DNA binding to human neutrophil surface.

Bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Neutrophil activation does not require DNA internalization, suggesting that it results from the interaction of bacterial DNA with a neutrophil surface receptor. The aim of this study was to characterize the interaction of bacterial DNA with the neutrophil surface. Bacterial DNA binding showed saturation and was inhibited by unlabeled DNA but not by other polyanions like yeast tRNA and poly-A. Resembling the conditions under which bacterial DNA triggers neutrophil activation, binding was not modified in the presence or absence of calcium, magnesium or serum. Treatment of neutrophils with proteases not only dramatically reduced bacterial DNA binding but also inhibited neutrophil activation induced by bacterial DNA. Experiments performed with DNA samples of different lengths obtained after digestion of bacterial DNA with DNase showed that only DNA fragments greater than approximately 170-180 nucleotides competed bacterial DNA binding and retained the ability to trigger cell activation. Treatment of neutrophils with chemoattractants or conventional agonists significantly increased bacterial DNA binding. Moreover, neutrophils that underwent transmigration through human endothelial cell monolayers even in the absence of chemoattractants, exhibited higher binding levels of bacterial DNA. Together, our findings provide evidence that binding of bacterial DNA to neutrophils is a receptor-mediated process that conditions the ability of DNA to trigger cell activation. We speculate that neutrophil recognition of bacterial DNA might be modulated by the balance of agonists present at inflammatory foci. This effect might be relevant in bacterial infections with a biofilm etiology, in which extracellular DNA could function as a potent neutrophil agonist.

The early protective thymus-independent antibody response to foot-and-mouth disease virus is mediated by splenic CD9+ B lymphocytes.

Infection of mice with cytopathic foot-and-mouth disease virus (FMDV) induces a rapid and specific thymus-independent (TI) neutralizing antibody response that promptly clears the virus. Herein, it is shown that FMDV-infected dendritic cells (DCs) directly stimulate splenic innate-like CD9(+) B lymphocytes to rapidly (3 days) produce neutralizing anti-FMDV immunoglobulin M antibodies without T-lymphocyte collaboration. In contrast, neither follicular (CD9(-)) B lymphocytes from the spleen nor B lymphocytes from lymph nodes efficiently respond to stimulation with FMDV-infected DCs. The production of these protective neutralizing antibodies is dependent on DC-derived interleukin-6 (IL-6) and on CD9(+) cell-derived IL-10 secretion. In comparison, DCs loaded with UV-inactivated FMDV are significantly less efficient in directly stimulating B lymphocytes to secrete TI antibodies. A critical role of the spleen in the early production of anti-FMDV antibodies in infected mice was also demonstrated in vivo. Indeed, either splenectomy or functional disruption of the marginal zone of the spleen delays and reduces the magnitude of the TI anti-FMDV antibody response in infected mice. Together, these results indicate that in addition to virus localization, the FMDV-mediated modulation of DC functionality is a key parameter that collaborates in the induction of a rapid and protective TI antibody response against this virus.

Neutrophil signaling pathways activated by bacterial DNA stimulation.

We have previously shown that bacterial DNA activates human neutrophils in a CpG-independent manner. In this study, we have characterized the signaling pathways involved in the activation mechanism. We found that p38 MAPK, ERK1/2, and JNK pathways, as well as the PI3K/Akt pathway, are activated by bacterial DNA. We also determined that bacterial DNA induces NF-kappaB and AP-1 activation. When analyzing the role of these pathways on neutrophil functions, we observed that up-regulation of CD11b triggered by bacterial DNA was decreased by pharmacological inhibitors of the p38 MAPK, ERK1/2, and JNK, whereas stimulation of IL-8 release was dependent on p38, ERK1/2, and NF-kappaB. Moreover, we found that IL-8 production was markedly enhanced by inhibition of JNK, suggesting that this pathway negatively modulates NF-kappaB-dependent transcription. We also observed that bacterial DNA stimulated IL-1R-associated kinase-1 kinase activity and its partial degradation. Finally, we determined that bacterial DNA stimulated CD11b up-regulation in TLR9(-/-) but not in MyD88(-/-) mouse neutrophils, supporting that bacterial DNA induces neutrophil activation through a TLR9-independent and MyD88-dependent pathway.

Impairment of thymus-dependent responses by murine dendritic cells infected with foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) is a cytopathic virus that experimentally infects mice, inducing a thymus-independent neutralizing Ab response that rapidly clears the virus. In contrast, vaccination with UV-inactivated virus induces a typical thymus-dependent (TD) response. In this study we show that dendritic cells (DCs) are susceptible to infection with FMDV in vitro, although viral replication is abortive. Infected DCs down-regulate the expression of MHC class II and CD40 molecules and up-regulate the expression of CD11b. In addition, infected DCs exhibit morphological and functional changes toward a macrophage-like phenotype. FMDV-infected DCs fail to stimulate T cell proliferation in vitro and to boost an Ab response in vivo. Moreover, infection of DCs in vitro induces the secretion of IFN-gamma and the suppressive cytokine IL-10 in cocultures of DCs and splenocytes. High quantities of these cytokines are also detected in the spleens of FMDV-infected mice, but not in the spleens of vaccinated mice. The peak secretion of IFN-gamma and IL-10 is concurrent with the suppression of Con A-mediated proliferation of T cells obtained from the spleens of infected mice. Furthermore, the secretion of these cytokines correlates with the suppression of the response to OVA, a typical TD Ag. Thus, infection of DCs with FMDV induces suppression of TD responses without affecting the induction of a protective thymus-independent response. Later, T cell responses are restored, setting the stage for the development of a long-lasting protective immunity.