Neutrophils as Trojan Horse Vehicles for Brucella abortus Macrophage Infection

Brucella abortus is a stealthy intracellular bacterial pathogen of animals and humans. This bacterium promotes the premature cell death of neutrophils (PMN) and resists the killing action of these leukocytes. B. abortus-infected PMNs presented phosphatidylserine (PS) as “eat me” signal on the cell surface. This signal promoted direct contacts between PMNs and macrophages (Mϕs) and favored the phagocytosis of the infected dying PMNs. Once inside Mϕs, B. abortus replicated within Mϕs at significantly higher numbers than when Mϕs were infected with bacteria alone. The high levels of the regulatory IL-10 and the lower levels of proinflammatory TNF-α released by the B. abortus-PMN infected Mϕs, at the initial stages of the infection, suggested a non-phlogistic phagocytosis mechanism. Thereafter, the levels of proinflammatory cytokines increased in the B. abortus-PMN-infected Mϕs. Still, the efficient bacterial replication proceeded, regardless of the cytokine levels and Mϕ type. Blockage of PS with Annexin V on the surface of B. abortus-infected PMNs hindered their contact with Mϕs and hampered the association, internalization, and replication of B. abortus within these cells. We propose that B. abortus infected PMNs serve as “Trojan horse” vehicles for the efficient dispersion and replication of the bacterium within the host

N-Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils

Brucella abortus is an intracellular pathogen of monocytes, macrophages, dendritic cells, and placental trophoblasts. This bacterium causes a chronic disease in bovines and in humans. In these hosts, the bacterium also invades neutrophils; however, it fails to replicate and just resists the killing action of these leukocytes without inducing significant activation or neutrophilia. Moreover, B. abortus causes the premature cell death of human neutrophils. In the murine model, the bacterium is found within macrophages and dendritic cells at early times of infection but seldom in neutrophils. Based on this observation, we explored the interaction of mouse neutrophils with B. abortus. In contrast to human, dog, and bovine neutrophils, naive mouse neutrophils fail to recognize smooth B. abortus bacteria at early stages of infection. Murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. Alternatively, mouse normal serum is capable of opsonizing rough Brucella mutants. Despite this, neutrophils still fail to kill Brucella, and the bacterium induces cell death of murine leukocytes. In addition, mouse serum does not opsonize Yersinia enterocolitica O:9, a bacterium displaying the same surface polysaccharide antigen as smooth B. abortus. Therefore, the lack of murine serum opsonization and absence of murine neutrophil recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides. Although the mouse is a valuable model for understanding the immunobiology of brucellosis, direct extrapolation from one animal system to another has to be undertaken with caution.Fondo Especial de la Educación Superior/[UNA-SIA-0505-13]/FEES-CONARE/Costa RicaFondo Especial de la Educación Superior/[UNA-SIA-0504-13]/FEES-CONARE/Costa RicaFondo Especial de la Educación Superior/[UNA-SIA-0248-13]/FEES-CONARE/Costa RicaFondo Especial de la Educación Superior/[UNA-SIA-0434-14]/FEES-CONARE/Costa RicaConsejo Nacional para Investigaciones Científicas y Tecnológicas/[FV-0004-13]/CONICIT-FORINVES/Costa RicaThe International Center for Genomic Engineering and Biotechnology/[CRP/12/007]/ICGEB/IndiaUniversidad de Costa Rica/[803-B3-761]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.Fondo Especial de la Educación Superior/[0500-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0504-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0505-13]/FEES/Costa RicaFondo Especial de la Educación Superior/[0248-13]/FEES/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Interaction of murine neutrophils with Brucella abortus

La brucelosis es una enfermedad zoonótica con distribución mundial. La enfermedad se caracteriza por ocasionar fiebre ondulante en humanos y abortos en animales domésticos como vacas, ovejas, cabras, cerdos y perros. Brucella abortus ha evolucionado como un patógeno furtivo capaz de eludir las respuestas proinflamatorias, incluyendo a los neutrófilos polimorfonucleares (PMNs), que son las principales células efectoras del sistema inmune innato. El eje central de esta tesis explora la interacción de los PMNs con Brucella abortus. Debido a que el modelo experimental central de esta tesis se centró en los modelos de infección bacteriana y depleción de PMNs murinos, la primera parte de esta tesis incluye una revisión sobre estos modelos y el uso de ratones neutropénicos en diferentes infecciones bacterianas. El primer artículo que publicamos contrasta la interacción de los PMNs de ratones con aquellos de humanos, perros y bovinos, mostrando que estos últimos no reconocen a las bacterias lisas de B. abortus en las primeras etapas de la infección. Los componentes del suero normal murino no opsonizan las cepas lisas de B. abortus, y la fagocitosis de PMNs se logra solo después de la aparición de anticuerpos. La falta de opsonización de suero murino y la ausencia de reconocimiento de PMN murino son específicos, y las moléculas responsables del camuflaje de B. abortus son los homopolisacáridos de superficie de N-formil-perosamina que incluye la cadena O del lipopolisacárido y los haptenos nativos. Posteriormente exploramos cómo los PMNs modulan la inmunidad adaptativa durante las etapas iniciales de la infección aguda en la brucelosis murina. En ratones, la depleción de los PMNs al inicio de la inmunidad adaptativa favorece la tasa de eliminación de B. abortus. También se demuestra que los PMNs modulan activamente el curso de la infección de B. abortus, aun cuando la respuesta adaptativa está desarrollada. Bajo una línea de trabajo afín, mostramos que Brucella tiene la capacidad de inducir la muerte prematura de PMNs humanos, lo que sugiere que Brucella podría disminuir la presencia de PMNs infectados en los órganos diana y promover la neutropenia durante la brucelosis crónica. Esta muerte celular no proinflamatoria de los PMNs infectados por B. abortus concuerda con la propuesta de que estos leucocitos sirven como "caballos de Troya" ya que sirven como vehículos para infectar células fagocíticas profesionales sin inducir activación. Finalmente, describimos la persistencia de Brucella en las células de la médula ósea de los ratones y proponemos que este tejido es esencial en el establecimiento de infecciones crónicas de larga duración. Así mismo, demostramos que los PMNs infectados con Brucella son fagocitados fácilmente por macrófagos murinos de una manera no proinflamatoria, y que las bacterias liberadas a través de PMNs se replican ampliamente dentro de macrófagos. Por lo tanto, se proporciona una prueba de concepto de que los PMNs sirven como "caballos de Troya" o vehículo para la dispersión y replicación de B. abortus en el hospedero. Esta tesis demuestra que los PMN tienen un papel importante en la patogénesis de Brucella, así como en la modulación de la respuesta inmune del hospedero.Brucellosis is a zoonotic disease with a worldwide distribution, which causes undulant fever in humans and abortions in domestic animals, such as cows, sheep, goats, pigs, and dogs. Brucella abortus has evolved as a stealthy pathogen capable of circumventing pro-inflammatory responses, including PMNs, which are the main effector cells of the innate immune system. In this thesis, we explore the interaction of PMNs with B. abortus. The first part of this thesis includes a review of the depletion model of murine PMNs and the use of neutropenic mice in various bacterial infections. The first published paper compares the interaction of naïve murine PMNs with human, dog, and bovine PMNs, showing that murine PMNs fail to recognize smooth B. abortus cells at early stages of infection. The murine normal serum components do not opsonize smooth Brucella strains, and neutrophil phagocytosis is achieved only after the appearance of antibodies. The lack of murine serum opsonization and absence of murine PMN recognition are specific, and the molecules responsible for the Brucella camouflage are N-formyl-perosamine surface homopolysaccharides of lipopolysaccharide and native haptens. Afterward, we explored how PMNs modulate adaptive immunity in the initial stages of the acute murine Brucella infection. The removal of PMNs influences adaptive immunity at the onset of B. abortus infection, enhancing bacterial elimination from the target organs of mice. We demonstrate that PMNs have an active role modulating the course of B. abortus infection after the adaptive immune response already developed. In a related manuscript, we show that B. abortus can prematurely kill human PMNs suggesting Brucella may hamper the presence of infected PMNs in the target organs and promote neutropenia during chronic brucellosis. This non-phlogistic cell death of Brucella infected PMNs agrees with the proposal of these leukocytes function as “Trojan horse” vehicles for infecting phagocytic cells without promoting activation. Finally, we describe the persistence of B. abortus in cells of the mice bone marrow and propose this tissue as essential in the establishment of long-lasting chronic infections. Here we demonstrate that murine macrophages readily phagocyte Brucella-infected PMNs in a non-phlogistic mxr and that bacteria delivered through PMNs, extensively replicate inside macrophages. Following this we provide a proof of concept for the “Troyan horse” hypothesis that proposes that B. abortus infected PMNs function as vehicles for the dispersion and replication of the bacteria inside the host, modulating the host immune response.Ministerio de Ciencia, Tecnología y Telecomunicaciones/[PND-014-2015-1]/MICITT/Costa RicaInternational Centre for Genetic Engineering and Biotechnology/[CRP/16/005]/ICGEB/ItaliaUniversidad de Costa Rica/[803-B7-341]/UCR/Costa RicaUniversidad de Costa Rica/[SEP-2497-2019]/UCR/Costa RicaUniversidad de Costa Rica/[SEP-1391-2018]/UCR/Costa RicaUniversidad de Costa Rica/[SEP-5212-2018]/UCR/Costa RicaUniversidad de Costa Rica/[SEP-1611-2019]/UCR/Costa RicaUniversidad de Costa Rica/[SEP-5196-2015]/UCR/Costa RicaUniversidad de Costa Rica/[SEP-685-2017]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)UCR::Vicerrectoría de Investigación::Sistema de Estudios de Posgrado::Interdisciplinarias::Doctorado Académico en Ciencia

Neutrophils dampen adaptive immunity in Brucellosis

Brucella organisms are intracellular stealth pathogens of animals and humans. The bacteria overcome the assault of innate immunity at early stages of an infection. Removal of polymorphonuclear neutrophils (PMNs) at the onset of adaptive immunity against Brucella abortus favored bacterial elimination in mice. This was associated with higher levels of interferon gamma (IFN-γ) and a higher proportion of cells expressing interleukin 6 (IL-6) and inducible nitric oxide synthase (iNOS), compatible with M1 macrophages, in PMN-depleted B. abortus-infected (PMNd-Br) mice. At later times in the acute infection phase, the amounts of IFN-γ fell while IL-6, IL-10, and IL-12 became the predominant cytokines in PMNd-Br mice. IL-4, IL-1β, and tumor necrosis factor alpha (TNF-α) remained at background levels at all times of the infection. Depletion of PMNs at the acute stages of infection promoted the premature resolution of spleen inflammation. The efficient removal of bacteria in the PMNd-Br mice was not due to an increase of antibodies, since the immunoglobulin isotype responses to Brucella antigens were dampened. Anti-Brucella antibodies abrogated the production of IL-6, IL-10, and IL-12 but did not affect the levels of IFN-γ at later stages of infection in PMNd-Br mice. These results demonstrate that PMNs have an active role in modulating the course of B. abortus infection after the adaptive immune response has already developed.Los organismos Brucella son patógenos intracelulares sigilosos de animales y humanos. La bacteria supera el ataque de la inmunidad innata en las primeras etapas de una infección. La eliminación de los neutrófilos polimorfonucleares (PMN) al inicio de la inmunidad adaptativa contra Brucella abortus favoreció la eliminación bacteriana en los ratones. Esto se asoció con niveles más altos de interferón gamma (IFN-γ) y una mayor proporción de células que expresan interleucina 6 (IL-6) y óxido nítrico sintasa inducible (iNOS), compatible con macrófagos M1, en ratones infectados con PMN depleted B. abortus (PMNd-Br). Más tarde, en la fase de infección aguda, las cantidades de IFN-γ disminuyeron mientras que las IL-6, IL-10 e IL-12 se convirtieron en las citoquinas predominantes en los ratones PMNd-Br. IL-4, IL-1β, y el factor de necrosis tumoral alfa (TNF-α) permanecieron en los niveles de fondo en todo momento de la infección. El agotamiento de las PMN en las etapas agudas de la infección promovió la resolución prematura de la inflamación del bazo. La eliminación eficiente de las bacterias en los ratones PMNd-Br no se debió a un aumento de los anticuerpos, ya que las respuestas del isotipo de inmunoglobulina a los antígenos de Brucella se vieron amortiguadas. Los anticuerpos anti-Brucella abrogaron la producción de IL-6, IL-10 y IL-12 pero no afectaron los niveles de IFN-γ en las etapas posteriores de la infección en los ratones PMNd-Br. Estos resultados demuestran que los PMN tienen un papel activo en la modulación del curso de la infección por B. abortus después de que la respuesta inmune adaptativa ya se ha desarrollado.Universidad Nacional, Costa RicaEscuela Medicina Veterinari

Neutrophils as Trojan Horse Vehicles for Brucella abortus Macrophage Infection

Brucella abortus is a stealthy intracellular bacterial pathogen of animals and humans. This bacterium promotes the premature cell death of neutrophils (PMN) and resists the killing action of these leukocytes. B. abortus-infected PMNs presented phosphatidylserine (PS) as “eat me” signal on the cell surface. This signal promoted direct contacts between PMNs and macrophages (Mϕs) and favored the phagocytosis of the infected dying PMNs. Once inside Mϕs, B. abortus replicated within Mϕs at significantly higher numbers than when Mϕs were infected with bacteria alone. The high levels of the regulatory IL-10 and the lower levels of proinflammatory TNF-α released by the B. abortus-PMN infected Mϕs, at the initial stages of the infection, suggested a non-phlogistic phagocytosis mechanism. Thereafter, the levels of proinflammatory cytokines increased in the B. abortus-PMN-infected Mϕs. Still, the efficient bacterial replication proceeded, regardless of the cytokine levels and Mϕ type. Blockage of PS with Annexin V on the surface of B. abortus-infected PMNs hindered their contact with Mϕs and hampered the association, internalization, and replication of B. abortus within these cells. We propose that B. abortus infected PMNs serve as “Trojan horse” vehicles for the efficient dispersion and replication of the bacterium within the host.International Centre for Genomic Engineering and Biotechnology/[CRP/16/005]/ICGEB/ItalyFondo Institucional de Desarrollo Académico (FIDA)/[0087-17]/UNA/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[PNM-001-2015-1]/MICITT/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[PND-014-2015-1]/MICITT/Costa RicaEspacio Universitario de Estudios Avanzados (UCREA)/[803-B8-762]/UCR/Costa RicaUCR::Vicerrectoría de Docencia::Salud::Facultad de MicrobiologíaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Persistence of Brucella abortus in the bone marrow of infected mice

Brucellosis is a zoonotic bacterial infection that may persist for long periods causing relapses in antibiotic-treated patients. The ability of Brucella to develop chronic infections is linked to their capacity to invade and replicate within the mononuclear phagocyte system, including the bone marrow (BM). Persistence of Brucella in the BM has been associated with hematological complications such as neutropenia, thrombocytopenia, anemia, and pancytopenia in human patients. In the mouse model, we observed that the number of Brucella abortus in the BM remained constant for up to 168 days of postinfection. This persistence was associated with histopathological changes, accompanied by augmented numbers of BM myeloid GMP progenitors, PMNs, and CD4+ lymphocytes during the acute phase (eight days) of the infection in the BM. Monocytes, PMNs, and GMP cells were identified as the cells harboring Brucella in the BM. We propose that the BM is an essential niche for the bacterium to establish long-lasting infections and that infected PMNs may serve as vehicles for dispersion of Brucella organisms, following the Trojan horse hypothesis. Monocytes are solid candidates for Brucella reservoirs in the BM.La brucelosis es una infección bacteriana zoonótica que puede persistir durante largos períodos de tiempo y causar recaídas en pacientes tratados con antibióticos. La posibilidad de que los brucelosos desarrollen infecciones crónicas está vinculada a su capacidad de invadir y replicarse dentro del sistema mononuclear-fagocitario, incluyendo la médula ósea (BM). La persistencia de la Brucellaina la BM ha sido asociada con complicaciones hematológicas como la neutropenia, la trombocitopenia, la anemia y la pancitopenia en pacientes humanos. En el modelo de ratón, observamos que el número de Brucella abortusin la BM se mantuvo constante hasta 168 días después de la infección. Esta persistencia se asoció a cambios histopatológicos, acompañados de un aumento en el número de progenitores mieloides GMP de la BM, PMN y linfocitos CD4+ durante la fase aguda (ocho días) de la infección en la BM. Se identificaron los monocitos, PMNs y células GMP como las células que albergan a la Brucella en la BM. Proponemos que el BM es un nicho esencial para que la bacteria establezca infecciones duraderas y que las PMN infectadas pueden servir como vehículos para la dispersión de los organismos de Brucella, siguiendo la hipótesis del Caballo de Troya. Los monocitos son candidatos sólidos para los depósitos de Brucelos en el BM.Escuela Medicina Veterinari

<i>B</i>. <i>abortus</i> is partially resistant to the killing action of PMNs.

<p>(A) PMNs were isolated from blood and incubated with <i>B</i>. <i>abortus</i> or <i>S</i>. <i>enterica</i> (MOI 5) and CFUs determined at different time points. (B) Heparinized blood was incubated with <i>B</i>. <i>abortus</i>-GFP or fluorescent latex beads for two hours (MOI 10 or 100). Blood smears were then fixed and mounted with ProLong Gold Antifade Reagent with DAPI. At least 100 PMNs were counted per sample and the number of intracellular bacterial or latex particles determined in each PMN and the proportion expressed as % of phagocytized particles. (C) Human PMNs infected with different MOI of <i>B</i>. <i>abortus</i>-GFP and stained as in “B”. Microscope images are at 400 × magnification. Representative PMNs with DAPI-stained nuclei and intracellular green fluorescent <i>B</i>. <i>abortus</i> were photographed under the microscope using the appropriate color filter channel. Images were cut from microscope field, contrasted and saturated using Hue tool to obtain suitable color separation. Images were then merged using Adobe Photoshop 8 software. Experiments were repeated at least three times. Values of <i>p</i><0.01 (**) are indicated.</p

<i>Br-</i>LPS released inside cells is mostly found within vacuolar compartments of PMNs.

<p>Purified human PMNs 5 ×10⁶ were infected with <i>B</i>. <i>abortus</i> 2308 at MOI 20. After one hour incubation, infected cells were fixed and processed for immunogold staining and electron microscopy. Detection of <i>Br</i>-LPS was performed using mouse IgG anti Br-LPS in combination with protein-A/protein-G colloidal gold 15 nm. (A) PMN (n, nucleus of cell) with intracellular <i>B</i>. <i>abortus</i> (white asterisk) and immunogold detection of <i>Br</i>-LPS. (B) and (C) correspond to amplified sections pointed with arrows from “A” panel; <i>B</i>. <i>abortus</i> (white asterisk) and immunogold detection of <i>Br</i>-LPS inside vacuoles (pointed by black arrow heads). (D) <i>B</i>. <i>abortus</i> (white asterisk) within a phagosome (ph) and vacuoles containing immunogold labeled <i>Br</i>-LPS (black arrow heads). (E) PMN membrane ruffle showing immunogold detection of <i>Br</i>-LPS associated to the membrane (white arrow heads) and <i>B</i>. <i>abortus</i> (white asterisk) debris inside a phagosome (ph) and immunogold detection of <i>Br</i>-LPS inside vacuoles (black arrow heads). No colloidal gold particles were observed when IgG purified from normal mouse serum was used for controlling the specificity of the reaction. Bar represents 500 nm.</p

<i>B</i>. <i>abortus</i> infection induces PMN cell death in a dose dependent manner.

<p>(A) Heparinized blood was incubated with <i>B</i>. <i>abortus</i>-GFP (MOI 10) for two hours and PMNs population analyzed for cell death using AquaDead and Annexin V markers. GFP fluorescence intensity was used to differentiate among three categories: (a) low, (b) intermediate and (c) high infection. (B) Percentages of PMNs positive for any marker in relation to the number of internalized bacteria are shown. Experiments were repeated at least three times.</p