In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b+ F4/80+ MHC-II+ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS+ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis

Differential Muc2 and Muc5ac secretion by stimulated guinea pig tracheal epithelial cells in vitro

BACKGROUND: Mucus overproduction is a characteristic of inflammatory pulmonary diseases including asthma, chronic bronchitis, and cystic fibrosis. Expression of two mucin genes, MUC2 and MUC5AC, and their protein products (mucins), is modulated in certain disease states. Understanding the signaling mechanisms that regulate the production and secretion of these major mucus components may contribute significantly to development of effective therapies to modify their expression in inflamed airways. METHODS: To study the differential expression of Muc2 and Muc5ac, a novel monoclonal antibody recognizing guinea pig Muc2 and a commercially-available antibody against human MUC5AC were optimized for recognition of specific guinea pig mucins by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). These antibodies were then used to analyze expression of Muc2 and another mucin subtype (likely Muc5ac) in guinea pig tracheal epithelial (GPTE) cells stimulated with a mixture of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interferon- γ (IFN-γ)]. RESULTS: The anti-Muc2 (C4) and anti-MUC5AC (45M1) monoclonal antibodies specifically recognized proteins located in Muc2-dominant small intestinal and Muc5ac-dominant stomach mucosae, respectively, in both Western and ELISA experimental protocols. IHC protocols confirmed that C4 recognizes murine small intestine mucosal proteins while 45M1 does not react. C4 and 45M1 also stained specific epithelial cells in guinea pig lung sections. In the resting state, Muc2 was recognized as a highly expressed intracellular mucin in GPTE cells in vitro. Following cytokine exposure, secretion of Muc2, but not the mucin recognized by the 45M1 antibody (likely Muc5ac), was increased from the GPTE cells, with a concomitant increase in intracellular expression of both mucins. CONCLUSION: Given the tissue specificity in IHC and the differential hybridization to high molecular weight proteins by Western blot, we conclude that the antibodies used in this study can recognize specific mucin subtypes in guinea pig airway epithelium and in proteins from GPTE cells. In addition, Muc2 is highly expressed constitutively, modulated by inflammation, and secreted differentially (as compared to Muc5ac) in GPTE cells. This finding contrasts with expression patterns in the airway epithelium of a variety of mammalian species in which only Muc5ac predominates

Effects of rising temperature on pelagic biogeochemistry in mesocosm systems: a comparative analysis of the AQUASHIFT Kiel experiments

A comparative analysis of data, obtained during four indoor-mesocosm experiments with natural spring plankton communities from the Baltic Sea, was conducted to investigate whether biogeochemical cycling is affected by an increase in water temperature of up to 6 °C above present-day conditions. In all experiments, warming stimulated in particular heterotrophic bacterial processes and had an accelerating effect on the temporal development of phytoplankton blooms. This was also mirrored in the build-up and partitioning of organic matter between particulate and dissolved phases. Thus, warming increased both the magnitude and rate of dissolved organic carbon (DOC) build-up, whereas the accumulation of particulate organic carbon (POC) and phosphorus (POP) decreased with rising temperature. In concert, the observed temperature-mediated changes in biogeochemical components suggest strong shifts in the functioning of marine pelagic food webs and the ocean’s biological carbon pump, hence providing potential feedback mechanisms to Earth’s climate system

Brucella abortus Uses a Stealthy Strategy to Avoid Activation of the Innate Immune System during the Onset of Infection

To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity. Methodology/Principal Findings Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-α-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice. Conclusion/Significance We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections

Some environmental factors influencing phytoplankton in the Southern Ocean around South Georgia

Data on phytoplankton and zooplankton biomass, and physical and chemical variables, are combined with a published multivariate description of diatom species composition to interpret variation within an area around South Georgia surveyed during an austral summer. Large-scale species distributions could be equated to the different water masses which reflected the interaction of the Antarctic Circumpolar Current with the island and the Scotia Ridge. Small-scale factors were found to act at an interstation scale and imposed local variation on the biogeographic pattern. Nutrient depletion could be related to phytoplankton biomass but no single inorganic nutrient of those measured (NO 3 −N, PO 4 −P and silica) could be identified as important. The ratio Si:P appeared to be more important as an ecological factor. The impact of grazing by krill and other zooplankton could only be resolved as differences in phytoplankton biomass and phaeopigment content. Diatom species composition showed a relation to local krill abundance very different from that suggested by published studies, but could be explained as the effect of earlier grazing outside the study area. The effects of vertical mixing could not account for interstation differences as pycnocline depth was uniformly greater than euphotic depth, and vertical stability very low. Some comparison was made with data collected in 1926–31 by the Discovery Investigations. Significant differences in the distribution of certain taxa such as Chaetoceros criophilum and C. socialis were traced to major differences in hydrology.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46983/1/300_2004_Article_BF00443379.pd

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