Maiara S. Severo

Ixodes scapularis saliva mitigates inflammatory cytokine secretion during Anaplasma phagocytophilum stimulation of immune cells

BackgroundIxodes scapularis saliva enables the transmission of infectious agents to the mammalian host due to its immunomodulatory, anesthetic and anti-coagulant properties. However, how I. scapularis saliva influences host cytokine secretion in the presence of the obligate intracellular rickettsial pathogen Anaplasma phagocytophilum remains elusive.MethodsBone marrow derived macrophages (BMDMs) were stimulated with pathogen associated molecular patterns (PAMPs) and A. phagocytophilum. Cytokine secretion was measured in the presence and absence of I. scapularis saliva. Human peripheral blood mononuclear cells (PBMCs) were also stimulated with Tumor Necrosis Factor (TNF)-α in the presence and absence of I. scapularis saliva and interleukin (IL)-8 was measured.ResultsI. scapularis saliva inhibits inflammatory cytokine secretion by macrophages during stimulation of Toll-like (TLR) and Nod-like receptor (NLR) signaling pathways. The effect of I. scapularis saliva on immune cells is not restricted to murine macrophages because decreasing levels of interleukin (IL)-8 were observed after TNF-α stimulation of human peripheral blood mononuclear cells. I. scapularis saliva also mitigates pro-inflammatory cytokine response by murine macrophages during challenge with A. phagocytophilum.ConclusionsThese findings suggest that I. scapularis may inhibit inflammatory cytokine secretion during rickettsial transmission at the vector-host interface.

The tick salivary protein sialostatin L2 inhibits caspase-1-mediated inflammation during Anaplasma phagocytophilum infection.

ABSTRACT Saliva from arthropod vectors facilitates blood feeding by altering host inflammation. Whether arthropod saliva counters inflammasome signaling, a protein scaffold that regulates the activity of caspase-1 and cleavage of interleukin-1β (IL-1β) and IL-18 into mature molecules, remains elusive. In this study, we provide evidence that a tick salivary protein, sialostatin L2, inhibits inflammasome formation during pathogen infection. We show that sialostatin L2 targets caspase-1 activity during host stimulation with the rickettsial agent Anaplasma phagocytophilum. A. phagocytophilum causes macrophage activation and hemophagocytic syndrome features. The effect of sialostatin L2 in macrophages was not due to direct caspase-1 enzymatic inhibition, and it did not rely on nuclear factor κB or cathepsin L signaling. Reactive oxygen species from NADPH oxidase and the Loop2 domain of sialostatin L2 were important for the regulatory process. Altogether, our data expand the knowledge of immunoregulatory pathways of tick salivary proteins and unveil an important finding in inflammasome biology.

Anaplasma phagocytophilum: deceptively simple or simply deceptive?

Anaplasma phagocytophilum is an obligate intracellular rickettsial pathogen transmitted by ixodid ticks. This bacterium colonizes myeloid and nonmyeloid cells and causes human granulocytic anaplasmosis--an important immunopathological vector-borne disease in the USA, Europe and Asia. Recent studies uncovered novel insights into the mechanisms of A. phagocytophilum pathogenesis and immunity. Here, we provide an overview of the underlying events by which the immune system responds to A. phagocytophilum infection, how this pathogen counteracts host immunity and the contribution of the tick vector for microbial transmission. We also discuss current scientific gaps in the knowledge of A. phagocytophilum biology for the purpose of exchanging research perspectives.

The E3 Ubiquitin Ligase XIAP Restricts Anaplasma phagocytophilum Colonization of Ixodes scapularis Ticks

Ubiquitination is a posttranslational modification that regulates protein degradation and signaling in eukaryotes. Although it is acknowledged that pathogens exploit ubiquitination to infect mammalian cells, it remains unknown how microbes interact with the ubiquitination machinery in medically relevant arthropods. Here, we show that the ubiquitination machinery is present in the tick Ixodes scapularis and demonstrate that the E3 ubiquitin ligase named x-linked inhibitor of apoptosis protein (XIAP) restricts bacterial colonization of this arthropod vector. We provide evidence that xiap silencing significantly increases tick colonization by the bacterium Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis. We also demonstrate that (i) XIAP polyubiquitination is dependent on the really interesting new gene (RING) catalytic domain, (ii) XIAP polyubiquitination occurs via lysine (K)-63 but not K-48 residues, and (iii) XIAP-dependent K-63 polyubiquitination requires zinc for catalysis. Taken together, our data define a role for ubiquitination during bacterial colonization of disease vectors.

Anaplasma phagocytophilum Dihydrolipoamide Dehydrogenase 1 Affects Host-Derived Immunopathology during Microbial Colonization

ABSTRACT Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.

The ‘ubiquitous’ reality of vector immunology

Ubiquitination (ubiquitylation) is a common protein modification that regulates a multitude of processes within the cell. This modification is typically accomplished through the covalent binding of ubiquitin to a lysine residue onto a target protein and is catalysed by the presence of three enzymes: an activating enzyme (E1), ubiquitin‐conjugating enzyme (E2) and ubiquitin‐protein ligase (E3). In recent years, ubiquitination has risen as a major signalling regulator of immunity and microbial pathogenesis in the mammalian system. Still, little is known about how ubiquitin relates specifically to vector immunology. Here, we provide a brief overview of ubiquitin biochemistry and describe how ubiquitination regulates immune responses in arthropods of medical relevance. We also discuss scientific gaps in the literature and suggest that, similar to mammals, ubiquitin is a major regulator of immunity in medically important arthropods.

Effect of Mercurius solubilis on the bacteriological response in the alveolitis process in rats

OBJECTIVE The purpose of this study was to assess the bacteriological response in alveolitis in rats treated with the homeopathic medicine Merc solubilis (Merc sol.) 12 cH. METHODS The study was randomized and observer blind. The animals were anesthetized and the upper right incisor extracted resulting in alveolitis. Animals were randomly assigned to groups (n=18/group): Water control, Alcohol control and Merc sol. 12 cH. These groups were subsequently divided into 3 subgroups (n=6/subgroup): Early Euthanasia (EE), Mid Euthanasia (ME) and Late Euthanasia (LE), killed at the 6th, 15th and 21st days respectively. The perialveolar microbiota was collected by swab in Brain Heart Infusion (BHI) for seeding and bacterioscopy. After seeding, the Petri dishes were incubated at 37 degrees C for 48 h. RESULTS Quantitative and qualitative changes were observed in the perialveolar microbiota when the groups were compared. Water control and Alcohol control had the highest counts of pathogenic bacteria, the microbiotica of the Merc sol. group remained closer to normal. CONCLUSIONS Merc sol. 12 cH did not reduce bacterial growth, but the microbiotica remained within the parameters of normality, obtaining the best results at 21 days after treatment.

Decoding the ubiquitin-mediated pathway of arthropod disease vectors.

Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means for disease intervention has been already adopted in the clinic. In this study, we employed a bioinformatics approach to uncover the ubiquitin-mediated pathway in the genomes of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Ixodes scapularis, Pediculus humanus and Rhodnius prolixus. We observed that (1) disease vectors encode a lower percentage of ubiquitin-related genes when compared to Drosophila melanogaster, Mus musculus and Homo sapiens but not Saccharomyces cerevisiae; (2) overall, there are more proteins categorized as E3 ubiquitin ligases when compared to E2-conjugating or E1-activating enzymes; (3) the ubiquitin machinery within the three mosquito genomes is highly similar; (4) ubiquitin genes are more than doubled in the Chagas disease vector (R. prolixus) when compared to other arthropod vectors; (5) the deer tick I. scapularis and the body louse (P. humanus) genomes carry low numbers of E1-activating enzymes and HECT-type E3 ubiquitin ligases; (6) R. prolixus have low numbers of RING-type E3 ubiquitin ligases; and (7) C. quinquefasciatus present elevated numbers of predicted F-box E3 ubiquitin ligases, JAB and UCH deubiquitinases. Taken together, these findings provide novel opportunities to study the interaction between a pathogen and an arthropod vector.

Long-term effects of neonatal malnutrition on microbicide response, production of cytokines, and survival of macrophages infected by Staphylococcus aureus sensitive/resistant to methicillin

OBJECTIVE: To assess microbicide function and macrophage viability after in vitro cellular infection by methicillin-sensitive/resistant Staphylococcus aureus in nourished rats and rats subjected to neonatal malnutrition. METHODS: Male Wistar rats (n=40) were divided in two groups: Nourished (rats suckled by dams consuming a 17% casein diet) and Malnourished (rats suckled by dams consuming an 8% casein diet). Macrophages were recovered after tracheotomy, by bronchoalveolar lavage. After mononuclear cell isolation, four systems were established: negative control composed exclusively of phagocytes; positive control composed of macrophages plus lipopolysaccharide; and two testing systems, macrophages plus methicillin-sensitive Staphylococcus aureus and macrophages plus methicillin-resistant Staphylococcus aureus. The plates were incubated in a humid atmosphere at 37 degrees Celsius containing 5% CO2 for 24 hours. After this period tests the microbicidal response, cytokine production, and cell viability were analyzed. The statistical analysis consisted of analysis of variance (p<0.05). RESULTS: Malnutrition reduced weight gain, rate of phagocytosis, production of superoxide anion and nitric oxide, and macrophage viability. Production of nitrite and interleukin 18, and viability of macrophages infected with methicillin-resistant Staphylococcus aureus were lower. CONCLUSION: The neonatal malnutrition model compromised phagocyte function and reduced microbicidal response and cell viability. Interaction between malnutrition and the methicillin-resistant strain decreased the production of inflammatory mediators by effector cells of the immune response, which may compromise the immune systems defense ability.

Immunological parameters of macrophages infected by methicillin resistant/sensitive Staphylococcus aureus

INTRODUCAO: Modificacoes nas paredes celulares das cepas de Staphylococcus aureus relacionadas com o fenotipo de multirresistencia poderiam influenciar seus mecanismos de evasao frente a resposta imune? OBJETIVO: Avaliar a resposta microbicida e a sobrevivencia de macrofagos alveolares apos infeccao in vitro com Staphylococcus aureus meticilina sensivel/resistente. MATERIAL E METODOS: Utilizaram-se 20 ratos adultos, machos, albinos e da linhagem Wistar. Os macrofagos alveolares foram obtidos apos procedimento cirurgico de traqueostomia, por meios da coleta do lavado broncoalveolar. Os macrofagos alveolares foram cultivados na proporcao de 1:1 celulas/ml de Roswell Park Memorial Institute (RPMI)/poco e isolados pela capacidade de adesao a placa. Para avaliacao de parâmetros imunologicos, foram estabelecidos quatro sistemas: controle negativo, controle positivo, S. aureus sensivel a meticilina (MSSA) e S. aureus resistente a meticilina (MRSA). RESULTADOS: Ao comparar os sistemas de MSSA e MRSA, nao foi observada diferenca no indice de aderencia, na taxa de fagocitose, na producao do ânion superoxido e na viabilidade dos macrofagos. Ao analisar a cinetica de oxido nitrico, houve menor producao media desse radical para o MRSA quando comparado com o MSSA, no periodo de 4 a 10 horas de incubacao das culturas celulares. Entretanto, apos 12 horas, nao foi detectada divergencias entre esses sistemas. CONCLUSAO: Sugere-se que a resistencia a meticilina podera ser um fator que influenciara a capacidade de evasao da bacteria a resposta microbicida dos macrofagos. Apesar dos resultados de alguns parâmetros imunologicos terem sido similares entre os sistemas analisados, as oscilacoes ocorridas durante a producao do oxido nitrico poderao contribuir de forma importante para a sobrevivencia da bacteria Staphylococcus aureus.