Carbon Black CB-EDA Nanoparticles in Macrophages: Changes in the Oxidative Stress Pathway and in Apoptosis Signaling

The influence of black carbon nanoparticles on J774.A1 murine cells was investigated with the objective of exploring the cytotoxicity of black carbon functionalized with ethylenediamine CB-EDA. The results showed that CB-EDA has a cytotoxic profile for J774.A1 macrophages in a time- and dose-dependent manner. When phagocytosed by the macrophage, CB-EDA triggers a mechanism that leads to apoptosis. In this process, there is an increase in oxidative stress pathways due to the activation of nitric oxide and then ROS. This causes an imbalance in redox function and a disruption of membrane integrity that occurs due to high levels of LDH, in addition to favoring the release of the pro-inflammatory cytokines IL-6, IL-12, and tumor necrosis factor (TNF) in an attempt to modulate the cell. However, these stimuli are not sufficient to repair the cell and the level of mitochondrial integrity is affected, causing a decrease in cell viability. This mechanism may be correlated with the activation of the caspasse-3 pathway, which, when compromised, cleaves and induces cells death via apoptosis, either through early or late apoptosis. In view of this, the potential for cell damage was investigated by analyzing the oxidative and inflammatory profile in the macrophage lineage J774.A1 and identifying potential mechanisms and metabolic pathways connected to these processes when cells were exposed to NP CB-EDA for both 24 h and 48 h.The authors would like to thank Márcia Regina Cominetti (Departamento de Gerontologia, Universidade Federal de São Carlos, São Carlos, SP, Brazil) for availability of equipment. M.A. was supported by the Margarita Salas postdoctoral contract MGS/2021/21 (UP2021-021) financed by the European Union—Next Generation EU. The authors would also like to express their gratitude for the financial aid from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (2013/07296-2), the Financiadora de Estudos e Projetos—FINEP, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES (Financial Code 001) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq

HGPRT and PNP: Recombinant Enzymes from <i>Schistosoma mansoni</i> and Their Role in Immunotherapy during Experimental Murine Schistosomiasis

Schistosomiasis is a parasitic infection caused by trematode worms (also called blood flukes) of the genus Schistosoma sp., which affects over 230 million people worldwide, causing 200,000 deaths annually. There is no vaccine or new drugs available, which represents a worrying aspect, since there is loss of sensitivity of the parasite to the medication recommended by the World Health Organization, Praziquantel. The present study evaluated the effects of the recombinant enzymes of S. mansoni Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT), Purine Nucleoside Phosphorylase (PNP) and the MIX of both enzymes in the immunotherapy of schistosomiasis in murine model. These enzymes are part of the purine salvage pathway, the only metabolic pathway present in the parasite for this purpose, being essential for the synthesis of DNA and RNA. Female mice of Swiss and BALB/c strains were infected with cercariae and treated, intraperitoneally, with three doses of 100 µg of enzymes. After the immunotherapy, the eggs and adult worms were counted in the feces; the number of eosinophils from the fluid in the peritoneal cavity and peripheral blood was observed; and the quantification of the cytokine IL-4 and the production of antibodies IgE was analyzed. The evaluation of the number of granulomas and collagen deposition via histological slides of the liver was performed. The results demonstrate that immunotherapy with the enzyme HGPRT seems to stimulate the production of IL-4 and promoted a significant reduction of granulomas in the liver in treated animals. The treatment with the enzyme PNP and the MIX was able to reduce the number of worms in the liver and in the mesenteric vessels of the intestine, to reduce the number of eggs in the feces and to negatively modulate the number of eosinophils. Therefore, immunotherapy with the recombinant enzymes of S. mansoni HGPRT and PNP might contribute to the control and reduction of the pathophysiological aspects of schistosomiasis, helping to decrease the morbidity associated with the infection in murine model

The <i>pkcA</i>^G579R mutant is sensitive to antifungal drugs.

<p>Antifungal susceptibility using E-test gradient strips for voriconazole (A) and caspofungin (B).</p

Growth phenotypes of the <i>pkcA</i>^G579R mutant strain in the presence of oxidative damage.

<p>The strains were grown in liquid MM in 24-well plates supplemented with in the indicated concentrations of paraquat (A) or menadione (B) during 48 hours at 37°C.</p

The <i>pkcA</i>^G579R mutant strain is defective in activating the CWI pathway.

<p>Conidia of the wild-type and mutant strains were grown overnight in liquid YG medium and cell wall stress was induced by exposure to CR for 0, 15, 30 and 60 minutes. Samples were collected at the indicated time points for western blot preparation. Phosphorylated and the total fraction of MpkA were detected using anti-phospho p44/42 and anti-p44/42 MAPK antibodies, respectively. γ-tubulin antibody and Coomassie Brilliant Blue stained gel were used as loading sample controls (A). Densitometry analysis of western blots using the ImageJ software presenting the difference in the phosphorylated MpkA/non-phosphorylated MpkA ratio in the wild-type and mutant strain expressed as percentage in the histogram (B).</p

<i>pkcA</i>^G579R strain presents no virulence attenuation in a mouse model but activates innate immunity against <i>A</i>. <i>fumigatus</i>.

<p>(A) Secretion of TNF-α from bone marrow derived macrophages (BMDM) after co-incubation with <i>A</i>. <i>fumigatus</i> hyphae of wild-type, <i>pkcA</i>^G579R and complementing strains. TNF-α levels were quantified by ELISA from the culture supernatant after 18 hours of BMDMs infection. Data show average ± standard deviation (* p ≤ 0.005). NI: Non−infected; LPS: lipopolysaccharide (positive control). (B) Phagocytosis index is increased in the <i>pkcA</i>^G579R mutant strain (average ± standard deviation, *p ≤ 0.01 compared to the wild−type and the complemented strain). (C) Comparative analysis of wild type, <i>pkcA</i>^G579R and complemented strains in a neutropenic murine model of invasive pulmonary aspergillosis.</p

Chelerythrine treatment leads to a fungicidal effect of <i>A</i>. <i>fumigatus pkcA</i>^G579R mutant.

<p>(A) 2x10³ conidia from each strain were grown for 24 hours in liquid MM at 30°C in the presence of the indicated concentration of chelerythrine. After growth, cells were centrifuged, washed in pre-warmed medium and allow to recover in fresh MM medium for an additional 24 hours. After this time, survival was determined via the MTT assay comparing the formanzan salt formation at each time point in comparison to the untreated control. * p ≤ 0.01. (B) Western blot for MpkA phosphorylation. Wild-type and <i>pkcA</i>^G579R strains were grown for 16 h in YG at 37°C and then chelerythrine (25 μM) was added, or not (control), to the medium for 120 minutes. Anti-phospho-p44/42 MAPK antibody was used to detect the phosphorylation of MpkA. The γ-tubulin antibody and Coomassie Brilliant Blue stained gels were used as loading sample controls. Signal intensity was quantified using the ImageJ software by dividing the intensity of phosphorylatred MpkA/γ-tubulin and expressed as arbitrary units.</p

Transcriptional analysis of chitin synthase genes.

<p>The wild-type, and <i>pkcA</i>^G579R strains were grown for 24 hours in YG medium and transferred to fresh pre-warmed YG with either 0 or 300 μg/ml of CR and grown for a further 15, 30 and 60 minutes. mRNA abundance for each gene was assessed by real time RT-PCR and normalized to β-tubulin. Fold increase in each strain represents the normalized mRNA abundance relative to the wild-type strain at time point 0 (i. e. prior to CR exposure). Data represent the average value of at least three biological replicates, each repeated in duplicate in the same run and standard deviation. * p ≤ 0.05.</p

Growth phenotypes of the <i>pkcA</i>^G579R mutant.

<p>(A) 1x10⁵ conidia of each strain were inoculated onto the center of a solid YG plates supplemented with different cell wall perturbing agents: Congo Red (CR); Calcofluor White (CFW); caffeine (CAF); anidulafungin (AF); Sodium Dodecyl Sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). (B) The indicated number of conidia was spotted onto solid YG plates supplemented with nikkomycin Z (NKZ) and fluconazole (FLUC). The plates were incubated for 3 days (A) or 2 days (B) at 37°C.</p

PkcA contributes to thermotolerant growth and germination in <i>A</i>. <i>fumigatus</i>.

<p>1x10⁴ conidia of each strain were inoculated onto the center of a YAG medium and radial growth was measured after 3 days at the indicated temperatures (A-B). 1x10⁶ conidia of each strain were inoculated in 2 ml liquid YG culture and incubated at 37°C and 45°C during 2, 4, 6 and 8 hours before the percentage of germination was evaluated (C). Experiments were performed in triplicate and the results are the mean ± standard deviation. * Statistically significant by Student’s T-test (p ≤ 0.01).</p