Abstract
Background
The role of myeloid-derived suppressor cells (MDSCs) in patients with severe tuberculosis who suffer from uncontrolled pulmonary inflammation caused by hypervirulent mycobacterial ...infection remains unclear.
Methods
This issue was addressed using C57BL/6 mice infected with highly virulent Mycobacterium bovis strain MP287/03.
Results
CD11b+GR1int population increased in the bone marrow, blood and lungs during advanced disease. Pulmonary CD11b+GR1int (Ly6GintLy6Cint) cells showed granularity similar to neutrophils and expressed immature myeloid cell markers. These immature neutrophils harbored intracellular bacilli and were preferentially located in the alveoli. T-cell suppression occurred concomitantly with CD11b+GR1int cell accumulation in the lungs. Furthermore, lung and bone marrow GR1+ cells suppressed both T-cell proliferation and interferon γ production in vitro. Anti-GR1 therapy given when MDSCs infiltrated the lungs prevented expansion and fusion of primary pulmonary lesions and the development of intragranulomatous caseous necrosis, along with increased mouse survival and partial recovery of T-cell function. Lung bacterial load was reduced by anti-GR1 treatment, but mycobacteria released from the depleted cells proliferated extracellularly in the alveoli, forming cords and clumps.
Conclusions
Granulocytic MDSCs massively infiltrate the lungs during infection with hypervirulent mycobacteria, promoting bacterial growth and the development of inflammatory and necrotic lesions, and are promising targets for host-directed therapies.
Granulocytic myeloid-derived suppressor cells (MDSCs) act as a permissive niche for intracellular mycobacterial growth and spread bacilli throughout lung tissue in advanced MP287/03 infection. Massive death of infected MDSCs is apparently decisive for the development of pulmonary necrotic lesions.
Main conclusion
The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA ...validation, gene activation) associated with nematode bioassays.
Agrobacterium rhizogenes
-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by
A. rhizogenes
from petioles of detached soybean leaves. The soybean
GmPR10
and
GmGST
genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of
Meloidogyne incognita
-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous
GmExpA
and
GmExpLB
genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.
The detection of extracellular adenosine triphosphate (eATP) by P2X7 receptor promotes the development of severe tuberculosis. This study shows that adenosine, a byproduct of eATP degradation, ...impairs the CD4+ T-cell response in the lung parenchyma and contributes to disease progression.
Abstract
Background
Tuberculous pneumonia, necrotic granulomatous lesions, and bacterial dissemination characterize severe forms of mycobacterial infection.
Methods
To evaluate the pulmonary CD4+ T-cell response during severe tuberculosis, C57BL/6 mice were infected with approximately 100 bacilli of 3 hypervirulent mycobacterial isolates (Mycobacterium tuberculosis strain Beijing 1471 and Mycobacterium bovis strains B2 and MP287/03) or the H37Rv M tuberculosis strain as reference for mycobacterial virulence. Because high expression of both CD39 and CD73 ectonucleotidases was detected on parenchymal CD4+ T cells, we investigated whether CD4+ T-cell suppression in the context of severe disease was due to the extracellular adenosine accumulation that resulted from tissue damage.
Results
Lowest expression of CD69, which is an activation marker implicated in maintaining cells in tissues, was observed in lungs from mice displaying the most severe pulmonary pathology. Reduced interferon (IFN)γ-producing CD4+ T cells were also found in the lung of these mice. Intranasal administration of the adenosine receptor antagonist caffeine substantially enhanced the frequency and number of parenchymal CD4+ T cells as well as both CD69 expression and IFNγ production.
Conclusions
These results indicate that adenosine, which may be generated by extracellular adenosine triphosphate degradation, impairs the parenchymal CD4+ T-cell response and contributes to the development of severe tuberculosis.
The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell ...death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R-/- mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R-/- mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) that in most cases induces irreversible necrosis of lung tissue as a result of excessive inflammatory ...reactions. The murine model of TB in resistant C57BL/6 mice infected with reference Mtb strains is widely used in TB studies; however, these mice do not show a necrotic pathology, which restricts their use in studies of irreversible tissue damage. Recently, we demonstrated that necrotic lung lesions could be induced in the C57BL/6 mice by highly virulent Mtb strains belonging to the modern Beijing sublineage. However, the pathogenic mechanisms leading to necrosis in this model were not elucidated. In this study, we investigated the dynamics of lung lesions in mice infected with highly virulent Beijing Mtb strain M299, compared with those infected with laboratory Mtb strain H37Rv. The data demonstrate that necrotic lung lesions in mice infected by the strain M299 were associated with enhanced recruitment of myeloid cells, especially neutrophils, and increased levels of proinflammatory cytokines, consistent with exacerbated inflammation. High levels of IFN-γ production contributed to the control of bacterial growth. Further progression to chronic disease was associated with a reduction in the levels of inflammatory mediators in the lungs, the accumulation of foamy macrophages and partial healing of the necrotic tissue by fibrosis. At a late stage of disease, degradation of foamy cells resulted in the liberation of accumulated lipids and persisting bacilli and further activation of inflammation, which promoted lung consolidation. Overall, our studies show that C57BL/6 mice infected with highly virulent Mtb strain may serve as a TB model reproducing an exacerbated inflammatory response in a resistant host to hypervirulent mycobacteria, leading to irreversible necrotic lung lesions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•The construction of a chimeric protein leads to enhanced immune protection against Ascaris infection.•Vaccination with chimeric protein induced greater than 70% reduction in larval load after ...challenge.•Vaccine protection is associated with IgG response and reduced lung inflammation compared to controls.
An estimated 400 million people are infected by parasites of the genus Ascaris and the existing control measures are inefficient. Vaccine development using B cell antigens is a promising strategy for increased protection against this parasite. The present study aimed at developing a chimeric protein capable of conferring protection against infection by Ascaris sp. For this purpose, we performed B-cell epitope predictions on previously described vaccine candidate proteins from Ascaris suum and the corresponding peptides were used to construct a chimeric protein. Female BALB / c mice were immunized subcutaneously in three doses at 10 day intervals with a vaccine formulation comprised of the chimeric protein together with monophosphoryl lipid A (MPLA). Control groups included protein alone, MPLA, or PBS. After challenge infection, animals vaccinated with chimeric protein plus MPLA showed a reduction of 73.54% of larval load in the lung compared to control group animals. Animals immunized with chimeric protein plus MPLA also display higher IgG response and a reduction in lung inflammation. Our study highlights how chimeric proteins containing more than one B cell epitope can enhance immune protection against helminthic infection and offer new approaches to the development of Ascaris vaccines.
Among non-tuberculous mycobacteria,
Mycobacterium kansasii
is one of the most pathogenic, able to cause pulmonary disease indistinguishable from tuberculosis in immunocompetent susceptible adults. ...The lack of animal models that reproduce human-like lung disease, associated with the necrotic lung pathology, impairs studies of
M. kansasii
virulence and pathogenicity. In this study, we examined the ability of the C57BL/6 mice, intratracheally infected with highly virulent
M. kansasii
strains, to produce a chronic infection and necrotic lung pathology. As a first approach, we evaluated ten
M. kansasii
strains isolated from Brazilian patients with pulmonary disease and the reference strain
M. kansasii
ATCC 12478 for virulence-associated features in macrophages infected
in vitro
; five of these strains differing in virulence were selected for
in vivo
analysis. Highly virulent isolates induced progressive lung disease in mice, forming large encapsulated caseous granulomas in later stages (120–150 days post-infection), while the low-virulent strain was cleared from the lungs by day 40. Two strains demonstrated increased virulence, causing premature death in the infected animals. These data demonstrate that C57BL/6 mice are an excellent candidate to investigate the virulence of
M. kansasii
isolates. We observed considerable heterogeneity in the virulence profile of these strains, in which the presence of highly virulent strains allowed us to establish a clinically relevant animal model. Comparing public genomic data between Brazilian isolates and isolates from other geographic regions worldwide demonstrated that at least some of the highly pathogenic strains isolated in Brazil display remarkable genomic similarities with the ATCC strain 12478 isolated in the United States 70 years ago (less than 100 SNPs of difference), as well as with some recent European clinical isolates. These data suggest that few pathogenic clones have been widely spread within
M. kansasii
population around the world.
Strains of the Beijing genotype family of Mycobacterium tuberculosis are a cause of particular concern because of their increasing dissemination in the world and their association with drug ...resistance. Phylogenetically, this family includes distinct ancient and modern sublineages. The modern strains, contrary to the ancestral counterparts, demonstrated increasing prevalence in many world regions that suggest an enhanced bacterial pathogenicity. We therefore evaluated virulence of modern versus ancient Beijing strains with similar epidemiological and genotype characteristics. For this, we selected six strains that had very similar 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing profiles and belonged to the region of difference 181 (RD181) subgroup but differed using markers (mutT2 and mutT4 genes and NTF locus) that discriminate between modern and ancient Beijing sublineages. The strains were isolated from native patients in Brazil and Mozambique, countries with a low prevalence of Beijing strains. The virulence levels of these strains were determined in models of pulmonary infection in mice and in vitro macrophage infection and compared with that of a strain from Russia, part of the epidemic and hypervirulent Beijing clone B0/W148, and of the laboratory strain H37Rv. The results showed that two of the three modern Beijing strains were highly pathogenic, exhibiting levels of virulence comparable with that of the epidemic Russian strain. In contrast, all isolates of the ancient sublineage displayed intermediate or low virulence. The data obtained demonstrate that the strains of the modern Beijing sublineage are more likely to exhibit highly virulent phenotypes than ancient strains and suggest that genetic alterations characteristic of the modern Beijing sublineage favor selection of highly virulent bacteria.
Computer vision system (CVSs) are effective tools that enable large‐scale phenotyping with a low‐cost and non‐invasive method, which avoids animal stress. Economically important traits, such as rib ...and loin yield, are difficult to measure; therefore, the use of CVS is crucial to accurately predict several measures to allow their inclusion in breeding goals by indirect predictors. Therefore, this study aimed (1) to validate CVS by a deep learning approach and to automatically predict morphometric measurements in tambaqui and (2) to estimate genetic parameters for growth traits and body yield. Data from 365 individuals belonging to 11 full‐sib families were evaluated. Seven growth traits were measured. After biometrics, each fish was processed in the following body regions: head, rib, loin, R + L (rib + loin). For deep learning image segmentation, we adopted a method based on the instance segmentation of the Mask R‐CNN (Region‐based Convolutional Neural Networks) model. Pearson's correlation values between measurements predicted manually and automatically by the CVS were high and positive. Regarding the classification performance, visible differences were detected in only about 3% of the images. Heritability estimates for growth and body yield traits ranged from low to high. The genetic correlations between the percentage of body parts and morphometric characteristics were favorable and highly correlated, except for percentage head, whose correlations were unfavorable. In conclusion, the CVS validated in this image dataset proved to be resilient and can be used for large‐scale phenotyping in tambaqui. The weight of the rib and loin are traits under moderate genetic control and should respond to selection. In addition, standard length and pelvis length can be used as an efficient and indirect selection criterion for body yield in this tambaqui population.
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