Type 2 inflammation occurs in a large subgroup of asthmatics, and novel cytokine-directed therapies are being developed to treat this population. In mouse models, interleukin-33 (IL-33) activates ...lung resident innate lymphoid type 2 cells (ILC2s) to initiate airway type 2 inflammation. In human asthma, which is chronic and difficult to model, the role of IL-33 and the target cells responsible for persistent type 2 inflammation remain undefined. Full-length IL-33 is a nuclear protein and may function as an “alarmin” during cell death, a process that is uncommon in chronic stable asthma. We demonstrate a previously unidentified mechanism of IL-33 activity that involves alternative transcript splicing, which may operate in stable asthma. In human airway epithelial cells, alternative splicing of the IL-33 transcript is consistently present, and the deletion of exons 3 and 4 (Δ exon 3,4) confers cytoplasmic localization and facilitates extracellular secretion, while retaining signaling capacity. In nonexacerbating asthmatics, the expression of Δ exon 3,4 is strongly associated with airway type 2 inflammation, whereas full-length IL-33 is not. To further define the extracellular role of IL-33 in stable asthma, we sought to determine the cellular targets of its activity. Comprehensive flow cytometry and RNA sequencing of sputum cells suggest basophils and mast cells, not ILC2s, are the cellular sources of type 2 cytokines in chronic asthma. We conclude that IL-33 isoforms activate basophils and mast cells to drive type 2 inflammation in chronic stable asthma, and novel IL-33 inhibitors will need to block all biologically active isoforms.
Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently ...reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs.
The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants.
All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical.
TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.
Female transgenic mice that constitutively overexpress the transcription factor ATF3 in the basal epithelium of the mammary gland develop mammary carcinomas with high frequency, but only if allowed ...to mate and raise pups early in life. This transgenic mouse model system reproduces some features of human breast cancer in that about 20% of human breast tumor specimens exhibit overexpression of ATF3 in the tumor cells. The ATF3-induced mouse tumors are phenotypically similar to mammary tumors induced by overexpression of activating Wnt/β-catenin pathway genes. We now show that the Wnt/β-catenin pathway is indeed activated in ATF3-induced tumors. β-catenin is transcriptionally up-regulated in the tumors, and high levels of nuclear β-catenin are seen in tumor cells. A reporter gene for Wnt/β-catenin pathway activity, TOPGAL, is up-regulated in the tumors and several downstream targets of Wnt signaling, including Ccnd1, Jun, Axin2 and Dkk4, are also expressed at higher levels in ATF3-induced tumors compared to mammary glands of transgenic females. Several positive-acting ligands for this pathway, including Wnt3, Wnt3a, Wnt7b, and Wnt5a, are significantly overexpressed in tumor tissue, and mRNA for Wnt3 is about 5-fold more abundant in transgenic mammary tissue than in non-transgenic mammary tissue. Two known transcriptional targets of ATF3, Snai1 and Snai2, are also overexpressed in the tumors, and Snail and Slug proteins are found to be located primarily in the nuclei of tumor cells. In vitro knockdown of Atf3 expression results in significant decreases in expression of Wnt7b, Tcf7, Snai2 and Jun, suggesting that these genes may be direct transcriptional targets of ATF3 protein. By chromatin immunoprecipitation analysis, both ATF3 and JUN proteins appear to bind to a particular subclass of AP-1 sites upstream of the transcriptional start sites of each of these genes.
Basal-like breast cancer (BLBC) is an aggressive and deadly subtype of human breast cancer that is highly metastatic, displays stem-cell like features, and has limited treatment options. Therefore, ...developing and characterizing preclinical mouse models with tumors that resemble BLBC is important for human therapeutic development.
is a potent oncogene that is aberrantly expressed in most human breast cancers. In the BK5.ATF3 mouse model, overexpression of
in the basal epithelial cells of the mammary gland produces tumors that are characterized by activation of the Wnt/β-catenin signaling pathway. Here, we used RNA-Seq and microRNA (miRNA) microarrays to better define the molecular features of BK5.ATF3-derived mammary tumors. These analyses showed that these tumors share many characteristics of human BLBC including reduced expression of
,
, and
and increased expression of
,
, and the genes encoding keratins 5, 6, and 17. An analysis of miRNA expression revealed reduced levels of
and
, leading to the upregulation of their target genes including both the pluripotency factors
and
as well as the cancer stem-cell-related gene
. Finally, we show through knock-down experiments that
may directly modulate
expression. Taken together, our results indicate that the
mouse mammary tumor model could provide a powerful model to define the molecular mechanisms leading to BLBC, identify the factors that contribute to its aggressiveness, and, ultimately, discover specific genes and gene networks for therapeutic targeting.
Human innate immunity against most African trypanosomes, including Trypanosoma brucei brucei, is mediated by a minor subclass of toxic serum HDL, called trypanosome lytic factor-1 (TLF-1). This HDL ...contains two primate specific proteins, apolipoprotein L-1 and haptoglobin (Hp)-related protein, as well as apolipoprotein A-1. These assembled proteins provide a powerful defense against trypanosome infection. Trypanosoma brucei rhodesiense causes human African sleeping sickness because it has evolved an inhibitor of TLF-1, serum resistance-associated (SRA) protein. Trypanosoma brucei gambiense lacks the SRA gene, yet it infects humans. As transfection of T. b. gambiense (group 1) is not possible, we initially used in vitro-selected TLF-1-resistant T. b. brucei to examine SRA-independent mechanisms of TLF-1 resistance. Here we show that TLF-1 resistance in T. b. brucel is caused by reduced expression of the Hp/Hb receptor gene (TbbHpHbR). Importantly, T. b. gambiense (group 1) also showed a marked reduction in uptake of TLF-1 and a corresponding decrease in expression of T. b. gambiense Hp/Hb receptor (TbgHpHbR). Ectopic expression of TbbHpHbR in TLF-1-resistant T. b. brucei rescued TLF-1 uptake, demonstrating that decreased TbbHpHbR expression conferred TLF-1 resistance. Ectopic expression of TbgHpHbR in TLF-1-resistant T. b. brucei failed to rescue TLF-1 killing, suggesting that coding sequence changes altered Hp/Hb receptor binding affinity for TLF-1. We propose that the combination of coding sequence mutations and decreased expression of TbgHpHbR directly contribute to parasite evasion of human innate immunity and infectivity of group 1 T. b. gambiense.
The late cornified envelope (LCE) gene cluster within the epidermal differentiation complex on human chromosome one (mouse chromosome three) contains multiple conserved genes encoding stratum-corneum ...proteins. Within the LCE cluster, genes form “groups” based on chromosomal position and protein homology. We link a recently accepted nomenclature for the LCE cluster (formerly XP5, small proline-rich-like, late-envelope protein genes) to gene structure, groupings, and chromosomal organization, and carry out a pan-cluster quantitative expression analysis in a variety of tissues and environmental conditions. This analysis shows that (i) the cluster organizes into two “skin” expressing groups and a third group with low-level, tissue-specific expression patterns in all barrier-forming epithelia tested, including internal epithelia; (ii) LCE genes respond “group-wise” to environmental stimuli such as calcium levels and ultraviolet (UV) light, highlighting the functional significance of groups; (iii) in response to UV stimulation there is massive upregulation of a single, normally quiescent, non-skin LCE gene; and (iv) heterogeneity occurs between individuals with one individual lacking expression of an LCE skin gene without overt skin disease, suggesting LCE genes affect subtle attributes of skin function. This quantitative and pan-cluster expression analysis suggests that LCE groups have distinct functions and that within groups regulatory diversification permits specific responsiveness to environmental challenge.
O26 is the commonest non-O157 Shiga toxin (
)-producing
serogroup reported in human infections worldwide. Ruminants, particularly cattle, are the primary reservoir source for human infection. In this ...study, we compared the whole genomes and virulence profiles of O26:H11 strains (
= 99) isolated from Scottish cattle with strains from human infections (
= 96) held by the Scottish
O157/STEC Reference Laboratory, isolated between 2002 and 2020. Bovine strains were from two national cross-sectional cattle surveys conducted between 2002-2004 and 2014-2015. A maximum likelihood phylogeny was constructed from a core-genome alignment with the O26:H11 strain 11368 reference genome. Genomes were screened against a panel of 2,710 virulence genes using the Virulence Finder Database. All
-positive bovine O26:H11 strains belonged to the ST21 lineage and were grouped into three main clades. Bovine and human source strains were interspersed, and the
subtype was relatively clade-specific. Highly pathogenic
2a-only ST21 strains were identified in two herds sampled in the second cattle survey and in human clinical infections from 2010 onwards. The closest pairwise distance was 9 single-nucleotide polymorphisms (SNPs) between Scottish bovine and human strains and 69 SNPs between the two cattle surveys. Bovine O26:H11 was compared to public EnteroBase ST29 complex genomes and found to have the greatest commonality with O26:H11 strains from the rest of the UK, followed by France, Italy, and Belgium. Virulence profiles of
-positive bovine and human strains were similar but more conserved for the
subtype. O26:H11
-negative ST29 (
= 17) and ST396 strains (
= 5) were isolated from 19 cattle herds; all were
-positive, and 10 of these herds yielded strains positive for
,
, and
, gene markers suggestive of enterohaemorrhagic potential. There was a significant association (
< 0.001) between nucleotide sequence percent identity and
status for the bacteriophage insertion site genes
for
and
for
. Acquired antimicrobial resistance genes were identified
in 12.1% of bovine and 17.7% of human O26:H11 strains, with s
,
,
and
being most common. This study describes the diversity among Scottish bovine O26:H11 strains and investigates their relationship to human STEC infections.
The postgenomic era has revolutionized approaches to defining host-pathogen interactions and the investigation of the influence of genetic variation in either protagonist upon infection outcome. We ...analyzed pathology induced by infection with two genetically distinct Trypanosoma brucei strains and found that pathogenesis is partly strain specific, involving distinct host mechanisms. Infections of BALB/c mice with one strain (927) resulted in more severe anemia and greater erythropoietin production compared to infections with the second strain (247), which, contrastingly, produced greater splenomegaly and reticulocytosis. Plasma interleukin-10 (IL-10) and gamma interferon levels were significantly higher in strain 927-infected mice, whereas IL-12 was higher in strain 247-infected mice. To define mechanisms underlying these differences, expression microarray analysis of host genes in the spleen at day 10 postinfection was undertaken. Rank product analysis (RPA) showed that 40% of the significantly differentially expressed genes were specific to infection with one or the other trypanosome strain. RPA and pathway analysis identified LXR/RXR signaling, IL-10 signaling, and alternative macrophage activation as the most significantly differentially activated host processes. These data suggest that innate immune response modulation is a key determinant in trypanosome infections, the pattern of which can vary, dependent upon the trypanosome strain. This strongly suggests that a parasite genetic component is responsible for causing disease in the host. Our understanding of trypanosome infections is largely based on studies involving single parasite strains, and our results suggest that an integrated host-parasite approach is required for future studies on trypanosome pathogenesis. Furthermore, it is necessary to incorporate parasite variation into both experimental systems and models of pathogenesis.
Overexpression of the bZip transcription factor, ATF3, in basal epithelial cells of transgenic mice under the control of the bovine cytokeratin-5 (CK5) promoter has previously been shown to induce ...epidermal hyperplasia, hair follicle anomalies and neoplastic lesions of the oral mucosa including squamous cell carcinomas. CK5 is known to be expressed in myoepithelial cells of the mammary gland, suggesting the possibility that transgenic BK5.ATF3 mice may exhibit mammary gland phenotypes.
Mammary glands from nulliparous mice in our BK5.ATF3 colony, both non-transgenic and transgenic, were examined for anomalies by histopathology and immunohistochemistry. Nulliparous and biparous female mice were observed for possible mammary tumor development, and suspicious masses were analyzed by histopathology and immunohistochemistry. Human breast tumor samples, as well as normal breast tissue, were similarly analyzed for ATF3 expression.
Transgenic BK5.ATF3 mice expressed nuclear ATF3 in the basal layer of the mammary ductal epithelium, and often developed squamous metaplastic lesions in one or more mammary glands by 25 weeks of age. No progression to malignancy was seen in nulliparous BK5.ATF3 or non-transgenic mice held for 16 months. However, biparous BK5.ATF3 mice developed mammary carcinomas with squamous metaplasia between 6 months and one year of age, reaching an incidence of 67%. Cytokeratin expression in the tumors was profoundly disturbed, including expression of CK5 and CK8 (characteristic of basal and luminal cells, respectively) throughout the epithelial component of the tumors, CK6 (potentially a stem cell marker), CK10 (a marker of interfollicular epidermal differentiation), and mIRSa2 and mIRSa3.1 (markers of the inner root sheath of hair follicles). Immunohistochemical studies indicated that a subset of human breast tumors exhibit high levels of nuclear ATF3 expression.
Overexpression of ATF3 in CK5-expressing cells of the murine mammary gland results in the development of squamous metaplastic lesions in nulliparous females, and in mammary tumors in biparous mice, suggesting that ATF3 acts as a mammary oncogene. A subset of human breast tumors expresses high levels of ATF3, suggesting that ATF3 may play an oncogenic role in human breast tumorigenesis, and therefore may be useful as either a biomarker or therapeutic target.
African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this ...development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda) and Southern (Malawi) Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics.
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