Lung Microbiota Changes Associated with Chronic Pseudomonas aeruginosa Lung Infection and the Impact of Intravenous Colistimethate Sodium.

BACKGROUND: Exacerbations associated with chronic lung infection with Pseudomonas aeruginosa are a major contributor to morbidity, mortality and premature death in cystic fibrosis. Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density. This potentially suggests a role for the latter in the pathogenesis of exacerbations. However, other data suggesting that changes in P. aeruginosa sputum culture status may not reliably predict an improvement in clinical status, and data indicating no significant changes in either total bacterial counts or in P. aeruginosa numbers in sputum samples collected prior to pulmonary exacerbation sheds doubt on this assumption. We used our recently developed lung segmental model of chronic Pseudomonas infection in sheep to investigate the lung microbiota changes associated with chronic P. aeruginosa lung infection and the impact of systemic therapy with colistimethate sodium (CMS). METHODOLOGY/PRINCIPAL FINDINGS: We collected protected specimen brush (PSB) samples from sheep (n = 8) both prior to and 14 days after establishment of chronic local lung infection with P aeruginosa. Samples were taken from both directly infected lung segments (direct) and segments spatially remote to such sites (remote). Four sheep were treated with daily intravenous injections of CMS between days 7 and 14, and four were treated with a placebo. Necropsy examination at d14 confirmed the presence of chronic local lung infection and lung pathology in every direct lung segment. The predominant orders in lung microbiota communities before infection were Bacillales, Actinomycetales and Clostridiales. While lung microbiota samples were more likely to share similarities with other samples derived from the same lung, considerable within- and between-animal heterogeneity could be appreciated. Pseudomonadales joined the aforementioned list of predominant orders in lung microbiota communities after infection. Whilst treatment with CMS appeared to have little impact on microbial community composition after infection, or the change undergone by communities in reaching that state, when Gram negative organisms (excluding Pseudomonadales) were considered together as a group there was a significant decrease in their relative proportion that was only observed in the sheep treated with CMS. With only one exception the reduction was seen in both direct and remote lung segments. This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment. CONCLUSIONS/SIGNIFICANCE: The proportional increase in Pseudomonadales relative to other Gram negative bacteria in the lungs of sheep treated with systemic CMS highlights the potential for such therapies to inadvertently select or create a niche for bacteria seeding from a persistent source of chronic infection.

Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer.

Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs.

A lung segmental model of chronic Pseudomonas infection in sheep.

BACKGROUND: Chronic lung infection with Pseudomonas aeruginosa is a major contributor to morbidity, mortality and premature death in cystic fibrosis. A new paradigm for managing such infections is needed, as are relevant and translatable animal models to identify and test concepts. We sought to improve on limitations associated with existing models of infection in small animals through developing a lung segmental model of chronic Pseudomonas infection in sheep. METHODOLOGY/PRINCIPAL FINDINGS: Using local lung instillation of P. aeruginosa suspended in agar beads we were able to demonstrate that such infection led to the development of a suppurative, necrotising and pyogranulomatous pneumonia centred on the instilled beads. No overt evidence of organ or systemic compromise was apparent in any animal during the course of infection. Infection persisted in the lungs of individual animals for as long as 66 days after initial instillation. Quantitative microbiology applied to bronchoalveolar lavage fluid derived from infected segments proved an insensitive index of the presence of significant infection in lung tissue (>10(4) cfu/g). CONCLUSIONS/SIGNIFICANCE: The agar bead model of chronic P. aeruginosa lung infection in sheep is a relevant platform to investigate both the pathobiology of such infections as well as novel approaches to their diagnosis and therapy. Particular ethical benefits relate to the model in terms of refining existing approaches by compromising a smaller proportion of the lung with infection and facilitating longitudinal assessment by bronchoscopy, and also potentially reducing animal numbers through facilitating within-animal comparisons of differential therapeutic approaches.

Trichinella spiralis induces de novo expression of group IVC phospholipase A2 in the intestinal epithelium.

Phospholipase A2 (PLA2) enzymes play a central role in the initiation, propagation and resolution of inflammation. Here, we describe de novo expression of group IVC PLA2 (PLA2g4c) within the intestinal epithelium of Trichinella spiralis parasitised mice. This mouse mast cell protease-1 sensitive, calcium-independent PLA2 is not detectable in the jejunal epithelium of uninfected mice but becomes highly expressed within the epithelial compartment within days of nematode establishment. We propose that epithelial PLA2g4c accounts for the increased lysophospholipase activity observed during intestinal nematodiasis and that it plays a major role in the inflammatory response to nematodes.

Aberrant mucosal mast cell protease expression in the enteric epithelium of nematode-infected mice lacking the integrin alphavbeta6, a transforming growth factor-beta1 activator.

Infection of mice with the nematode Trichinella spiralis triggers recruitment and differentiation of intraepithelial intestinal mucosal mast cells expressing mouse mast cell protease 1 (Mcpt-1), which contributes to expulsion of the parasite. Expression of Mcpt-1 is transforming growth factor (TGF)-beta1-dependent in vitro. TGF-beta1, which is secreted within tissues as a biologically inactive complex with latency-associated peptide, requires extracellular modification to become functionally active. The integrin-alpha(nu)beta(6) mediates local activation of TGF-beta(1) in association with epithelia. Using T. spiralis-infected beta(6)(-/-) mice, we show accumulation of mucosal mast cells in the lamina propria of the small intestine with minimal recruitment into the epithelial compartment. This was accompanied by a coordinate reduction in expression of both Mcpt-1 and -2 in the jejunum and increased tryptase expression, whereas Mcpt-9 became completely undetectable. In contrast, the cytokine stem cell factor, a regulator of mast cell differentiation and survival, was significantly up-regulated in T. spiralis-infected beta(6)(-/-) mice compared with infected beta(6)(+/+) controls. Despite these changes, beta(6)(-/-) mice still appeared to expel the worms normally. We postulate that compromised TGF-beta(1) activation within the gastrointestinal epithelial compartment is a major, but not the only, contributing factor to the observed changes in mucosal mast cell protease and epithelial cytokine expression in beta(6)(-/-) mice.

Lymphocyte-independent connective tissue mast cells populate murine synovium.

OBJECTIVE: Mast cells (MCs) are a heterogeneous population of tissue-resident bone marrow-derived cells; distinct MC subpopulations are situated at specific microanatomic locations. The phenotype of the murine synovial MC remains undefined. Since MCs have been implicated in the pathogenesis of inflammatory arthritis, we sought to define the phenotype of the murine synovial MC population in normal and arthritic joints. We also examined the contribution of lymphocytes to synovial MC physiology. METHODS: The MC phenotype in healthy and K/BxN serum transfer-induced arthritic synovial tissue was defined using immunohistochemical staining of prototypic MC-specific proteases (murine MC proteases [mMCP] 1, 2, 4, 5, 6, and 7) (chymases and tryptases). MC numbers and density were determined by histomorphometry in healthy and arthritic synovia. The lymphocyte contribution to MC populations was assessed using RAG-null mice. RESULTS: We found that synovial MCs display a connective tissue mast cell (CTMC) phenotype in both normal and arthritic synovial tissue, which expresses mMCP-4, -5, -6, and -7, but not mMCP-1 or mMCP-2. In addition, MC hyperplasia was seen in the arthritic synovium. In RAG-null mice, the phenotype and degree of MC hyperplasia were identical to those observed in normal mice with and without arthritis. Furthermore, in contrast to skin CTMCs, all synovial MCs expressed mMCP-6, demonstrating discrete differences between synovial CTMCs and other anatomic CTMC populations. CONCLUSION: Our findings demonstrate that the murine synovial MC population is composed of lymphocyte-independent CTMCs and identify arthritic synovium as a model system by which to gain insight into the poorly understood physiology of CTMCs in chronic inflammation.

Integrin-alphavbeta6, a putative receptor for foot-and-mouth disease virus, is constitutively expressed in ruminant airways.

Evolved functions of integrin-alpha(v)beta(6) include roles in epithelial cell-extracellular matrix protein interactions and in the binding and activation of latent TGF-beta(1). Integrin-alpha(v)beta(6) is also exploited as a receptor by foot-and-mouth disease virus (FMDV) and may play a significant role in its transmission and pathogenesis. The ovine beta(6) integrin subunit was cloned and sequenced (EMBL accession no. AJ439062). Screening of normal ovine tissues by RT-PCR and immunocytochemistry confirmed that integrin-alphavbeta6 is restricted to sheep epithelial cells. Integrin-alphavbeta6 expression was detected in epithelia of the airways, oral cavity, gastrointestinal tract, kidney, sweat glands, hair follicle sheaths, and the epidermis of pedal coronary band (PB) but not of normal skin. Consistent with FMDV tropism, integrin-alphavbeta6 was detected within the basal layers of the stratified squamous epithelium of the oral mucosa and PB. In addition, integrin-alphavbeta6 appears to be constitutively expressed in the normal airways of both cattle and sheep. The latter finding suggests that ruminant airway epithelium presents a highly accessible target for initiation of infection with FMDV by inhalation.

Expression of integrin-alphaE by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth factor-beta1-activating integrin alphavbeta6.

Peak intestinal mucosal mast cell (MMC) recruitment coincides with expulsion of Trichinella spiralis, at a time when the majority of the MMCs are located within the epithelium in BALB/c mice. Although expression of integrin-alpha(E)beta(7) by MMCs has not been formally demonstrated, it has been proposed as a potential mechanism to account for the predominantly intraepithelial location of MMCs during nematode infection. Co-expression of integrin-alpha(E)beta(7) and the MMC chymase mouse mast cell protease-1, by mouse bone marrow-derived mast cells, is strictly regulated by transforming growth factor (TGF)-beta(1). However, TGF-beta(1) is secreted as part of a latent complex in vivo and subsequent extracellular modification is required to render it biologically active. We now show, for the first time, that intraepithelial MMCs express integrin-alpha(E)beta(7) in Trichinella-infected BALB/c and S129 mice. In S129 mice that lack the gene for the integrin-beta(6) subunit and, as consequence, do not express the epithelial integrin-alpha(v)beta(6), integrin-alpha(E) expression is virtually abolished and recruitment of MMCs into the intestinal epithelium is dramatically reduced despite significant overall augmentation of the MMC population. Because a major function of integrin-alpha(v)beta(6) is to activate latent TGF-beta(1,) these findings strongly support a role for TGF-beta(1) in both the recruitment and differentiation of murine MMCs during nematode infection.