Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.

Development of a refined ex vivo model of peritoneal adhesion formation, and a role for connexin 43 in their development.

Despite many advances across the surgical sciences, post-surgical peritoneal adhesions still pose a considerable risk in modern-day procedures and are highly undesirable. We have developed a novel mouse peritoneal strip ex vivo adhesion model which may serve to bridge the gap between single cell culture systems and in vivo animal drug testing for the assessment of potential anti-adhesion agents, and study of causality of the process. We investigated the optimal conditions for adhesion formation with mouse peritoneal tissue strips by modifying an existing ex vivo rat model of peritoneal adhesions. We assessed the impact of the following conditions on the formation of adhesions: contact pressure, abrasions, and the presence of clotted blood. Macroscopic adhesions were detected in all mouse peritoneal strips exposed to specific conditions, namely abrasions and clotted blood, where peritoneal surfaces were kept in contact with pressure using cotton gauze in a tissue cassette. Adhesions were confirmed microscopically. Interestingly, connexin 43, a gap junction protein, was found to be upregulated at sites of adhesions. Key features of this model were the use of padding the abraded tissue with gauze and the use of a standardised volume of clotted blood. Using this model, peritoneal strips cultured with clotted blood between abraded surfaces were found to reproducibly develop adhesion bands at 72 h. Our goal is to develop a model that can be used in genetically modified mice in order to dissect out the role of particular genes in adhesion formation and to test drugs to prevent adhesion formation.

Connexin43 in Post-Surgical Peritoneal Adhesion Formation.

OBJECTIVE: Post-surgical peritoneal adhesions are a serious problem for the quality of life and fertility. Yet there are no effective ways of preventing their occurrence. The gap junction protein Cx43 is known to be involved in fibrosis in several different organs and disease conditions often associated with inflammation. Here we examined the Cx43 dynamic expression in an ischemic button model of surgical adhesions. METHODS: Using the mouse ischemic button model, Cx43 antisense was delivered in Pluronic gel to attenuate Cx43 expression. The severity of button formation and immunofluorescence analysis of Cx43 and TGF-ß1 were performed. The concentration of tissue plasminogen activator via ELISA was also performed. RESULTS: As early as 6 h after button formation, the Cx43 levels were elevated in and around the button and some weak adhesions were formed. By 24 h Cx43 levels had increased further and adhesions were more defined. At 7 days the adhesions were much more robust, opaque, and vascularized, requiring blunt or sharp dissection to break them. Cx43 antisense attenuated its upregulation and, reduced the number and severity of adhesions that formed. CONCLUSION: Targeting Cx43 after surgical procedures may be a potential therapeutic strategy for preventing adhesion formation or at least reducing their severity.

Connexins in endothelial barrier function - novel therapeutic targets countering vascular hyperpermeability.

Prolonged vascular hyperpermeability is a common feature of many diseases. Vascular hyperpermeability is typically associated with changes in the expression patterns of adherens and tight junction proteins. Here, we focus on the less-appreciated contribution of gap junction proteins (connexins) to basal vascular permeability and endothelial dysfunction. First, we assess the association of connexins with endothelial barrier integrity by introducing tools used in connexin biology and relating the findings to customary readouts in vascular biology. Second, we explore potential mechanistic ties between connexins and junction regulation. Third, we review the role of connexins in microvascular organisation and development, focusing on interactions of the endothelium with mural cells and tissue-specific perivascular cells. Last, we see how connexins contribute to the interactions between the endothelium and components of the immune system, by using neutrophils as an example. Mounting evidence of crosstalk between connexins and other junction proteins suggests that we rethink the way in which different junction components contribute to endothelial barrier function. Given the multiple points of connexin-mediated communication arising from the endothelium, there is great potential for synergism between connexin-targeted inhibitors and existing immune-targeted therapeutics. As more drugs targeting connexins progress through clinical trials, it is hoped that some might prove effective at countering vascular hyperpermeability.