Regulation of neutrophil senescence by microRNAs

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease

A Requirement for Argonaute 4 in Mammalian Antiviral Defense

While interferon (IFN) responses are critical for mammalian antiviral defense, induction of antiviral RNA interference (RNAi) is evident. To date, individual functions of the mammalian RNAi and micro RNA (miRNA) effector proteins Argonautes 1-4 (AGO1-AGO4) during virus infection remain undetermined. AGO2 was recently implicated in mammalian antiviral defense, so we examined antiviral activity of AGO1, AGO3, or AGO4 in IFN-competent immune cells. Only AGO4-deficient cells are hyper-susceptible to virus infection. AGO4 antiviral function is both IFN dependent and IFN independent, since AGO4 promotes IFN but also maintains antiviral capacity following prevention of IFN signaling or production. We identified AGO-loaded virus-derived short interfering RNAs (vsiRNAs), a molecular marker of antiviral RNAi, in macrophages infected with influenza or influenza lacking the IFN and RNAi suppressor NS1, which are uniquely diminished without AGO4. Importantly, AGO4-deficient influenza-infected mice have significantly higher burden and viral titers in vivo. Together, our data assign an essential role for AGO4 in mammalian antiviral defense

Integration of multi-omics data from re-wilded laboratory mice to identify key parameters that determine immune heterogeneity and activation

Abstract Current research on immunology are largely based on clean laboratory mice kept in specific pathogen free (SPF) environments. They have the advantage of being inbred, genetically homogeneous and can be manipulated genetically in disease models. However, humans as with all free-living mammals face a more complex environment and are exposed to various food resources, microbial experiences, thermogenic changes, and must constantly respond to environmental challenges. We have established a unique experimental system in which laboratory mice are “re-wilded” through release into an outdoor enclosure facility. Here, we use multi-parameter assays including 16S/Metagenomic sequencing, intestinal RNA-sequencing, and 34 parameters of flow cytometry to explain heterogeneity of immune responses from exposure to the wild environment. Increased immune activation of re-wilded mice occur in the absence of viral, bacterial or parasitic pathogen exposure in this enclosure, including increase of CD4+ and CD8+ memory T cells, effector T cells, innate lymphoid cells (ILCs), circulating neutrophils, reduction in naïve T cells, and increased expression of costimulatory molecules on antigen presenting cells. When we examined cytokine production in response to ex vivo stimulation of mesenteric lymph node cells with 8 bacterial and fungal stimuli, the mesenteric lymph node cells from re-wilded mice are poised for different responses via robust induction of cytokines than laboratory mice. By using machine learning models, we are integrating these multi-omic datasets to better predict the association between environmental and host genetic factors on driving heterogeneity of immune responses in the re-wilded mice.</jats:p

Housing laboratory mice deficient for <i>Nod2</i> and <i>Atg16l1</i> in a natural environment uncovers genetic and environmental contributions to immune variation

Abstract The relative contributions of genetic and environmental factors to variation in immune responses are still poorly understood. Here, we performed a deep phenotypic analysis of immunological parameters of laboratory mice released into an outdoor enclosure, carrying susceptibility genes (Nod2 and Atg16l1) implicated in the development of inflammatory bowel diseases. Variations of immune cell populations were largely driven by environment, whereas cytokine production in response to stimulation was affected more by genetic mutations. Multi-omic models identified transcriptional signatures associated with differences in T cell populations. Subnetworks associated with responses against Clostridium perfringens, Candida albicans and Bacteroides vulgatus were also coupled with rewilding. Hence, exposing laboratory mice carrying different genetic mutations to a natural environment uncovered important contributors to immune variation.</jats:p

Housing laboratory mice deficient for <i>Nod2</i> and <i>Atg16l1</i> in a natural environment uncovers genetic and environmental contributions to immune variation

SUMMARYThe relative contributions of genetic and environmental factors to variation in immune responses are still poorly understood. Here, we performed a deep phenotypic analysis of immunological parameters of laboratory mice released into an outdoor enclosure, carrying susceptibility genes (Nod2 and Atg16l1) implicated in the development of inflammatory bowel diseases. Variations of immune cell populations were largely driven by environment, whereas cytokine production in response to stimulation was affected more by genetic mutations. Multi-omic models identified transcriptional signatures associated with differences in T cell populations. Subnetworks associated with responses against Clostridium perfringens, Candida albicans and Bacteroides vulgatus were also coupled with rewilding. Hence, exposing laboratory mice carrying different genetic mutations to a natural environment uncovered important contributors to immune variation.One sentence summaryNatural environment exposure in laboratory mice primarily promotes variation in population frequencies of immune cells, whereas cytokine responses to stimulation are affected more by genetic susceptibility to inflammatory bowel disease.</jats:sec

Altered immunity of laboratory mice in the natural environment is associated with fungal colonization

AbstractThe immune systems of free-living mammals such as humans and wild mice display a heightened degree of activation compared with laboratory mice maintained under artificial conditions. Here, we demonstrate that releasing inbred laboratory mice into an outdoor enclosure to mimic life in a natural environment alters the state of immunity. In addition to enhancing the differentiation of T cell populations previously associated with pathogen exposure, we found that outdoor release of mice led to an increase in circulating granulocytes. However, rewilded mice were not infected by pathogens previously implicated in immune activation. Rather, changes to the immune system were associated with an altered composition of the microbiota, and fungi isolated from rewilded mice were sufficient to increase circulating granulocytes. These findings establish an experimental procedure to investigate the impact of the natural environment on immune development and identify a role for sustained fungal exposure in determining granulocyte numbers.</jats:p